Reprint of "Role of G protein-coupled estrogen receptor (GPER/GPR30) in maintenance of meiotic arrest in fish oocytes".

PubMed

Thomas, Peter

2018-02-01

An essential role for GPER (formerly known as GPR30) in regulating mammalian reproduction has not been identified to date, although it has shown to be involved in the regulation a broad range of other estrogen-dependent functions. In contrast, an important reproductive role for GPER in the maintenance of oocyte meiotic arrest has been identified in teleost fishes, which is briefly reviewed here. Recent studies have clearly shown that ovarian follicle production of estradiol-17β (E 2 ) maintains meiotic arrest in several teleost species through activation of GPER coupled to a stimulatory G protein (G s ) on oocyte plasma membranes, resulting in stimulation of cAMP production and maintenance of elevated cAMP levels. Studies with denuded zebrafish oocytes and with microinjection of GPER antisense oligonucleotides into oocytes have demonstrated the requirement for both ovarian follicle production of estrogens and expression of GPER on the oocyte surface for maintenance of meiotic arrest. This inhibitory action of E 2 on the resumption of meiosis is mimicked by the GPER-selective agonist G-1, by the GPER agonists and nuclear ER antagonists, ICI 182,780 and tamoxifen, and also by the xenoestrogen bisphenol-A (BPA) and related alkylphenols. GPER also maintains meiotic arrest of zebrafish oocytes through estrogen- and BPA-dependent GPER activation of epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (MAPK) signaling. Interestingly, progesterone receptor component 1 (PGRMC1) is also involved in estrogen maintenance of meiotic arrest through regulation of EGFR expression on the oocyte plasma membrane. The preovulatory surge in LH secretion induces the ovarian synthesis of progestin hormones that activate a membrane progestin receptor alpha (mPRα)/inhibitory G protein (Gi) pathway. It also increases ovarian synthesis of the catecholestrogen, 2-hydroxy-estradiol-17β (2-OHE 2 ) which inhibits the GPER/Gs/adenylyl cyclase pathway. Both of these LH actions cause declines in oocyte cAMP levels resulting in the resumption of meiosis. GPER is also present on murine oocytes but there are no reports of studies investigating its possible involvement in maintaining meiotic arrest in mammals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Imaging the ovary.

PubMed

Feng, Yi; Tamadon, Amin; Hsueh, Aaron J W

2018-05-01

During each reproductive cycle, the ovary exhibits tissue remodelling and cyclic vasculature changes associated with hormonally regulated folliculogenesis, follicle rupture, luteal formation and regression. However, the relationships among different types of follicles and corpora lutea are unclear, and the role of ovarian vasculature in folliculogenesis and luteal dynamics has not been extensively investigated. Understanding of ovarian physiology and pathophysiology relies upon elucidation of ovarian morphology and architecture. This paper summarizes the literature on traditional approaches to the imaging of ovarian structures and discusses recent advances in ovarian imaging. Traditional in-vivo ultrasound, together with histological and electron microscopic approaches provide detailed views of the ovary at organ, tissue and molecular levels. However, in-vivo imaging is limited to antral and larger follicles whereas histological imaging is mainly two-dimensional in nature. Also discussed are emerging approaches in the use of near-infrared fluorophores to image follicles in live animals to detect preantral follicles as well as visualizing ovarian structures using CLARITY in fixed whole ovaries to elucidate three-dimensional interrelationships among follicles, corpora lutea and ovarian vasculature. Advances in ovarian imaging techniques provide new understanding of ovarian physiology and allow for the development of better tools to diagnose ovarian pathophysiology. Copyright © 2018 Reproductive Healthcare Ltd. All rights reserved.

Short-term culture of adult bovine ovarian tissues: chorioallantoic membrane (CAM) vs. traditional in vitro culture systems.

PubMed

Beck, Kylie; Singh, Jaswant; Dar, Mohammad Arshud; Anzar, Muhammad

2018-03-09

A suitable culture system is important for follicle growth in adult bovine ovarian tissue. This study aimed to assess the avian chorioallantoic membrane (CAM) for short-term culture of adult bovine ovarian tissues compared with a traditional in vitro culture system. Ovarian cortical tissues (1-2 mm 3 ), collected from slaughtered adult cows, were randomly assigned to control, CAM or in vitro culture groups. In the control group, ovarian tissues were fixed with paraformaldehyde without culture. In CAM and in vitro culture groups, the ovarian tissues were cultured for up to 5 days and then fixed. Ovarian tissues were examined on culture days 0, 1, 3 and 5 for angiogenesis, follicle morphology and growth. In all groups, primordial and growing (healthy and atretic) follicle densities were determined. In the CAM culture, the avian blood vessel density increased (p < 0.01) over time with a decline (p < 0.001) in the bovine blood vessel density. Healthy primordial, atretic primordial and healthy growing follicle densities were higher (p < 0.05) in CAM-cultured ovarian tissues than in vitro-cultured tissues. Regardless of the culture system, the density of healthy primordial follicles decreased (p < 0.001) over time with an increase in healthy growing follicles on day 3 (p < 0.01) and an increase in atretic (primordial and growing) follicles during the 5-day culture period (p < 0.001). The proportions of healthy primordial and atretic growing follicles were also affected by culture day (p < 0.001). The CAM culture in chick embryos supported the bovine ovarian tissue grafts for 3 days demonstrating that CAM can be used as a satisfactory short-term culture system to assess ovarian tissue health, and to study follicle activation and development.

In Vitro Culture of Ovarian Follicles from Peromyscus

PubMed Central

He, Xiaoming; Toth, Thomas L.

2016-01-01

The ovarian follicle is the fundamental functional tissue unit of mammalian ovary. Each ovarian follicle contains one single oocyte. Isolation and in vitro culture of ovarian follicles to obtain fertilizable oocytes have been regarded as a promising strategy for women to combat infertility. The follicles from Peromyscus are considered as a better model than that from inbred mice for studying follicle culture. This is because Peromyscus mice are outbred (as with humans) with an increased life span. In this article, we reviewed studies on this subject conducted using Peromyscus follicles. These studies show that the conventional 2D micro-drop and 3D hanging-drop approaches established for in vitro culture of early preantral follicles from inbred mice are not directly applicable for cultivating the follicles from Peromyscus However, the efficiency could be significantly improved by culturing multiple early preantral follicles in one hanging drop of Peromyscus ovarian cell-conditioned medium. It is further revealed that the mechanical heterogeneity in the extracellular matrix of ovary is crucial for developing early preantral follicles to the antral stage and for the subsequent ovulation to release cumulus-oocyte complex. These findings may provide valuable guidance for furthering the technology of in vitro follicle culture to restore fertility in the clinic. PMID:27397871

Accuracy and safety verification of ovarian reserve assessment technique for ovarian tissue transplantation using optical coherence tomography in mice ovary

NASA Astrophysics Data System (ADS)

Takae, Seido; Tsukada, Kosuke; Sato, Yorino; Okamoto, Naoki; Kawahara, Tai; Suzuki, Nao

2017-03-01

Except for histological study, there are currently no suitable techniques available for the detection and identification of primordial follicles in ovary of primary ovarian insufficiency patients who have undetectable AMH levels. Also, the ability to locate and quantify follicles on ovarian cortex strips, without fixation, is valuable for patients who could undergo subsequent successful ovarian tissue transplantation. Although optical coherence tomography (OCT) is a well-established high resolution imaging technique without fixation commonly applied in biomedicine, few reports are available on ovarian tissue imaging. In present study, we established standard OCT follicle images at each developmental stage, including the primordial follicle, and demonstrated the efficacy of OCT to estimate IVF outcome in transplanted mice ovary like ovarian reserve tests. Unfortunately, the current commercial OCT could not be used to accurate follicle count the number of follicles for whole ovary, because the maximum depth of examination was 100 μm. And we demonstrated the safety of OCT examination, it did not affect IVF outcome and birth defect rate, and reproductive ability. Although there is room for improvement, these findings will be first step to bring OCT examination a step closer to clinical application for measuring true ovarian reserve and localizing follicles.

Disordered follicle development

PubMed Central

Chang, R. Jeffrey; Cook-Andersen, Heidi

2013-01-01

Alterations of ovarian follicle morphology and function have been well documented in women with PCOS. These include increased numbers of growing preantral follicles, failure of follicle growth beyond the mid-antral stage, evidence of granulosa call degeneration, and theca cell hyperplasia. Functional abnormalities include paradoxical granulosa cell hyperresponsiveness to FSH which is clinically linked to ovarian hyperstimulation during ovulation induction. In addition, there is likely a primary theca cell defect that accounts for the majority of excess androgen production in this disorder. The precise mechanisms responsible for altered follicle function are not completely clear. However, several factors appear to influence normal advancement of follicle development as well as impair ovarian steroidogenesis. These include intra- as well as extraovarian influences that distort normal ovarian growth and disrupt steroid production by follicle cells. PMID:22874072

A putative role for anti-Müllerian hormone (AMH) in optimising ovarian reserve expenditure.

PubMed

Pankhurst, Michael W

2017-04-01

The mammalian ovary has a finite supply of oocytes, which are contained within primordial follicles where they are arrested in a dormant state. The number of primordial follicles in the ovary at puberty is highly variable between females of the same species. Females that enter puberty with a small ovarian reserve are at risk of a shorter reproductive lifespan, as their ovarian reserve is expected to be depleted faster. One of the roles of anti-Müllerian hormone (AMH) is to inhibit primordial follicle activation, which slows the rate at which the ovarian reserve is depleted. A simple interpretation is that the function of AMH is to conserve ovarian reserve. However, the females with the lowest ovarian reserve and the greatest risk of early reserve depletion have the lowest levels of AMH. In contrast, AMH apparently strongly inhibits primordial follicle activation in females with ample ovarian reserve, for reasons that remain unexplained. The rate of primordial follicle activation determines the size of the developing follicle pool, which in turn, determines how many oocytes are available to be selected for ovulation. This review discusses the evidence that AMH regulates the size of the developing follicle pool by altering the rate of primordial follicle activation in a context-dependent manner. The expression patterns of AMH across life are also consistent with changing requirements for primordial follicle activation in the ageing ovary. A potential role of AMH in the fertility of ageing females is proposed herein. © 2017 Society for Endocrinology.

Embryonic exposure to benzo(a)pyrene inhibits reproductive capability in adult female zebrafish and correlation with DNA methylation.

PubMed

Gao, Dongxu; Lin, Jing; Ou, Kunlin; Chen, Ying; Li, Hongbin; Dai, Qinhua; Yu, Zhenni; Zuo, Zhenghong; Wang, Chonggang

2018-05-09

This study was conducted to investigate the effects of embryonic short-term exposure to benzo(a)pyrene (BaP), a model polycyclic aromatic hydrocarbon, on ovarian development and reproductive capability in adult female zebrafish. In 1-year-old fish after embryonic exposure to BaP for 96 h, the gonadosomatic indices and the percentage of mature oocytes were significantly decreased in the 0.5, 5 and 50 nmol/L treatments. The spawned egg number, the fertilization rate and the hatching success were significantly reduced, while the malformation rate of the F1 unexposed larvae were increased. The mRNA levels of follicle-stimulating hormone, luteinizing hormone, ovarian cytochrome P450 aromatase cyp19a1a and cyp19b, estrogen receptor esr1 and esr2, and hepatic vitellogenin vtg1 and vtg2 genes, were down-regulated in adult female zebrafish that were exposed to BaP during embryonic stage. Both 17β-estradiol and testosterone levels were reduced in the ovary of adult females. The methylation levels of the gonadotropin releasing hormone (GnRH) gene gnrh3 were significantly increased in the adult zebrafish brain, and those of the GnRH receptor gene gnrhr3 were elevated both in the larvae exposed to BaP and in the adult brain, which might cause the down-regulation of the mRNA levels of gnrh3 and gnrhr3. This epigenetic change caused by embryonic exposure to BaP might be a reason for physiological changes along the brain-pituitary-gonad axis. These results suggest that short-term exposure in early life should be included and evaluated in any risk assessment of pollutant exposure to the reproductive health of fish. Copyright © 2018 Elsevier Ltd. All rights reserved.

Advancing ovarian folliculometry with selective plane illumination microscopy

NASA Astrophysics Data System (ADS)

Lin, Hsiao-Chun Amy; Dutta, Rahul; Mandal, Subhamoy; Kind, Alexander; Schnieke, Angelika; Razansky, Daniel

2016-12-01

Determination of ovarian status and follicle monitoring are common methods of diagnosing female infertility. We evaluated the suitability of selective plane illumination microscopy (SPIM) for the study of ovarian follicles. The large field of view and fast acquisition speed of our SPIM system enables rendering of volumetric image stacks from intact whole porcine ovarian follicles, clearly visualizing follicular features including follicle volume and average diameter (70 μm-2.5 mm), their spherical asymmetry parameters, size of developing cumulus oophorus complexes (40 μm-110 μm), and follicular wall thickness (90 μm-120 μm). Follicles at all developmental stages were identified. A distribution of the theca thickness was measured for each follicle, and a relationship between these distributions and the stages of follicular development was discerned. The ability of the system to non-destructively generate sub-cellular resolution 3D images of developing follicles, with excellent image contrast and high throughput capacity compared to conventional histology, suggests that it can be used to monitor follicular development and identify structural abnormalities indicative of ovarian ailments. Accurate folliculometric measurements provided by SPIM images can immensely help the understanding of ovarian physiology and provide important information for the proper management of ovarian diseases.

The effect of ovarian follicle size on pituitary and ovarian responses to copulation in domesticated South American camelids.

PubMed

Bravo, P W; Stabenfeldt, G H; Lasley, B L; Fowler, M E

1991-10-01

The relation of ovarian follicle size to pituitary and ovarian responses to copulation was studied in domesticated South American camelids (llamas and alpacas). Females from each species were divided into four groups according to follicle size: small (4-5 mm), growing (6-7 mm), mature (8-12 mm), and regressing (10-7 mm). The pituitary response to copulation was determined by analysis of LH and FSH concentrations in plasma. The ovarian response to copulation was determined by ultrasonography and by analysis of estrone sulfate (follicular status) and pregnanediol glucuronide (luteal status) concentrations in urine. Females with small follicles (4-5 mm) released less LH after copulation than did those with larger follicles, and ovulation was not induced. Females with growing and mature follicles (7-12 mm) released LH in response to copulation that was adequate to induce ovulation and to initiate normal luteal activity. While copulation-induced LH release in females with regressing follicles was similar to that released in animals with growing and mature follicles, regressing follicles were luteinized instead of being ovulated. The luteal structure formed as a result of luteinization of follicles had a short life span, i.e., 5.1 days. Copulation-induced LH release was significantly higher in llamas vs. alpacas in animals with mature or regressing follicles, but not in those with small or growing follicles. Urinary estrone sulfate and pregnanediol glucuronide concentrations correlated positively with the presence of follicles and corpora lutea, respectively.

Bovine ovarian follicular growth and development correlate with lysophosphatidic acid expression.

PubMed

Sinderewicz, Emilia; Grycmacher, Katarzyna; Boruszewska, Dorota; Kowalczyk-Zięba, Ilona; Staszkiewicz, Joanna; Ślężak, Tomasz; Woclawek-Potocka, Izabela

2018-01-15

The basis of successful reproduction is proper ovarian follicular growth and development. In addition to prostaglandins and vascular endothelial growth factor, a number of novel factors are suggested as important regulators of follicular growth and development: PGES, TFG, CD36, RABGAP1, DBI and BTC. This study focuses on examining the expression of these factors in granulosa and thecal cells that originate from different ovarian follicle types and their link with the expression of lysophosphatidic acid (LPA), known local regulator of reproductive functions in the cow. Ovarian follicles were divided into healthy, transitional, and atretic categories. The mRNA expression levels for PGES, TFG, CD36, RABGAP1, DBI and BTC in granulosa and thecal cells in different follicle types were measured by real-time PCR. The correlations among expression of enzymes synthesizing LPA (autotaxin, phospholipase A2), receptors for LPA and examined factors were measured. Immunolocalization of PGES, TFG, CD36, RABGAP1, DBI and BTC was examined by immunohistochemistry. We investigated follicle-type dependent mRNA expression of factors potentially involved in ovarian follicular growth and development, both in granulosa and thecal cells of bovine ovarian follicles. Strong correlations among receptors for LPA, enzymes synthesizing LPA, and the examined factors in healthy and transitional follicles were observed, with its strongest interconnection with TFG, DBI and RABGAP1 in granulosa cells, and TFG in thecal cells; whereas no correlations in atretic follicles were detected. A greater number of correlations were found in thecal cells than in granulosa cells as well as in healthy follicles than in transitional follicles. These data indicate the role of LPA in the growth, development and physiology of the bovine ovarian follicle. Copyright © 2017 Elsevier Inc. All rights reserved.

Short-term transplantation of isolated human ovarian follicles and cortical tissue into nude mice.

PubMed

Dolmans, Marie-Madeleine; Martinez-Madrid, Belen; Gadisseux, Elodie; Guiot, Yves; Yuan, Wu Yuan; Torre, Antoine; Camboni, Alessandra; Van Langendonckt, Anne; Donnez, Jacques

2007-08-01

This study was designed to evaluate follicular survival and growth after short-term transplantation of fresh isolated human follicles and ovarian cortical tissue to nude mice. Ovarian biopsies were obtained from nine women undergoing laparoscopy. Twelve nude mice were xenografted with an ovarian cortical fragment in the right ovarian bursa, and a clot containing isolated follicles in the left, for a period of 7 days. One ungrafted fragment was used as a control. Histological sections were analyzed to determine follicle number and stage. The proliferative status of follicular cells was assessed by Ki-67 immunostaining. A total of 659 follicles was analyzed by histology and 545 follicles by immunohistochemistry. The percentage of primordial follicles was found to be markedly reduced 1 week post-grafting when compared with ungrafted tissue, while the percentage of primary follicles had significantly increased. Only 8% of follicles showed Ki-67-positive granulosa cells before grafting, whereas 1 week after grafting, 71% of follicles in fragments and 67% of isolated follicles were Ki-67-positive (P<0.001). Moreover, the histological aspect of isolated follicle grafts was similar to that of grafted fragments: follicles were surrounded by vimentin-positive stroma-like tissue of human origin, as confirmed by fluorescent in situ hybridization with human-specific probes. Our results demonstrate, for the first time, that isolated human follicles are able to survive and grow after xenografting. This study also shows massive in vivo follicular activation after transplantation of grafted fragments and isolated follicles. One week after grafting, well-structured stroma-like tissue of human origin was observed around the isolated follicles. The potential origin of this stroma is discussed.

A prospective evaluation of antral follicle function in women with 46,XX spontaneous primary ovarian insufficiency

PubMed Central

Hubayter, Ziad R; Popat, Vaishali; Vanderhoof, Vien H; Ndubizu, Obioma; Johnson, Diane; Mao, Edie; Calis, Karim A; Troendle, James F.; Nelson, Lawrence M.

2010-01-01

Objective To assess ovarian follicle function in women with 46,XX spontaneous primary ovarian insufficiency Design Case-control with nested prospective cohort Setting Clinical Research Center, National Institutes of Health Patients Women with primary ovarian insufficiency without estrogen replacement for two weeks (N=97) and regularly menstruating control women (N=42) Interventions Single injection of 300 IU hrFSH Main outcome measures Change in serum estradiol at 24 hours Results Antral follicles ≥ 3 mm were detected in 73% (69/95) of patients; both serum estradiol and progesterone levels correlated significantly with maximum follicle diameter in these women. Patients with a maximum follicle diameter ≥ 8 mm had significantly higher serum estradiol and progesterone levels and significantly lower FSH and LH levels as compared to patients without such follicles. In controls estradiol levels increased significantly after FSH administration but in patients this was not the case despite the presence of an antral follicle ≥ 8 mm. Conclusion Most women with 46,XX spontaneous primary ovarian insufficiency have antral follicles detectable by ultrasound, suggesting that down-regulation of FSH receptors is not the predominant mechanism of follicle dysfunction. Evidence of progesterone secretion by antral follicles ≥ 8 mm in these patients is consistent with prior histologic evidence that follicle luteinization is the predominant mechanism of follicle dysfunction in this condition. Prospective controlled investigation designed to improve ovulatory function and fertility in these women is indicated. PMID:19939372

Eliminating malignant cells from cryopreserved ovarian tissue is possible in leukaemia patients.

PubMed

Soares, Michelle; Saussoy, Pascale; Maskens, Mathilde; Reul, Hélène; Amorim, Christiani A; Donnez, Jacques; Dolmans, Marie-Madeleine

2017-07-01

Reimplantation of cryopreserved ovarian tissue (OT) can successfully restore ovarian function in young cancer patients after gonadotoxic treatment. However, for patients with leukaemia, there is a risk of malignant cell transmission. Our objective was to evaluate minimal disseminated disease in OT from leukaemia patients and test a follicle isolation technique to obtain disease-free follicle suspensions. Cryopreserved OT from 12 leukaemia patients was thawed and analysed by histology and long-term xenografting in immunosuppressed mice. In 10 patients, follicles were isolated from OT, and polymerase chain reaction (PCR) was performed on tissue, digested ovarian suspensions and isolated follicle suspensions to investigate leukaemic cell presence. Mean patient age was 17·1 years. An average of 3·2 follicles were isolated per mm² of cortex. Xenografting of OT induced leukaemic masses in 2/12 mice. PCR identified leukaemic cell presence in 66% of OT. Malignant cells were also detected in digested ovarian suspensions. However, none of the follicle samples (>2300 follicles tested) showed any malignant cell presence after washing. This study demonstrates that it is possible to recover large numbers of viable follicles from cryopreserved OT of leukaemia patients. All isolated and washed follicle suspensions tested negative for leukaemic cells, giving leukaemia patients genuine hope of fertility restoration. © 2017 John Wiley & Sons Ltd.

Ovine secondary follicles vitrified out the ovarian tissue grow and develop in vitro better than those vitrified into the ovarian fragments.

PubMed

Lunardi, Franciele Osmarini; de Aguiar, Francisco Leo Nascimento; Duarte, Ana Beatriz Graça; Araújo, Valdevane Rocha; de Lima, Laritza Ferreira; Ribeiro de Sá, Naiza Arcângela; Vieira Correia, Hudson Henrique; Domingues, Sheyla Farhayldes Souza; Campello, Cláudio Cabral; Smitz, Johan; de Figueiredo, José Ricardo; Ribeiro Rodrigues, Ana Paula

2016-04-15

Cryopreservation of preantral follicles is a promising technique to preserve female fertility. The aim of this study was to evaluate the effect of vitrification on the development of secondary follicles included in ovarian tissue or isolated after microdissection. An important end point included is the capacity of grown oocytes to resume meiosis. Sheep ovarian cortexes were cut into fragments and split into three different groups: (1) fresh (control): secondary follicles isolated without any previous vitrification; (2) follicle-vitrification (follicle-vit): secondary follicles vitrified in isolated form; and (3) tissue-vitrification (tissue-vit): secondary follicles vitrified within fragments of ovarian tissue (in situ former) and subsequently subjected to isolation. From the three groups, isolated secondary follicles were submitted to IVC for 18 days. After IVC, cumulus-oocyte complexes (COCs) were harvested from follicles. As an additional control group, in vivo grown, in vivo-grown COCs were collected from antral ovarian follicles. All, recovered COCs were matured and the chromatin configuration was evaluated. Data were analyzed by ANOVA, and the means were compared by Student-Newman-Keuls test, and by chi-square. Differences were considered to be significant when P < 0.05. Isolated preantral follicles from all treatments had normal morphology, antrum formation, and low follicle degeneration after IVC. The growth rate between control and follicle-vit did not differ (P > 0.05), and was higher (P < 0.05) than for tissue-vit. The percentage of follicles that decreased diameter during IVC was significantly higher in tissue-vit than the in follicle-vit. Recovery rate of oocytes from normal follicles was higher in follicle-vit than in tissue-vit. Furthermore, oocyte viability was lower in tissue-vit than other treatments, and follicle-vit did not differ from control and in vivo grown. The percentage of oocytes meiosis resuming was not different between treatments except for in vivo grown. After vitrification, only follicle-vit showed metaphase I oocyte. We conclude that secondary follicles vitrified after isolation displayed a better follicular growth rate, oocyte viability, percentage of oocytes reaching the metaphase I stage, and fewer follicles with decreased diameter after IVC. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of cryopreservation and transplantation on the expression of kit ligand and anti-Mullerian hormone in human ovarian tissue.

PubMed

David, Anu; Van Langendonckt, Anne; Gilliaux, Sébastien; Dolmans, Marie-Madeleine; Donnez, Jacques; Amorim, Christiani A

2012-04-01

Although cryopreservation and transplantation of ovarian tissue represent a promising alternative to safeguard fertility in cancer patients, low recovery rates of oocytes aspirated from antral follicles and a significant number of empty follicles have been observed in women with transplanted frozen-thawed ovarian tissue. In order to understand how freezing and/or grafting may affect follicular development, the follicular expression of kit ligand (KL) and anti-Müllerian hormone (AMH), two key factors activating and inhibiting follicle growth, were assessed after long-term grafting in severe combined immunodeficient (SCID) mice. Ovarian biopsies from eight patients were used for fresh and frozen-thawed tissue xenografting in 13 SCID mice for a period of 28 weeks, including 2 weeks of gonadotrophin stimulation. KL, AMH and proliferating cell nuclear antigen (PCNA) immunostaining were quantified before and after grafting in the two treatment groups (fresh and frozen-thawed grafted ovarian tissue). Lower expression of KL was found in primordial and primary follicles after grafting of both fresh and frozen-thawed tissue. Consistent expression of AMH was found in most growing follicles at a similar rate in both graft types. In fresh and frozen-thawed grafts, 13-14% of primordial follicles were PCNA-positive, indicating a similar maintenance of quiescent follicles despite follicle activation. Grafting and/or gonadotrophin stimulation appear to affect the follicular expression of KL, which may alter oocyte quality. AMH expression in growing follicles after ovarian tissue transplantation may be one of the factors contributing to the preservation of resting follicles in 28-week-old grafts.

Positive effect of resveratrol against preantral follicles degeneration after ovarian tissue vitrification.

PubMed

Rocha, Carina Diniz; Soares, Mayara Mafra; de Cássia Antonino, Deize; Júnior, Jairo Melo; Freitas Mohallem, Renata Ferreira; Ribeiro Rodrigues, Ana Paula; Figueiredo, José Ricardo; Beletti, Marcelo Emílio; Jacomini, José Octavio; Alves, Benner Geraldo; Alves, Kele Amaral

2018-07-01

This study aimed to evaluate whether the addition of resveratrol to vitrification/thawing medium improves the cryotolerance of preantral follicles enclosed in bovine ovarian fragments. Ovarian fragments were obtained from bovine fetuses and distributed to the following groups: fresh ovarian fragments (control), vitrified (VIT), and vitrified with resveratrol (VIT + RESV). Overall, the mean percentage of normal follicles was greater (P < 0.05) in the VIT + RESV compared to the VIT group. Moreover, the probability of finding normal follicles was 2.5 greater (P < 0.05) in the VIT + RESV group. In class comparison, the primordial and transitional follicles have ∼3.0 times (P < 0.05) greater odds of being normal after vitrification compared to the secondary follicles. Additionally, a negative association (P < 0.05) was observed between the proportion of viable follicles and the stage of follicular development. ROS levels were similar (P > 0.05) between the VIT and VIT + RESV groups, and both were lower (P < 0.05) than the control group. The tissue viability in the VIT + RESV group was similar (P > 0.05) to the control group. In summary, the resveratrol provided greater ovarian tissue viability and has a positive effect against degeneration of preantral follicles enclosed in ovarian fragments. Copyright © 2018 Elsevier Inc. All rights reserved.

Engineering the Follicle Microenvironment

PubMed Central

West, Erin R.; Shea, Lonnie D.; Woodruff, Teresa K.

2008-01-01

In vitro ovarian follicle culture provides a tool to investigate folliculogenesis, and may one day provide women with fertility-preservation options. The application of tissue engineering principles to ovarian follicle maturation may enable the creation of controllable microenvironments that will coordinate the growth of the multiple cellular compartments within the follicle. Three-dimensional culture systems can preserve follicle architecture, thereby maintaining critical cell–cell and cell–matrix signaling lost in traditional two-dimensional attached follicle culture systems. Maintaining the follicular structure while manipulating the biochemical and mechanical environment will enable the development of controllable systems to investigate the fundamental biological principles underlying follicle maturation. This review describes recent advances in ovarian follicle culture, and highlights the tissue engineering principles that may be applied to follicle culture, with the ultimate objective of germline preservation for females facing premature infertility. PMID:17594609

Light-sheet microscopy for quantitative ovarian folliculometry

NASA Astrophysics Data System (ADS)

Lin, Hsiao-Chun Amy; Dutta, Rahul; Mandal, Subhamoy; Kind, Alexander; Schnieke, Angelika; Razansky, Daniel

2017-02-01

Determination of ovarian status and follicle monitoring are common methods of diagnosing female infertility. We evaluated the suitability of selective plane illumination microscopy (SPIM) for the study of ovarian follicles. Owing to the large field of view and fast acquisition speed of our newly developed SPIM system, volumetric image stacks from entire intact samples of pig ovaries have been rendered demonstrating clearly discernible follicular features like follicle diameters (70 μm - 2.5 mm), size of developing Cumulus oophorus complexes (COC ) (40 μm - 110 μm), and follicular wall thicknesses (90 μm-120 μm). The observation of clearly distinguishable COCs protruding into the follicular antrum was also shown possible, and correlation with the developmental stage of the follicles was determined. Follicles of all developmental stages were identified, and even the small primordial follicle clusters forming the egg nest could be observed. The ability of the system to non-destructively generate sub-cellular resolution 3D images of developing follicles, with excellent image contrast and high throughput capacity compared to conventional histology, suggests that it can be used to monitor follicular development and identify structural abnormalities indicative of ovarian ailments. Accurate folliculometric measurements provided by SPIM images can immensely help the understanding of ovarian physiology and provide important information for the proper management of ovarian diseases.

Expression of TNF-α system members in bovine ovarian follicles and the effects of TNF-α or dexamethasone on preantral follicle survival, development and ultrastructure in vitro.

PubMed

Silva, A W B; Ribeiro, R P; Menezes, V G; Barberino, R S; Passos, J R S; Dau, A M P; Costa, J J N; Melo, L R F; Bezerra, F T G; Donato, M A M; Peixoto, C A; Matos, M H T; Gonçalves, P B D; van den Hurk, R; Silva, J R V

2017-07-01

This study was conducted to detect the protein expression of TNF-α system members (TNF-α/TNFR1/TNFR2) in bovine ovarian follicles and to evaluate the effects of TNF-α or dexamethasone on the survival and growth of primordial follicles in vitro, as well as on gene expression in cultured ovarian tissue. It was hypothesized that TNF-α induces follicular atresia in ovarian tissues cultured in vitro, and that dexamethasone suppresses the production of endogenous TNF-α, which can improve follicle viability in vitro. Ovarian fragments were cultured for 6days in α-MEM + supplemented with TNF-α (0, 1, 10, 100 or 200ng/ml) or dexamethasone (0, 1, 10, 100 or 200ng/ml). After culture, the expression of mRNAs for BCL-2, BAX, P53, TNF-α, and CASP3 and CASP6 were evaluated. Immunohistochemical results showed that the TNF-α system members, were detected in bovine preantral and antral follicles. After 6days, the TNF-α (10ng/ml) treatment reduced the percentage of normal preantral follicles and increased the number of TUNEL-positive cells in cultured tissue. Dexamethasone (10ng/ml) during 6days of culture did maintain the percentage of normal follicles and the ultrastructure of follicles, while the presence of TNF-α or dexamethasone did not influence primordial follicle activation. However, TNF-α or dexamethasone had no effect on the levels of mRNA for P53, BCL-2, BAX and CASP6, in cultured tissues, but the presence of dexamethasone reduced the levels of CASP3 compared to ovarian slices cultured in control medium (α-MEM + ). In conclusion, proteins of the TNF-α system are expressed at different bovine follicle stages. The addition of TNF-α in culture reduces follicle survival and increases the number of apoptotic cells in ovarian tissue, while the presence of dexamethasone maintains follicle ultrastructure in cultured tissue. Copyright © 2017. Published by Elsevier B.V.

Fertility rescue and ovarian follicle growth promotion by bone marrow stem cell infusion.

PubMed

Herraiz, Sonia; Buigues, Anna; Díaz-García, César; Romeu, Mónica; Martínez, Susana; Gómez-Seguí, Inés; Simón, Carlos; Hsueh, Aaron J; Pellicer, Antonio

2018-05-01

To assess if infusion of human bone marrow-derived stem cells (BMDSCs) could promote follicle development in patients with impaired ovarian functions. Experimental design. University research laboratories. Immunodeficient NOD/SCID female mice. Human BMDSCs were injected into mice with chemotherapy-induced ovarian damage and into immunodeficient mice xenografted with human cortex from poor-responder patients (PRs). Follicle development, ovulation, and offspring. Apoptosis, proliferation, and vascularization were evaluated in mouse and human ovarian stroma. Fertility rescue and spontaneous pregnancies were achieved in mice ovaries mimicking PRs and ovarian insufficiency, induced by chemotherapy, after BMDSC infusion. Furthermore, BMDSC treatment resulted in production of higher numbers of preovulatory follicles, metaphase II oocytes, 2-cell embryos, and healthy pups. Stem cells promoted ovarian vascularization and cell proliferation, along with reduced apoptosis. In xenografted human ovarian tissues from PRs, infusion of BMDSCs and their CD133+ fraction led to their engraftment close to follicles, resulting in promotion of follicular growth, increases in E 2 secretion, and enhanced local vascularization. Our results raised the possibility that promoting ovarian angiogenesis by BMDSC infusion could be an alternative approach to improve follicular development in women with impaired ovarian function. NCT02240342. Copyright © 2018 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Activated ovarian endothelial cells promote early follicular development and survival.

PubMed

Kedem, Alon; Aelion-Brauer, Anate; Guo, Peipei; Wen, Duancheng; Ding, Bi-Sen; Lis, Raphael; Cheng, Du; Sandler, Vladislav M; Rafii, Shahin; Rosenwaks, Zev

2017-09-19

New data suggests that endothelial cells (ECs) elaborate essential "angiocrine factors". The aim of this study is to investigate the role of activated ovarian endothelial cells in early in-vitro follicular development. Mouse ovarian ECs were isolated using magnetic cell sorting or by FACS and cultured in serum free media. After a constitutive activation of the Akt pathway was initiated, early follicles (50-150 um) were mechanically isolated from 8-day-old mice and co-cultured with these activated ovarian endothelial cells (AOEC) (n = 32), gel (n = 24) or within matrigel (n = 27) in serum free media for 14 days. Follicular growth, survival and function were assessed. After 6 passages, flow cytometry showed 93% of cells grown in serum-free culture were VE-cadherin positive, CD-31 positive and CD 45 negative, matching the known EC profile. Beginning on day 4 of culture, we observed significantly higher follicular and oocyte growth rates in follicles co-cultured with AOECs compared with follicles on gel or matrigel. After 14 days of culture, 73% of primary follicles and 83% of secondary follicles co-cultured with AOEC survived, whereas the majority of follicles cultured on gel or matrigel underwent atresia. This is the first report of successful isolation and culture of ovarian ECs. We suggest that co-culture with activated ovarian ECs promotes early follicular development and survival. This model is a novel platform for the in vitro maturation of early follicles and for the future exploration of endothelial-follicular communication. In vitro development of early follicles necessitates a complex interplay of growth factors and signals required for development. Endothelial cells (ECs) may elaborate essential "angiocrine factors" involved in organ regeneration. We demonstrate that co-culture with ovarian ECs enables culture of primary and early secondary mouse ovarian follicles.

Triennial Reproduction Symposium: The ovarian follicular reserve in cattle: What regulates its formation and size?1,2

PubMed Central

Fortune, J. E.; Yang, M. Y.; Allen, J. J.; Herrick, S. L.

2017-01-01

The ovarian follicular reserve has been linked to fertility in cattle. Young adult cattle with low vs. high numbers of antral follicles ≥ 3 mm in diameter in follicular waves also have fewer preantral follicles and decreased fertility. This underscores the importance of understanding the factors that regulate early follicular development and establish the ovarian follicular reserve, but little is known about how the follicular reserve is first established. In ruminants and humans, follicles form during fetal life, but there is a gap (about 50 d in cattle) between the appearance of the first primordial follicles and the first growing, primary follicles. In this review we present evidence that in cattle, fetal ovarian steroids (i.e., estradiol and progesterone) are negative regulators of both follicle formation and of the acquisition by newly formed follicles of the capacity to activate (i.e., initiate growth). The results indicate that capacity to activate is linked to the completion of meiotic prophase I by the oocyte. The inhibitory effects of estradiol on follicle activation were found to be reversible and correlated with inhibition of the progression of meiotic prophase I. Fetal bovine ovaries produce steroid hormones and production varies considerably during gestation and in a pattern consistent with the hypothesis that they inhibit follicle formation and capacity of newly formed follicles to activate in vivo. However, little was known about how steroid production is regulated. In our studies, both LH and FSH stimulated progesterone and estradiol production by ovarian pieces in vitro. The addition of testosterone to the culture medium enhanced estradiol production, especially when FSH was also present, but inhibited progesterone production, even in the presence of gonadotropins. Evidence is also presented for effects of maternal nutrition and health and for potential effects of estrogenic endocrine-disrupting chemicals on the size of the ovarian follicular reserve established during fetal life. In summary, fetal ovarian steroids may be important regulators of the early stages of follicular development in cattle. Therefore, external factors that alter steroid production or action may affect the size of the ovarian follicular reserve. PMID:23736047

Short-term storage of canine preantral ovarian follicles using a powdered coconut water (ACP)-based medium.

PubMed

Lima, G L; Costa, L L M; Cavalcanti, D M L P; Rodrigues, C M F; Freire, F A M; Fontenele-Neto, J D; Silva, A R

2010-07-01

The objective was to investigate the use of powdered coconut water (ACP)-based medium for short-term preservation of canine preantral follicles. Pairs of ovaries from mongrel bitches (n=9) were divided into fragments. One ovarian fragment, treated as a fresh control, was immediately fixed for histological analysis, whereas the other six ovarian fragments were stored either in phosphate-buffered saline (PBS; control group) or ACP medium in isothermal Styrofoam boxes containing biological ice packs. The boxes were sealed and opened only after 12, 24, or 36h. After opening each box, the ovarian fragments were submitted to histological analysis. In total, 12,302 preantral follicles were evaluated, with 64.5% primordial, 33.3% primary, and 2.3% secondary follicles. There were multiple oocytes in 1.3% of the follicles analyzed. At 24h, ACP was more efficient in preserving follicular morphology than PBS (P<0.05). Compared with the fresh control group, a significant reduction in the percentage of morphologically normal ovarian follicles was observed for PBS, starting at 24h; however, the decline started only at 36h for the ACP medium. During the experiment, the temperature inside the isothermal boxes increased from 3 to 9 degrees C (P<0.05), despite a constant room temperature. In conclusion, powdered coconut water (ACP) was an appropriate medium for short-term storage of canine preantral ovarian follicles.

First transplantation of isolated murine follicles in alginate.

PubMed

Vanacker, Julie; Dolmans, Marie-Madeleine; Luyckx, Valérie; Donnez, Jacques; Amorim, Christiani A

2014-01-01

Our aim is to develop an artificial ovary allowing survival and growth of isolated follicles and ovarian cells, to restore fertility in women diagnosed with pathologies at high risk of ovarian involvement. For this, alginate beads containing isolated preantral follicles and ovarian cells were autografted to immunocompetent mice. One week after grafting, the beads were invaded by proliferating murine cells (12.1%) and capillaries. The recovery rate of follicles per graft ranged from 0% to 35.5%. Of the analyzed follicles, 77% were Ki67-positive and 81%, TUNEL-negative. Three antral follicles were also identified, evidencing their ability to grow in the matrix. Our results suggest that an artificial ovary is now conceivable, opening new perspectives to restore fertility in women.

Parabens Accelerate Ovarian Dysfunction in a 4-Vinylcyclohexene Diepoxide-Induced Ovarian Failure Model

PubMed Central

Lee, Jae-Hwan; Lee, Myeongho; Ahn, Changhwan; Kang, Hee Young; Tran, Dinh Nam; Jeung, Eui-Bae

2017-01-01

Parabens are widely used preservatives in basic necessities such as cosmetic and pharmaceutical products. In previous studies, xenoestrogenic actions of parabens were reported in an immature rat model and a rat pituitary cell line (GH3 cells). The relationship between parabens and ovarian failure has not been described. In the present study, the influence of parabens on ovarian folliculogenesis and steroidogenesis was investigated. A disruptor of ovarian small pre-antral follicles, 4-vinylcyclohexene diepoxide (VCD, 40 mg/kg), was used to induce premature ovarian failure (POF). Methylparaben (MP, 100 mg/kg), propylparaben (PP, 100 mg/kg), and butylparaben (BP, 100 mg/kg) dissolved in corn oil were treated in female 8-week-old Sprague-Dawley rat for 5 weeks. Estrus cycle status was checked daily by vaginal smear test. Ovarian follicle development and steroid synthesis were investigated through real-time PCR and histological analyses. Diestrus phases in the VCD, PP, and BP groups were longer than that in the vehicle group. VCD significantly decreased mRNA level of folliculogenesis-related genes (Foxl2, Kitl and Amh). All parabens significantly increased the Amh mRNA level but unchanged Foxl2 and Kitlg acting in primordial follicles. VCD and MP slightly increased Star and Cyp11a1 levels, which are related to an initial step in steroidogenesis. VCD and parabens induced an increase in FSH levels in serum and significantly decreased the total number of follicles. Increased FSH implies impairment in ovarian function due to VCD or parabens. These results suggest that VCD may suppress both formation and development of follicles. In particular, combined administration of VCD and parabens accelerated inhibition of the follicle-developmental process through elevated AMH level in small antral follicles. PMID:28208728

Mathematical modelling of decline in follicle pool during female reproductive ageing.

PubMed

Thilagam, Alagu

2016-03-01

The factors which govern the subtle links between follicle loss and mammalian female reproductive ageing remain unclear despite extensive studies undertaken to understand the critical physiological and biochemical mechanisms that underly the accelerated decline in follicle numbers in women older than 37 years. It is not certain whether there is a sole control by the ovary or whether other factors which affect ageing also intersect with the ovarian effect. There is convincing experimental evidence for an interplay of several processes that seem to influence the follicle loss-female reproductive ageing links, with specific hormones (follicle-stimulating hormone, anti-Müllerian hormone, dehydroepiandrosterone) noted to play important roles in follicular dynamics and ovarian ageing. In this work, we examine the subtle links between the rate of follicular decline with ageing and the role of hormones via a series of non-autonomous equations. Simulation results based on the time evolution of the number of ovarian follicles and biochemical changes in the ovarian environment influenced by hormone levels is compared with empirical data based on follicle loss-reproductive ageing correlation studies. © Crown copyright 2015.

Loss of PUMA protects the ovarian reserve during DNA-damaging chemotherapy and preserves fertility.

PubMed

Nguyen, Quynh-Nhu; Zerafa, Nadeen; Liew, Seng H; Morgan, F Hamish; Strasser, Andreas; Scott, Clare L; Findlay, Jock K; Hickey, Martha; Hutt, Karla J

2018-05-23

Female gametes are stored in the ovary in structures called primordial follicles, the supply of which is non-renewable. It is well established that DNA-damaging cancer treatments can deplete the ovarian reserve of primordial follicles, causing premature ovarian failure and infertility. The precise mechanisms underlying this chemotherapy-driven follicle loss are unclear, and this has limited the development of targeted ovarian-protective agents. To address this fundamental knowledge gap, we used gene deletion mouse models to examine the role of the DNA damage-induced pro-apoptotic protein, PUMA, and its transcriptional activator TAp63, in primordial follicle depletion caused by treatment with cyclophosphamide or cisplatin. Cyclophosphamide caused almost complete destruction of the primordial follicle pool in adult wild-type (WT) mice, and a significant destructive effect was also observed for cisplatin. In striking contrast, Puma -/- mice retained 100% of their primordial follicles following either genotoxic treatment. Furthermore, elimination of PUMA alone completely preserved fertility in cyclophosphamide-treated mice, indicating that oocytes rescued from DNA damage-induced death can repair themselves sufficiently to support reproductive function and offspring health. Primordial follicles were also protected in TAp63 -/- mice following cisplatin treatment, but not cyclophosphamide, suggesting mechanistic differences in the induction of apoptosis and depletion of the ovarian reserve in response to these different chemotherapies. These studies identify PUMA as a crucial effector of apoptosis responsible for depletion of primordial follicles following exposure to cyclophosphamide or cisplatin, and this indicates that inhibition of PUMA may be an effective ovarian-protective strategy during cancer treatment in women.

Female Reproductive Aging Is Master-Planned at the Level of Ovary

PubMed Central

Banerjee, Sayani; Banerjee, Sutapa; Saraswat, Ghungroo; Bandyopadhyay, Soma Aditya; Kabir, Syed N.

2014-01-01

The ovary receives a finite pool of follicles during fetal life. Atresia remains the major form of follicular expenditure at all stages since development of ovary. The follicular reserve, however, declines at an exponential rate leading to accelerated rate of decay during the years preceding menopause. We examined if diminished follicle reserve that characterizes ovarian aging impacts the attrition rate. Premature ovarian aging was induced in rats by intra-embryonic injection of galactosyltransferase-antibody on embryonic day 10. On post-natal day 35 of the female litters, either a wedge of fat (sham control) or a wild type ovary collected from 25-day old control rats, was transplanted under the ovarian bursa in both sides. Follicular growth and atresia, and ovarian microenvironment were evaluated in the follicle-deficient host ovary and transplanted ovary by real time RT-PCR analysis of growth differentiation factor-9, bone morphogenetic protein 15, and kit ligand, biochemical evaluation of ovarian lipid peroxidation, superoxide dismutase (SOD) and catalase activity, and western blot analysis of ovarian pro- and anti-apoptotic factors including p53, bax, bcl2, and caspase 3. Results demonstrated that the rate of follicular atresia, which was highly preponderant in the follicle-deficient ovary of the sham-operated group, was significantly prevented in the presence of the transplanted ovary. As against the follicle-deficient ovary of the sham-operated group, the follicle-deficient host ovary as well as the transplanted ovary in the ovary-transplanted group exhibited stimulated follicle growth with increased expression of anti-apoptotic factors and down regulation of pro-apoptotic factors. Both the host and transplanted ovaries also had significantly lower rate of lipid peroxidation with increased SOD and catalase activity. We conclude that the declining follicular reserve is perhaps the immediate thrust that increases the rate of follicle depletion during the final phase of ovarian life when the follicle reserve wanes below certain threshold size. PMID:24788203

Quantitative expression patterns of GDF9 and BMP15 genes in sheep ovarian follicles grown in vivo or cultured in vitro.

PubMed

Kona, S S R; Praveen Chakravarthi, V; Siva Kumar, A V N; Srividya, D; Padmaja, K; Rao, V H

2016-01-15

Quantitative patterns of expression of the growth differentiation factor 9 (GDF9) and bone morphogenic protein 15 (BMP15) genes in different development stages of in vivo and in vitro grown ovarian follicles in sheep were studied for the first time. Both GDF9 and BMP15 were expressed in the cumulus cells and oocytes at all the development stages of in vivo and in vitro grown ovarian follicles. Growth differentiation factor 9 and bone morphogenic protein 15 exhibited stage-specific undulations in the expression in the cumulus cells and oocytes isolated from in vivo grown ovarian follicles. These undulations could be related to discrete development events during the ovarian follicle development. The expression of GDF9 and BMP15 was highest (3.38 ± 0.02 and 2.69 ± 0.06, respectively; P ≤ 0.05) in the primordial follicles compared with preantral, early antral, antral, and large antral stages. Similarly, GDF9 and BMP15 expression in the cumulus cells (0 ± 0.16 and 0 ± 0.07) and oocytes (1.47 ± 0.07 and 1.32 ± 0.03) was lowest (P ≤ 0.05) in the in vivo grown antral follicles. In the cultured follicles, the stage-specific undulations observed in the expression of GDF9 and BMP15 in the in vivo grown follicles were either different or abolished. For example, in the oocytes from in vitro grown follicles, the expression of BMP15 did not change as the development progressed all the way from preantral to large antral follicle stage although in the oocytes from in vivo grown follicles BMP15 expression exhibited stage-specific variations. It is concluded that GDF9 and BMP15 follow a stage-specific pattern of expression during the in vivo development of ovarian follicles in sheep, and in vitro culture altered the stage-specific changes in the expression of these two genes. Copyright © 2016 Elsevier Inc. All rights reserved.

Prediction of Poor Ovarian response by Biochemical and Biophysical Markers: A Logistic Regression Model.

PubMed

Jaiswar, S P; Natu, S M; Sujata; Sankhwar, P L; Manjari, Gupta

2015-12-01

To study correlation between ovarian reserve with biophysical markers (antral follicle count and ovarian volume) and biochemical markers (S. FSH, S. Inhibin B, and S. AMH) and use these markers to predict poor ovarian response to ovarian induction. This is a prospective observational study. One hundred infertile women attending the Obst & Gynae Dept, KGMU were recruited. Blood samples were collected on day 2/day 3 for assessment of S. FSH, S. Inhibin B, and S. AMH and TVS were done for antral follicle count and ovarian volume. Clomephene citrate 100 mg 1OD was given from day 2 to 6, and patients were followed up with serial USG measurements. The numbers of dominant follicles (> or = 14 mm) at the time of hCG administration were counted. Patients with <3 follicles in the 1st cycle were subjected to the 2nd cycle of clomephene 100 mg 1OD from day 2 to day 6 with Inj HMG 150 IU given i.m. starting from day 8 and every alternate day until at least one leading follicle attained ≥18 mm. Development of <3 follicles at end of the 2nd cycle was considered as poor response. Univariate analyses showed that s. inhibin B presented the highest (ROCAUC = 0.862) discriminating potential for predicting poor ovarian response, In multivariate logistic regression model, the variables age, FSH, AMH, INHIBIN B, and AFC remained significant, and the resulting model showed a predicted accuracy of 84.4 %. A derived multimarker computation by a logistic regression model for predicting poor ovarian response was obtained through this study. Thus, potential poor responders could be identified easily, and appropriate ovarian stimulation protocol could be devised for such pts.

Polycystic ovary morphology is associated with insulin resistance in women with polycystic ovary syndrome.

PubMed

Hong, So-Hyeon; Sung, Yeon-Ah; Hong, Young Sun; Jeong, Kyungah; Chung, Hyewon; Lee, Hyejin

2017-10-01

Polycystic ovary syndrome (PCOS) is a heterogeneous disorder characterized by chronic anovulation, hyperandrogenism, polycystic ovary morphology (PCOM) and metabolic disturbances including insulin resistance and type 2 diabetes mellitus. Although insulin resistance could be associated with PCOM, recent studies have shown controversial results. The aim of this study was to determine the relationship between PCOM and insulin resistance. This was a cross-sectional clinical study. A total of 679 women with PCOS who were diagnosed using the National Institute of Child Health and Human Disease (NICHD) criteria and 272 control women were analysed. We measured fasting glucose and insulin levels, 75 g oral glucose tolerance test-derived glucose and insulin levels, testosterone levels, ovarian volume and follicle number. Polycystic ovary morphology was described in 543 women (80.0%) with PCOS. Women with PCOS had significantly higher 2 hours postload glucose, fasting and 2 hours postload insulin levels, ovarian volume, ovarian follicle numbers and lower insulin sensitivity compared with those of the controls (all P<.01). In women with PCOS, ovarian volume and ovarian follicle number were negatively associated with the quantitative insulin sensitivity check index after adjusting for age, body mass index and total testosterone; however, this association was not observed in the controls. In the logistic regression analysis, increased ovarian follicle number was associated with decreased insulin sensitivity in women with PCOS. In PCOS, enlarged ovarian volume and follicle excess were associated with insulin resistance, and the number of ovarian follicles could be a predictor of insulin resistance. © 2017 John Wiley & Sons Ltd.

A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice.

PubMed

Laronda, Monica M; Rutz, Alexandra L; Xiao, Shuo; Whelan, Kelly A; Duncan, Francesca E; Roth, Eric W; Woodruff, Teresa K; Shah, Ramille N

2017-05-16

Emerging additive manufacturing techniques enable investigation of the effects of pore geometry on cell behavior and function. Here, we 3D print microporous hydrogel scaffolds to test how varying pore geometry, accomplished by manipulating the advancing angle between printed layers, affects the survival of ovarian follicles. 30° and 60° scaffolds provide corners that surround follicles on multiple sides while 90° scaffolds have an open porosity that limits follicle-scaffold interaction. As the amount of scaffold interaction increases, follicle spreading is limited and survival increases. Follicle-seeded scaffolds become highly vascularized and ovarian function is fully restored when implanted in surgically sterilized mice. Moreover, pups are born through natural mating and thrive through maternal lactation. These findings present an in vivo functional ovarian implant designed with 3D printing, and indicate that scaffold pore architecture is a critical variable in additively manufactured scaffold design for functional tissue engineering.

Preservation of human ovarian follicles within tissue frozen by vitrification in a xeno-free closed system using only ethylene glycol as a permeating cryoprotectant.

PubMed

Sheikhi, Mona; Hultenby, Kjell; Niklasson, Boel; Lundqvist, Monalill; Hovatta, Outi

2013-07-01

To study the preservation of follicles within ovarian tissue vitrified using only one or a combination of three permeating cryoprotectants. Experimental study. University hospital. Ovarian tissue was donated by consenting women undergoing elective cesarean section. Ovarian tissue was vitrified in closed sealed vials using either a combination of dimethyl sulfoxide, 1,2-propanediol, and ethylene glycol (EG), or only EG as permeating cryoprotectants. Ovarian tissue was vitrified with the use of two vitrification methods. Tissue from the same donor was used for comparison of two different solutions. The morphology of the follicles was evaluated after vitrification, warming, and culture by light microscopy and transmission electron microscopy. Apoptosis was assessed by immunohistochemistry for active caspase-3 in fresh and vitrified tissue. Light and electron microscopic analysis showed equally well preserved morphology of oocytes, granulosa cells, and ovarian stroma when either of the vitrification solutions was used. No apoptosis was observed in primordial and primary follicles. Using only EG as a permeating cryoprotectant in a closed tube gives as good ultrastructural preservation of ovarian follicles as a more complicated system using several cryoprotectants. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

A Simplified Method for Three-Dimensional (3-D) Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice

PubMed Central

Higuchi, Carolyn M.; Maeda, Yuuki; Horiuchi, Toshitaka; Yamazaki, Yukiko

2015-01-01

In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART). Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture) for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture). We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-), A (Membrane/activin+), M (Matrigel/activin-), and M+A (Matrigel/activin+). We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A). Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM) and in vitro fertilization (IVF). Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A) than with those grown in membrane culture (C, A). In particular, activin A treatment further improved 3-D culture (M+A) success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian culture system using a Matrigel drop and activin A supplementation (M+A) provides optimal and convenient conditions to support growth of developmentally competent oocytes in vitro. PMID:26571501

Developmental Programming: Gestational Exposure to Excess Testosterone Alters Expression of Ovarian Matrix Metalloproteases and Their Target Proteins.

PubMed

Puttabyatappa, Muraly; Irwin, Ashleigh; Martin, Jacob D; Mesquitta, Makeda; Veiga-Lopez, Almudena; Padmanabhan, Vasantha

2018-06-01

Prenatal testosterone (T)-treated sheep, similar to women with polycystic ovary syndrome (PCOS), manifests reproductive defects that include multifollicular ovarian phenotype. Women with PCOS manifest increased ovarian matrix metalloproteinases (MMPs) activity. We tested the hypothesis that gestational T excess in sheep would alter ovarian expression of MMPs, tissue inhibitors of MMP (TIMP) and their target proteins laminin B (LAMB), collagen, tumor necrosis factor alpha (TNF), and connexin 43 (GJA1) consistent with increased MMP activity and that these changes are developmentally regulated. The ovarian content of these proteins was quantified by immunohistochemistry in fetal day 90, 140, and adult (21 months of age) ovaries. Prenatal T excess lowered GJA1 protein content in stroma and granulosa cells of primary follicles from fetal day 90 ovaries and decreased stromal MMP9, TIMP1, and LAMB in fetal day 140 ovaries. In the adult, prenatal T-treatment (1) increased MMP9 in theca cells of large preantral follicles and stroma, TNF in granulosa cells of small and large preantral follicles and theca cells of large preantral and antral follicles, and GJA1 in stroma, theca cells of large preantral follicles, and granulosa cells of antral follicles and (2) reduced TIMP1 in stroma, theca cells of large preantral and antral follicles, LAMB in stroma and small prenatral follicles, and collagen content in stroma and around antral follicles. These findings suggest a net increase in MMP activity and its target proteins TNF and GJA1 in prenatal T-treated adult but not in fetal ovaries and their potential involvement in the development of multifollicular morphology.

Superficial ovarian cortex vascularization is inversely related to the follicle reserve in normal cycling ovaries and is increased in polycystic ovary syndrome.

PubMed

Delgado-Rosas, F; Gaytán, M; Morales, C; Gómez, R; Gaytán, F

2009-05-01

The superficial ovarian cortex constitutes the micro-environment where resting and early growing follicles reside. As small follicles do not possess an independent capillary network, both their survival and early growth depend on their proximity to the cortical vessels. Little is known about the possible changes in superficial ovarian cortex vascularization in normal women throughout reproductive life or in pathological conditions such as polycystic ovary syndrome (PCOS) involving abnormal early follicle growth. We studied the vascularization of the superficial and deep cortical stroma (DCS) in normal cycling ovaries from 21 to 50 years of age and in infertile women with PCOS. We used archival ovarian samples and specific CD34 immunostaining to determine blood vessel density and to analyse correlation with age and with the ovarian follicle reserve. Normal cycling ovaries showed an age-related increase in the superficial cortical stroma vascularization that was inversely correlated with the density of small (primordial and primary) follicles. In contrast, blood vessel density in the DCS significantly decreased in women aged >or=40 years. Ovaries from PCOS showed a 2-fold increase in blood vessel density in both superficial cortical stroma and DCS with respect to age-matched controls. The increased vascularization of the superficial cortical stroma in normal ovaries in relation to age and in ovaries from PCOS could have profound effects on cortical metabolic rate, primordial follicle survival/activation and early follicle growth, and may underline changes in follicle dynamics in mid-aged women and in PCOS.

Prolonged hypothyroidism severely reduces ovarian follicular reserve in adult rats.

PubMed

Meng, Li; Rijntjes, Eddy; Swarts, Hans J M; Keijer, Jaap; Teerds, Katja J

2017-03-16

There is substantial evidence both in humans and in animals that a prolonged reduction in plasma thyroid hormone concentration leads to reproductive problems, including disturbed folliculogenesis, impaired ovulation and fertilization rates, miscarriage and pregnancy complications. The objective of the present study is to examine the consequences of chronic hypothyroidism, induced in adulthood, for the size of the ovarian follicle pool. In order to investigate this, adult female rats were provided either a control or an iodide deficient diet in combination with perchlorate supplementation to inhibit iodide uptake by the thyroid. Sixteen weeks later animals were sacrificed. Blood was collected for hormone analyses and ovaries were evaluated histologically. At the time of sacrifice, plasma thyroid-stimulating hormone concentrations were 20- to 40-fold increased, thyroxine concentrations were negligible while tri-iothyronin concentrations were decreased by 40% in the hypothyroid group, confirming that the animals were hypothyroid. Primordial, primary and preantral follicle numbers were significantly lower in the hypothyroid ovaries compared to the euthyroid controls, while a downward trend in antral follicle and corpora lutea numbers was observed. Surprisingly the percentage of atretic follicles was not significantly different between the two groups, suggesting that the reduced preantral and antral follicle numbers were presumably not the consequence of increased degeneration of these follicle types in the hypothyroid group. Plasma anti-Müllerian hormone (AMH) levels showed a significant correlation with the growing follicle population represented by the total ovarian number of primary, preantral and antral follicles, suggesting that also under hypothyroid conditions AMH can serve as a surrogate marker to assess the growing ovarian follicle population. The induction of a chronic hypothyroid condition in adult female rats negatively affects the ovarian follicular reserve and the size of the growing follicle population, which may impact fertility.

Survival of human pre-antral follicles after cryopreservation of ovarian tissue, follicular isolation and in vitro culture in a calcium alginate matrix.

PubMed

Amorim, Christiani A; Van Langendonckt, Anne; David, Anu; Dolmans, Marie-Madeleine; Donnez, Jacques

2009-01-01

Ovarian tissue cryopreservation is a promising technique to safeguard fertility in cancer patients. However, in some types of cancer, there is a risk of transmitting malignant cells present in the cryopreserved tissue. To avoid such a risk, pre-antral follicles could be isolated from ovarian tissue and grown in vitro. On the basis of this assumption, the aim of our study was to investigate in vitro survival and growth of pre-antral follicles after cryopreservation of ovarian tissue and follicular isolation, followed by encapsulation in alginate beads. Ovarian biopsies from four patients were frozen and thawed. Pre-antral follicles were then isolated and embedded in an alginate matrix before in vitro culture for 7 days. Small pre-antral follicles (42.98 +/- 9.06 microm) from frozen-thawed tissue can survive and develop after enzymatic isolation and in vitro culture. A total of 159 follicles were incubated in a three-dimensional system (alginate hydrogel) and, after 7 days, all of them showed an increase in size (final size 56.73 +/- 13.10 microm). The survival rate of the follicles was 90% (oocyte and all granulosa cells viable). Our preliminary results indicate that alginate hydrogels may be a suitable system for in vitro culture of isolated human pre-antral follicles. However, more studies are required to establish whether follicular morphology and functionality can be maintained using this matrix.

Induction of a menopausal state alters the growth and histology of ovarian tumors in a mouse model of ovarian cancer.

PubMed

Laviolette, Laura A; Ethier, Jean-François; Senterman, Mary K; Devine, Patrick J; Vanderhyden, Barbara C

2011-05-01

Ovarian cancer is often diagnosed in women after menopause when the levels of the serum gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are increased because of the depletion of growing follicles within the ovary. The ability of FSH and LH to modulate the disease has not been well studied owing to a lack of physiologically relevant models of ovarian cancer. In this study, 4-vinylcyclohexene diepoxide (VCD) was used to deplete ovarian follicles and increase the levels of circulating FSH and LH in the tgCAG-LS-TAg mouse model of ovarian cancer. VCD-induced follicle depletion was performed either before or after induction of the oncogene SV40 large and small T-antigens in the ovarian surface epithelial cells of tgCAG-LS-TAg mice, which was mediated by the intrabursal delivery of an adenovirus expressing Cre recombinase (AdCre). tgCAG-LS-TAg mice injected with AdCre developed undifferentiated ovarian tumors with mixed epithelial and stromal components and some features of sex cord stromal tumors. Treatment with VCD before or after AdCre injection yielded tumors of similar histology, but with the unique appearance of Sertoli cell nests. In mice treated with VCD before the induction of tumorigenesis, the ovarian tumors tended to grow more slowly. The human ovarian cancer cell lines SKOV3 and OVCAR3 responded similarly to increased levels of gonadotropins in a second model of menopause, growing more slowly in ovariectomized mice compared with cycling controls. These results suggest that follicle depletion and increased gonadotropin levels can alter the histology and the rate of growth of ovarian tumors.

Follicle Depletion Provides a Permissive Environment for Ovarian Carcinogenesis

PubMed Central

Wang, Ying; Cai, Kathy Qi; Smith, Elizabeth R.; Yeasky, Toni M.; Moore, Robert; Ganjei-Azar, Parvin; Klein-Szanto, Andres J.; Godwin, Andrew K.; Hamilton, Thomas C.

2016-01-01

We modeled the etiology of postmenopausal biology on ovarian cancer risk using germ cell-deficient white-spotting variant (Wv) mice, incorporating oncogenic mutations. Ovarian cancer incidence is highest in peri- and postmenopausal women, and epidemiological studies have established the impact of reproductive factors on ovarian cancer risk. Menopause as a result of ovarian follicle depletion is thought to contribute to higher cancer risk. As a consequence of follicle depletion, female Wv mice develop ovarian tubular adenomas, a benign epithelial tumor corresponding to surface epithelial invaginations and papillomatosis frequently found in postmenopausal human ovaries. Lineage tracing using MISR2-Cre indicated that the tubular adenomas that developed in Wv mice were largely derived from the MISR2 lineage, which marked only a fraction of ovarian surface and oviduct epithelial cells in wild-type tissues. Deletion of p27, either heterozygous or homozygous, was able to convert the benign tubular adenomas into more proliferative tumors. Restricted deletion of p53 in Wv/Wv mice by either intrabursal injection of adenoviral Cre or inclusion of the MISR2-Cre transgene also resulted in augmented tumor growth. This finding suggests that follicle depletion provides a permissive ovarian environment for oncogenic transformation of epithelial cells, presenting a mechanism for the increased ovarian cancer risk in postmenopausal women. PMID:27354067

Progesterone regulation of primordial follicle assembly in bovine fetal ovaries.

PubMed

Nilsson, Eric E; Skinner, Michael K

2009-12-10

Fertility in mammals is dependant on females having an adequate primordial follicle pool to supply oocytes for fertilization. The formation of primordial follicles is called ovarian follicular assembly. In rats and mice progesterone and estradiol have been shown to inhibit follicle assembly with assembly occurring after birth when the pups are removed from the high-steroid maternal environment. In contrast, primordial follicle assembly in other species, such as cattle and humans, occurs during fetal development before birth. The objective of the current study is to determine if progesterone levels regulate primordial follicle assembly in fetal bovine ovaries. Ovaries and blood were collected from bovine fetuses. Interestingly, ovarian progesterone and estradiol concentrations were found to decrease with increasing fetal age and correlated to increased primordial follicle assembly. Microarray analysis of fetal ovary RNA suggests that progesterone membrane receptor and estrogen nuclear receptor are expressed. Treatment of fetal bovine ovary cultures with a higher progesterone concentration significantly decreased primordial follicle assembly. Observations indicate that progesterone affects ovarian primordial follicle assembly in cattle, as it does in rats and mice.

Progesterone Regulation of Primordial Follicle Assembly In Bovine Fetal Ovaries

PubMed Central

Nilsson, Eric E.; Skinner, Michael K.

2009-01-01

Fertility in mammals is dependant on females having an adequate primordial follicle pool to supply oocytes for fertilization. The formation of primordial follicles is called ovarian follicular assembly. In rats and mice progesterone and estradiol have been shown to inhibit follicle assembly with assembly occurring after birth when the pups are removed from the high-steroid maternal environment. In contrast, primordial follicle assembly in other species, such as cattle and humans, occurs during fetal development before birth. The objective of the current study is to determine if progesterone levels regulate primordial follicle assembly in fetal bovine ovaries. Ovaries and blood were collected from bovine fetuses. Interestingly, ovarian progesterone and estradiol concentrations were found to decrease with increasing fetal age and correlated to increased primordial follicle assembly. Microarray analysis of fetal ovary RNA suggests that progesterone membrane receptor and estrogen nuclear receptor are expressed. Treatment of fetal bovine ovary cultures with a higher progesterone concentration significantly decreased primordial follicle assembly. Observations indicate that progesterone affects ovarian primordial follicle assembly in cattle, as it does in rats and mice. PMID:19747959

Examination of the Ovarian Reserve after Generation of Unilateral Rudimentary Uterine Horns in Rats

PubMed Central

Toyganözü, Hasan; Nazik, Hakan; Narin, Raziye; Satar, Deniz; Narin, Mehmet Ali; Büyüknacar, Sinem; Api, Murat; Aytan, Hakan

2014-01-01

Objective. The purpose of this experimental rat model study is to evaluate the changes in the ovarian environment after excision of the rudimentary horn. Methods. Ten female Wistar albino rats were used in this study. One cm of right uterine horn length was excised in the first operation. Two months after the first operation, all animals were sacrificed to obtain ovaries for histological examination. Mann-Whitney U test and Student's t-test were used for statistical analysis purposes. Statistical significance was defined as P < 0.005. Results. The number of primordial follicles (P = 0.415), primary follicles (P = 0.959), preantral follicles (P = 0.645), antral follicles (P = 0.328), and Graafian follicles (P = 0.721) was decreased and the number of atretic follicles (P = 0.374) increased in the right ovarian side. Howeve,r this difference was not found to be statistically significant. Conclusion. The results of this experimental rat model study suggest that the excision of rudimentary horn could have negative effects on ipsilateral ovarian functions. PMID:24672393

A rapid technique for the histological examination of large ovarian follicles.

PubMed

Driancourt, M A; Mariana, J C; Palmer, E

1981-01-01

A rapid technique for counting and classifying large ovarian follicles of domestic animals is described. Using a cryostat, 250-micrograms thick sections were cut from the frozen ovary; an image of the surface of each ovarian section was recorded on videotape. By replaying the videotape, the largest profile of each follicle larger than 1 mm in diameter was readily identified and measured. The presence or absence of atresia was determined by applying standard histological methods to fragments of individual follicles taken from the frozen sections. The results obtained are similar to those found using previous methods and demand only one-quarter of the time.

Restoration of fresh cat ovarian tissue function by autografting to subcutaneous tissue: A pilot study.

PubMed

Leonel, Ellen C R; Vilela, Janice M V; Paiva, Raísa E G; Jivago, José L P R; Amaral, Rodrigo S; Lucci, Carolina M

2018-01-01

Ovarian tissue transplantation could be a valuable technique for the preservation of endangered animals. The domestic cat affords an adequate experimental model for studies aimed at wild felids due to its phylogenetic similarity. Thus, this pilot study evaluated the efficacy of cat ovarian tissue autotransplantation to a peripheral site. Three adult queens were submitted to ovariohysterectomy. The ovaries were fragmented into eight pieces; two were fixed as a control and six were transplanted to subcutaneous tissue of the dorsal neck. Grafts were monitored weekly by ultrasound and fecal samples collected daily in order to monitor estradiol levels. Grafts were recovered on Days: 7, 14, 28, 49 and 63 post-transplantation for histological analyses. One graft was maintained in one animal for 8 months. A total of 2466 ovarian follicles were analyzed: 1406 primordial and 1060 growing follicles. All animals presented antral follicles in one or more of the grafts. The percentage of morphologically normal primordial follicles was always higher than 80%, except for Day 7 transplants. Although the proportion of growing follicles increased after transplantation, there was a general decrease in the percentage of morphologically normal growing follicles from Day 7 onwards. All animals demonstrated at least three estradiol peaks during the 63-day period, and one animal exhibited estrous behaviour on three occasions. Hormonal peaks directly correlated with the visualization of antral follicles (by ultrasound and/or histology) and the observation of estrous behaviour. Long-term results on one female showed the concentration of 37.8 pg/mL of serum estradiol on Day 233 post-grafting and the female exhibited estrous behaviour on several occasions. This graft showed one antral follicle, one luteinized follicle and two preantral follicles. In conclusion, cat ovary autotransplantation to the subcutaneous tissue restored ovarian function, with hormone production and antral follicle development, over both short and long term periods. This could be a valuable technique in the evaluation of ovarian cryopreservation methods in felids. Once the technique is shown successful, it may be applied in allografts or xenografts between different feline species. Copyright © 2017 Elsevier Inc. All rights reserved.

Hypothyroidism Reduces the Size of Ovarian Follicles and Promotes Hypertrophy of Periovarian Fat with Infiltration of Macrophages in Adult Rabbits.

PubMed

Rodríguez-Castelán, J; Méndez-Tepepa, M; Carrillo-Portillo, Y; Anaya-Hernández, A; Rodríguez-Antolín, J; Zambrano, E; Castelán, F; Cuevas-Romero, E

2017-01-01

Ovarian failure is related to dyslipidemias and inflammation, as well as to hypertrophy and dysfunction of the visceral adipose tissue (VAT). Although hypothyroidism has been associated with obesity, dyslipidemias, and inflammation in humans and animals, its influence on the characteristics of ovarian follicles in adulthood is scarcely known. Control and hypothyroid rabbits were used to analyze the ovarian follicles, expression of aromatase in the ovary, serum concentration of lipids, leptin, and uric acid, size of adipocytes, and infiltration of macrophages in the periovarian VAT. Hypothyroidism did not affect the percentage of functional or atretic follicles. However, it reduced the size of primary, secondary, and tertiary follicles considered as large and the expression of aromatase in the ovary. This effect was associated with high serum concentrations of total cholesterol and low-density lipoprotein cholesterol (LDL-C). In addition, hypothyroidism induced hypertrophy of adipocytes and a major infiltration of CD68+ macrophages into the periovarian VAT. Our results suggest that the reduced size of ovarian follicles promoted by hypothyroidism could be associated with dyslipidemias, hypertrophy, and inflammation of the periovarian VAT. Present findings may be useful to understand the influence of hypothyroidism in the ovary function in adulthood.

Hypothyroidism Reduces the Size of Ovarian Follicles and Promotes Hypertrophy of Periovarian Fat with Infiltration of Macrophages in Adult Rabbits

PubMed Central

Rodríguez-Castelán, J.; Méndez-Tepepa, M.; Carrillo-Portillo, Y.; Anaya-Hernández, A.; Zambrano, E.

2017-01-01

Ovarian failure is related to dyslipidemias and inflammation, as well as to hypertrophy and dysfunction of the visceral adipose tissue (VAT). Although hypothyroidism has been associated with obesity, dyslipidemias, and inflammation in humans and animals, its influence on the characteristics of ovarian follicles in adulthood is scarcely known. Control and hypothyroid rabbits were used to analyze the ovarian follicles, expression of aromatase in the ovary, serum concentration of lipids, leptin, and uric acid, size of adipocytes, and infiltration of macrophages in the periovarian VAT. Hypothyroidism did not affect the percentage of functional or atretic follicles. However, it reduced the size of primary, secondary, and tertiary follicles considered as large and the expression of aromatase in the ovary. This effect was associated with high serum concentrations of total cholesterol and low-density lipoprotein cholesterol (LDL-C). In addition, hypothyroidism induced hypertrophy of adipocytes and a major infiltration of CD68+ macrophages into the periovarian VAT. Our results suggest that the reduced size of ovarian follicles promoted by hypothyroidism could be associated with dyslipidemias, hypertrophy, and inflammation of the periovarian VAT. Present findings may be useful to understand the influence of hypothyroidism in the ovary function in adulthood. PMID:28133606

Testosterone selectively increases primary follicles in ovarian cortex grafted onto embryonic chick membranes: relevance to polycystic ovaries.

PubMed

Qureshi, A I; Nussey, S S; Bano, G; Musonda, P; Whitehead, S A; Mason, H D

2008-08-01

Histological studies have demonstrated that polycystic ovaries (PCO) contain increased numbers of preantral follicles with a specific increase in primary follicles. Polycystic ovary syndrome is associated with hyperandrogenism and pre- and postnatal androgenization of primates increases the pool of growing follicles producing changes resembling PCO. In vitro studies could test the hypothesis that androgens alter early folliculogenesis, but conventional culture techniques for small follicles are generally unsuitable in non-rodent species. Our objective was to develop and use a method to investigate the effects of testosterone on early folliculogenesis. We adapted an in ovo technique in which lamb cortical ovarian fragments were grafted onto the chorioallantoic membrane of fertilised chick eggs. Optimal experimental conditions for vascularisation and survival of tissue were determined and the model then used to investigate the effects of testosterone on follicle growth. Eggs were inoculated with testosterone at the time of implantation of the ovarian tissue, which was retrieved 5 days later. Tissue was sectioned and follicles staged and counted. There was no wholesale initiation of primordial follicle growth over the 5-day in ovo culture. Importantly, the proportion of primordial, primary and secondary follicles remained similar to those in unimplanted tissue. Testosterone increased the number of primary follicles by 50% compared with controls, an effect that was largely due to a reduction in atresia. In conclusion, incubation of ovarian cortex with testosterone reproduces the changes in early folliculogenesis reported in histological studies of PCO.

Ovarian follicle dynamics of female Greater Scaup during egg production

USGS Publications Warehouse

Gorman, Kristen B.; Flint, Paul L.; Esler, Daniel N.; Williams, T.D.

2007-01-01

Studies of female waterfowl nutrient reserve use during egg production require a precise understanding of ovarian follicle dynamics to correctly interpret breeding status, and, therefore, derive proper inference. Concerns over numerical declines of North American scaup have increased the need to better understand the role of female condition in reproductive performance. We quantified ovarian follicle dynamics of female Greater Scaup (Aythya marila) breeding on the Yukon–Kuskokwim Delta, Alaska, using a method that accounts for within day variation in follicle size. We considered several models for describing changes in follicle growth with the best supported model estimating the duration of rapid follicle growth (RFG) to be 5.20 ± 0.52 days (±95% confidence intervals) for each developing follicle. Average diameter and dry mass of preovulatory follicles were estimated to be 9.36 mm and 0.26 g, respectively, at the onset of RFG, and these follicle characteristics were 41.47 mm and 15.57 g, respectively, at ovulation. The average diameter of postovulatory follicles immediately following ovulation was estimated to be 17.35 mm, regressing quickly over several days. In addition, we derived predictive equations using diameter and dry mass to estimate the number of days before, and after, ovulation for pre- and postovulatory follicles, as well as an equation to estimate dry mass of damaged follicles. Our results allow precise definition of RFG and nest initiation dates, clutch size, and the daily energetic and nutritional demands of egg production at the individual level. This study provides the necessary foundation for additional work on Greater Scaup reproductive energetics and physiology, and offers an approach for quantifying ovarian follicle dynamics in other species.

The Increased Expression of Connexin and VEGF in Mouse Ovarian Tissue Vitrification by Follicle Stimulating Hormone

PubMed Central

Yang, Yanzhou; Chen, Jie; Wu, Hao; Pei, Xiuying; Chang, Qing; Ma, Wenzhi; Ma, Huiming; Hei, Changchun; Zheng, Xiaomin; Cai, Yufang; Zhao, Chengjun; Yu, Jia; Wang, Yanrong

2015-01-01

Ovarian follicular damages were caused by cryoinjury during the process of ovarian vitrification and ischemia/reperfusion during the process of ovarian transplantation. And appropriate FSH plays an important role in antiapoptosis during ovarian follicle development. Therefore, in this study, 0.3 IU/mL FSH was administered into medium during mouse ovarian cryopreservation by vitrification to ascertain the function of FSH on ovarian vitrification and avascular transplantation. The results suggested that the expressions of Cx37, Cx43, apoptotic molecular caspase-3, and angiogenesis molecular VEGF were confirmed using immunohistochemistry, western blotting, and real-time PCR, and the results suggested that the treatment with FSH remarkably increased the number of morphologically normal follicles in vitrified/warmed ovaries by upregulating the expression of Cx37, Cx43, VEGF, and VEGF receptor 2, but downregulating the expression of caspase-3. In addition, the vitrified/warmed ovaries were transplanted, and the related fertility was analyzed, and the results suggested that the fertility, neoangiogenesis, and follicle reserve were remarkably increased in the FSH administrated group. Taken together, administration of 0.3 IU/mL FSH during ovarian cryopreservation by vitrification can maintain ovarian survival during ovarian vitrification and increases the blood supply with avascular transplantation via upregulation of Cx43, Cx37, and VEGF/VEGFR2, as well as through its antiapoptotic effects. PMID:26539488

Ultrastructural observations of previtellogenic ovarian follicles of dove.

PubMed

Zarnescu, Otilia

2004-11-01

Dove ovarian follicle is a complex structure composed of oocyte surrounded by a somatic compartment consisting of theca externa, theca interna and granulosa. The structure of ovarian follicle (1 and 2 mm) of dove was studied by electron microscopy. The granulosa was pseudostratified in the 1-mm-diameter follicles and stratified with two or three irregular rows of cells in the 2-mm-diameter follicles. In the larger follicle indentations between oocyte and granulosa cells become more numerous and the microvilli of granulosa cell elongated to form a zona radiata with similarly elongated oocyte microvilli. Lining bodies were present at the tips of granulosa microvilli and in the cortical region of the oocyte. In the oocyte cortex were observed coated pits, coated vesicles, dense tubules, multivesicular bodies and primordial yolk spheres. Primordial yolk spheres may contain lining bodies and were observed fused with dense tubules and multivesicular bodies or associated with smooth cisternae.

Metabolomic changes in follicular fluid induced by soy isoflavones administered to rats from weaning until sexual maturity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wenxiang; Zhang, Wenchang, E-mail: wenchang2002@sina.com; Liu, Jin

Female Wistar rats at 21 days of age were treated with one of three concentrations of soy isoflavones (SIF) (50, 100 or 200 mg/kg body weight, orally, once per day) from weaning until sexual maturity (3 months) in order to evaluate the influence of SIF on ovarian follicle development. After treatment, the serum sex hormone levels and enumeration of ovarian follicles of the ovary were measured. The metabolic profile of follicular fluid was determined using HPLC-MS. Principal component analysis (PCA) and partial least-squares-discriminant analysis (PLS-DA) was used to identify differences in metabolites and reveal useful toxic biomarkers. The results indicatedmore » that modest doses of SIF affect ovarian follicle development, as demonstrated by decreased serum estradiol levels and increases in both ovarian follicle atresia and corpora lutea number in the ovary. SIF treatment-related metabolic alterations in follicular fluid were also found in the PCA and PLS-DA models. The 24 most significantly altered metabolites were identified, including primary sex hormones, amino acids, fatty acids and metabolites involved in energy metabolism. These findings may indicate that soy isoflavones affect ovarian follicle development by inducing metabolomic variations in the follicular fluid. - Highlights: ► Modest doses of soy isoflavones (SIF) do affect ovarian follicle development. ► SIF treatment-related metabolic alterations in follicular fluid were found. ► The 24 most significantly altered metabolites were identified.« less

Altered Sex Hormone Concentrations and Gonadal mRNA Expression Levels of Activin Signaling Factors in Hatchling Alligators From a Contaminated Florida Lake

PubMed Central

MOORE, BRANDON C.; KOHNO, SATOMI; COOK, ROBERT W.; ALVERS, ASHLEY L.; HAMLIN, HEATHER J.; WOODRUFF, TERESA K.; GUILLETTE, LOUIS J.

2014-01-01

Activins and estrogens participate in regulating the breakdown of ovarian germ cell nests and follicle assembly in mammals. In 1994, our group reported elevated frequencies of abnormal, multioocytic ovarian follicles in 6 month old, environmental contaminant-exposed female alligators after gonadotropin challenge. Here, we investigated if maternal contribution of endocrine disrupting contaminants to the egg subsequently alters estrogen/inhibin/activin signaling in hatchling female offspring, putatively predisposing an increased frequency of multioocytic follicle formation. We quantified basal and exogenous gonadotropin-stimulated concentrations of circulating plasma steroid hormones and ovarian activin signaling factor mRNA abundance in hatchling alligators from the same contaminated (Lake Apopka) and reference (Lake Woodruff) Florida lakes, as examined in 1994. Basal circulating plasma estradiol and testosterone concentrations were greater in alligators from the contaminated environment, whereas activin/inhibin βA subunit and follistatin mRNA abundances were lower than values measured in ovaries from reference lake animals. Challenged, contaminant-exposed animals showed a more robust increase in plasma estradiol concentration following an acute follicle stimulating hormone (FSH) challenge compared with reference site alligators. Aromatase and follistatin mRNA levels increased in response to an extended FSH challenge in the reference site animals, but not in the contaminant-exposed animals. In hatchling alligators, ovarian follicles have not yet formed; therefore, these endocrine differences are likely to affect subsequent ovarian development, including ovarian follicle assembly. PMID:20166196

Monohaloacetic acid drinking water disinfection by-products inhibit follicle growth and steroidogenesis in mouse ovarian antral follicles in vitro.

PubMed

Jeong, Clara H; Gao, Liying; Dettro, Tyler; Wagner, Elizabeth D; Ricke, William A; Plewa, Michael J; Flaws, Jodi A

2016-07-01

Water disinfection greatly reduced the incidence of waterborne diseases, but the reaction between disinfectants and natural organic matter in water leads to the formation of drinking water disinfection by-products (DBPs). DBPs have been shown to be toxic, but their effects on the ovary are not well defined. This study tested the hypothesis that monohalogenated DBPs (chloroacetic acid, CAA; bromoacetic acid, BAA; iodoacetic acid, IAA) inhibit antral follicle growth and steroidogenesis in mouse ovarian follicles. Antral follicles were isolated and cultured with either vehicle or DBPs (0.25-1.00mM of CAA; 2-15μM of BAA or IAA) for 48 and 96h. Follicle growth was measured every 24h and the media were analyzed for estradiol levels at 96h. Exposure to DBPs significantly inhibited antral follicle growth and reduced estradiol levels compared to controls. These data demonstrate that DBP exposure caused ovarian toxicity in vitro. Copyright © 2016 Elsevier Inc. All rights reserved.

Monohaloacetic acid drinking water disinfection by-products inhibit follicle growth and steroidogenesis in mouse ovarian antral follicles in vitro

PubMed Central

Jeong, Clara H.; Gao, Liying; Dettro, Tyler; Wagner, Elizabeth D.; Ricke, William A.; Plewa, Michael J.; Flaws, Jodi A.

2016-01-01

Water disinfection greatly reduced the incidence of waterborne diseases, but the reaction between disinfectants and natural organic matter in water leads to the formation of drinking water disinfection by-products (DBPs). DBPs have been shown to be toxic, but their effects on the ovary are not well defined. This study tested the hypothesis that monohalogenated DBPs (chloroacetic acid, CAA; bromoacetic acid, BAA; iodoacetic acid, IAA) inhibit antral follicle growth and steroidogenesis in mouse ovarian follicles. Antral follicles were isolated and cultured with either vehicle or DBPs (0.25–1.00 mM of CAA; 2–15 µM of BAA or IAA) for 48 and 96 h. Follicle growth was measured every 24 h and the media were analyzed for estradiol levels at 96 h. Exposure to DBPs significantly inhibited antral follicle growth and reduced estradiol levels compared to controls. These data demonstrate that DBP exposure caused ovarian toxicity in vitro. PMID:27151372

Is oxygen availability a limiting factor for in vitro folliculogenesis?

PubMed Central

Sudhakaran, Sam; Barbato, Vincenza; Merolla, Anna; Braun, Sabrina; Di Nardo, Maddalena; Costanzo, Valentina; Ferraro, Raffaele; Iannantuoni, Nicola

2018-01-01

Transplantation of ovarian tissue for the preservation of fertility in oncological patients is becoming an accepted clinical practice. However, the risk of re-introducing tumour cells at transplantation has stirred an increased interest for complete in vitro folliculogenesis. This has not yet been achieved in humans possibly for the lack of knowledge on the environmental milieu that orchestrates folliculogenesis in vivo. The main aim of this study was to investigate the effect of oxygen availability on follicle health and growth during in vitro culture of ovarian tissue strips. To this end, a model was developed to predict the dissolved oxygen concentration in tissue under varying culture conditions. Ovarian cortical strips of bovine, adopted as an animal model, and human tissue were cultured in conventional (CD) and gas permeable (PD) dishes under different media column heights and gaseous oxygen tensions for 3, 6 and 9 days. Follicle quality, activation of primordial follicles to the primary stage, and progression to the secondary stage were analysed through histology. Follicle viability was assessed through a live-dead assay at the confocal scanning laser microscope. Findings showed a higher follicle quality and viability after culture of bovine ovarian strips in PD in adequate medium height and oxygen tensions. The best culture conditions found in the bovine were adopted for human ovarian strip culture and promoted a higher follicle quality, viability and progression. Overall, data demonstrated that modulation of oxygen availability in tissue plays a key role in maintaining follicles’ health and their ability to survive and progress to the secondary stage during ovarian tissue in vitro culture. Such culture conditions could increase the yield of healthy secondary follicles for subsequent dissection and individual culture to obtain competent oocytes. PMID:29425251

Vitrification and xenografting of human ovarian tissue.

PubMed

Amorim, Christiani Andrade; Dolmans, Marie-Madeleine; David, Anu; Jaeger, Jonathan; Vanacker, Julie; Camboni, Alessandra; Donnez, Jacques; Van Langendonckt, Anne

2012-11-01

To assess the efficiency of two vitrification protocols to cryopreserve human preantral follicles with the use of a xenografting model. Pilot study. Gynecology research unit in a university hospital. Ovarian biopsies were obtained from seven women aged 30-41 years. Ovarian tissue fragments were subjected to one of three cryopreservation protocols (slow freezing, vitrification protocol 1, and vitrification protocol 2) and xenografted for 1 week to nude mice. The number of morphologically normal follicles after cryopreservation and grafting and fibrotic surface area were determined by histologic analysis. Apoptosis was assessed by the TUNEL method. Morphometric analysis of TUNEL-positive surface area also was performed. Follicle proliferation was evaluated by immunohistochemistry. After xenografting, a difference was observed between the cryopreservation procedures applied. According to TUNEL analysis, both vitrification protocols showed better preservation of preantral follicles than the conventional freezing method. Moreover, histologic evaluation showed a significantly higher proportion of primordial follicles in vitrified (protocol 2)-warmed ovarian tissue than in frozen-thawed tissue. The proportion of growing follicles and fibrotic surface area was similar in all groups. Vitrification procedures appeared to preserve not only the morphology and survival of preantral follicles after 1 week of xenografting, but also their ability to resume folliculogenesis. In addition, vitrification protocol 2 had a positive impact on the quiescent state of primordial follicles after xenografting. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Preconception folic acid use modulates estradiol and follicular responses to ovarian stimulation.

PubMed

Twigt, John M; Hammiche, Fatima; Sinclair, Kevin D; Beckers, Nicole G; Visser, Jenny A; Lindemans, Jan; de Jong, Frank H; Laven, Joop S E; Steegers-Theunissen, Régine P

2011-02-01

Folate is a methyl donor. Availability of folate affects DNA methylation profiles and thereby gene expression profiles. We investigated the effects of low-dose folic acid use (0.4 mg/d) on the ovarian response to mild and conventional ovarian stimulation in women. In a randomized trial among subfertile women, 24 and 26 subjects received conventional and mild ovarian stimulation, respectively. Blood samples were taken during the early follicular phase of the cycle prior to treatment and on the day of human chorionic gonadotropin administration for determination of serum total homocysteine, anti-Müllerian hormone (AMH), estradiol, and folate. Folic acid use was validated by questionnaire and serum folate levels. Preovulatory follicles were visualized, counted, and diameters recorded using transvaginal ultrasound. The relation between folic acid use and ovarian response was assessed using linear regression analysis. Folic acid use modified the ovarian response to ovarian stimulation treatment. The estradiol response was higher in nonfolic acid users receiving conventional treatment [β(interaction) = 0.52 (0.07-0.97); P = 0.03], and this effect was independent of serum AMH levels and the preovulatory follicle count. In the conventional treatment, the mean follicle number was also greater in nonusers compared with the users group (14.1 vs. 8.9, P = 0.03). Low-dose folic acid use attenuates follicular and endocrine responses to conventional stimulation, independent of AMH and follicle count. The nature of this observation suggests that the effect of folic acid is most prominent during early follicle development, affecting immature follicles. Deleterious effects of folate deficiency, like DNA hypomethylation and oxidative stress, can help to explain our observations.

Laparoscopic ovarian biopsy pick-up method for goats.

PubMed

Brandão, Fabiana A S; Alves, Benner G; Alves, Kele A; Souza, Samara S; Silva, Yago P; Freitas, Vicente J F; Teixeira, Dárcio I A; Gastal, Eduardo L

2018-02-01

Biopsy pick-up (BPU) has been considered a safe method to harvest ovarian fragments from live animals. However, no studies have been reported on the use of BPU to collect in vivo ovarian tissue in goats. The goals of this study were: (i) to test different biopsy needle sizes to collect ovarian tissue in situ using the BPU method (Experiment 1), and (ii) to study ovarian tissue features such as preantral follicle density, morphology, class distribution, and stromal cell density in ovarian fragments obtained in vivo through a laparoscopic BPU method (Experiment 2). In Experiment 1, goat ovaries (n = 20) were collected in a slaughterhouse and subjected to in situ BPU. Three needles (16, 18, and 20G) were tested. In Experiment 2, the most efficient biopsy needle from Experiment 1 was used to perform laparoscopic BPU in goats (n = 8). In Experiment 1, the recovery rate was greater (P < 0.05; range 50-62%) with 16G and 18G needles than the 20G (17%) needle. The mean weight of ovarian fragments collected by the 16G needle was greater (P < 0.05) than the 18G and the 20G needle. In Experiment 2, 62 biopsy attempts were performed and 52 ovarian fragments were collected (90% success rate). Overall, 2054 preantral follicles were recorded in 5882 histological sections analyzed. Mean preantral follicular density was 28.4 ± 1.3 follicles per cm 2 . The follicular density differed (P < 0.05) among animals and ovarian fragments within the same animal. The mean stromal cell density in the ovarian fragments was 37.1 ± 0.5 cells per 2500 μm 2 , and differed (P < 0.05) among animals. Moreover, preantral follicle density and stromal cell density were associated (P < 0.001). The percentage of morphologically normal follicles was 70.1 ± 1.2, and differed (P < 0.05) among animals. The majority (79%) of the morphologically normal follicles was classified as primordial follicles, and differed (P < 0.05) among animals and between ovaries. In summary, a laparoscopic BPU method has been developed to harvest ovarian tissue in vivo with a satisfactory success rate in goats. Furthermore, this study described for the first time that goat ovarian biopsy fragments have a high heterogeneity in follicular density, morphology, class distribution, and stromal cell density. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of toxicological endpoints in female zebrafish after bisphenol A exposure.

PubMed

Molina, Ana M; Abril, Nieves; Morales-Prieto, Noelia; Monterde, José G; Lora, Antonio J; Ayala, Nahúm; Moyano, Rosario

2018-02-01

Given the importance of bisphenol A (BPA) as a xenoestrogen and its potential effects on human and animal health, we evaluated BPA exposure's short-term effects on follicular development, yolk protein vitellogenin (VTG) production and aromatase expression in female zebrafish. Histological modifications were observed along with increased presence of atretic follicles. Whole-body VTG concentration increased with the dose of BPA exposure. In contrast, expression of Cyp19a mRNA in the ovaries of BPA-exposed fish exhibited an apparent non-monotonic response curve, marked by downregulation at 1 μg/L BPA, upregulation at 10 μg/L BPA, and a return to downregulation at 100 μg/L BPA and higher doses. Ovaries only exhibited significant increases in follicular atresia and VTG concentration after exposure to 100 μg/L BPA and higher doses. Ovarian histopathology, aromatase Cyp19a transcript levels and whole-body VTG protein abundance may be good biomarkers for early detection of environmental BPA exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Short-Term PTEN Inhibition Improves In Vitro Activation of Primordial Follicles, Preserves Follicular Viability, and Restores AMH Levels in Cryopreserved Ovarian Tissue From Cancer Patients.

PubMed

Novella-Maestre, Edurne; Herraiz, Sonia; Rodríguez-Iglesias, Beatriz; Díaz-García, César; Pellicer, Antonio

2015-01-01

In vitro activation and growth of primordial dormant follicles to produce fertilizable oocytes would provide a useful instrument for fertility preservation. The employment of Phosphatase and TENsin homolog (PTEN) inhibitors, in combination with Protein kinase B (Akt) stimulating molecules, has been previously employed to increase follicular activation through the stimulation of the PTEN-Akt pathway. We aim to establish improved in vitro activation also for cancer patients whose ovarian tissue has already been cryopreserved. Fresh and previously cryopreserved human ovarian cortex were exposed to short-term, low-concentration and ovary-specific treatment with only a PTEN inhibitor. Our in vitro activation protocol enhances the activation mechanisms of primordial follicles in both fresh and cryopreserved samples, and enlarges growing populations without inducing apoptosis in either follicles or the surrounding stroma. Treatment augments estradiol secretion and restores the expression levels of the previously diminished Anti-Müllerian hormone by means of cryopreservation procedures. Genomic modulation of the relative expression of PTEN pathway genes was found in treated samples. The in vitro activation protocol offers new alternatives for patients with cryopreserved tissue as it increases the pool of viable activated follicles available for in vitro growth procedures. The combination of ovarian tissue cryopreservation and in vitro activation of primordial follicles, the main ovarian reserve component, will be a major advancement in fertility preservation.

Effect of PCB 126 on aryl hydrocarbon receptor 1 (AHR1) and AHR1 nuclear translocator 1 (ARNT1) mRNA expression and CYP1 monooxygenase activity in chicken (Gallus domesticus) ovarian follicles.

PubMed

Wójcik, Dagmara; Antos, Piotr A; Katarzyńska, Dorota; Hrabia, Anna; Sechman, Andrzej

2015-12-03

The aim of the experiment was to study the in vitro effect of 3,3',4,4',5-pentachlorobiphenyl (PCB 126; a coplanar PCB congener) on aryl hydrocarbon receptor (AHR1) and AHR1 nuclear translocator (ARNT1) mRNA expression and the activity of CYP1 family monooxygenases in chicken ovarian follicles. White (1-4 mm) and yellowish (4-8 mm) prehierarchical follicles as well as fragments of the theca and granulosa layers of the 3 largest preovulatory follicles (F3-F1) were incubated in a medium supplemented with 0 (control group), 1, 10 or 100 nM PCB 126. The incubation was carried out for 6 h or 24 h for determination of mRNA expression of AHR1 and ARNT1 genes (real-time qPCR) and CYP1 monooxygenase activity (EROD and MROD fluorometric assays), respectively. It was found that chicken ovarian follicles express mRNA of AHR1 and ARNT1 genes. A modulatory effect of PCB 126 on AHR1 and ARNT1 expression depended not only on the biphenyl concentration but also on the follicular layer and the maturational state of the follicle. EROD and MROD activities appeared predominantly in the granulosa layer of the yellow preovulatory follicles. PCB 126 induced these activities in a dose-dependent manner in all ovarian follicles. The obtained results suggest that ovarian follicles, especially the granulosa layer, are involved in the detoxification process of PCBs in the laying hen. Taking this finding into consideration it can be suggested that the granulosa layer of the yellow hierarchical follicles plays a key role in the protective mechanism which reduces the amount of transferred dioxin-like compounds into the yolk of the oocyte. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Comparing long term impact on ovarian reserve between laparoscopic ovarian cystectomy and open laprotomy for ovarian endometrioma

PubMed Central

2013-01-01

Objective To compare the long term impact on ovarian reserve between laparoscopic ovarian cystectomy with bipolar electrocoagulation and laparotomic cystectomy with suturing for ovarian endometrotic cyst. Patient and method(s) 121 patients with benign ovarian endometroitic cysts were randomised to either laparoscopic ovarian cystectomy using bipolar electrocoagulation (61 patients) or laparotomic ovarian cystectomy using sutures (60 patients). Serum follicle-stimulating hormone, Antimullerian hormon, Basal antral follicle Count, mean ovarian diameter, and ovarian stromal blood flow velocity were measured at 6, 12 and 18 months after surgery and compared in both groups. Result(s) A statistically significant increase of serum FSH was found in the laproscopic bipolar group at 6-, 12 and 18-month postoperativly compared to open laparotomy suture group. Also, a statistically significant decrease of the mean AMH value occurred in laproscopic bipolar group at 6-, 12 and 18-month follow- up compared to open laparotomy suture group. Basal antral follicle number, mean ovarian diameter and peak systolic velocity were significantly decreased during the 6-, 12,18 -month follow-up in laproscopic bipolar group compared to open laparotomy suture group. Conclusion(s) After laproscopic ovarian cystecomy for endometrioma all pareameter of ovarian reseve are significantly decreased on long term follow up as compared to open laprotomy. PMID:24180348

Ovotoxicity of cigarette smoke: A systematic review of the literature.

PubMed

Budani, Maria Cristina; Tiboni, Gian Mario

2017-09-01

This study reviews the scientific literature on the noxious effects of cigarette smoke on the ovarian follicle, and the cumulative data on the impact of smoking on in vitro fertilization (IVF) cycle outcome. There is a close association between tobacco smoke and accelerated follicle loss, abnormal follicle growth and impairment of oocyte morphology and maturation. There is an increasing amount of evidence indicating that smoke can directly derange folliculogenesis. Increased cellular apoptosis or autophagy, DNA damage and abnormal crosstalk between oocyte and granulosa cells have been implicated in the demise of ovarian follicles. It becomes increasingly clear that maternal smoking can exert multigenerational effects on the ovarian function of the progeny. Growing evidence suggests that cigarette smoke is associated with decreased results after IVF. Further research is needed to better define the molecular mechanisms behind smoking-induced ovarian disruption. Copyright © 2017 Elsevier Inc. All rights reserved.

Ultrasound features of polycystic ovaries relate to degree of reproductive and metabolic disturbance in polycystic ovary syndrome.

PubMed

Christ, Jacob P; Vanden Brink, Heidi; Brooks, Eric D; Pierson, Roger A; Chizen, Donna R; Lujan, Marla E

2015-03-01

To reexamine associations between polycystic ovarian morphology (PCOM) and degree of symptomatology in polycystic ovary syndrome (PCOS) using a well-defined PCOS population, newer ultrasound technology, and reliable offline assessments of sonographic parameters. Cross-sectional observational study. Academic hospital and clinical research unit. Forty-nine women with PCOS as defined by hyperandrogenism and oligoamenorrhea. None. Number of follicles per follicle size category, antral follicle count (AFC), ovarian volume (OV), follicle distribution pattern, stromal area, ovarian area, stromal to ovarian area ratio (S/A) and stromal echogenicity index (SI), total (TT), androstenedione, LH, FSH, cholesterol, triglycerides, low-density lipoprotein, high-density lipoprotein, C-reactive protein, glucose, insulin, and hemoglobin A1C, menstrual cycle length, hirsutism score, body mass index (BMI), waist:hip ratio, and blood pressure. AFC, but not OV, was positively associated with TT (ρ = .610), androstenedione (ρ = .490), and LH:FSH (ρ = .402). SI was positively associated with androgen markers and LH:FSH, while S/A was negatively associated with these variables. Follicles ≤4 mm were negatively associated with various metabolic markers, whereas larger follicles (5-8 mm) showed positive associations. Stromal markers were not associated with cardiometabolic measures. LH:FSH best predicted follicles ≤4 mm, and BMI predicted 5- to 9-mm follicles. Dominant follicles ≥10 mm were best predicted by age. AFC, and not OV, reflected the severity of reproductive dysfunction in PCOS. Associations among different sized follicles were consistent with recruitable sized follicles, which reflects the severity of metabolic dysfunction in PCOS. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Anti-Müllerian hormone inhibits activation and growth of bovine ovarian follicles in vitro and is localized to growing follicles.

PubMed

Yang, M Y; Cushman, R A; Fortune, J E

2017-05-01

Does anti-Müllerian hormone (AMH) inhibit activation (initiation of growth) of primordial follicles and attenuate the growth of primary follicles in cattle, an excellent animal model for human ovarian follicular development? AMH inhibited activation of bovine primordial follicles and attenuated the growth of activated follicles in vitro. In mice null mutant for AMH, the pool of primordial follicles is depleted prematurely and AMH inhibits follicle activation in vitro. Results of studies with human ovarian tissue in vitro were inconsistent. Our previous work provided indirect evidence that AMH inhibits follicle activation in bovine ovaries. Pieces of fetal bovine ovarian cortex (2 pieces/culture well), obtained during mid or late pregnancy, were cultured in control medium or with graded doses of AMH for 2, 10 or 12 days. Effects of treatment on follicle activation and growth were determined by histological morphometry; follicles in every 20th histological section were staged (primordial or primary), counted, and measured. In addition, AMH was immunolocalized in bovine ovaries obtained at various times during pregnancy (n = 20 ovaries). Bovine fetal ovaries at mid or late gestation were obtained at a commercial abattoir. Pieces of ovarian cortex were cultured without or with AMH and fixed for histological morphometry on Day 0 and at the end of culture. Treatments were applied to duplicate cultures from each of two or three fetuses. In 12-day cultures, addition of AMH was delayed until the third day. Histological analysis provided information about the types, numbers and sizes of follicles in cortical pieces before and after treatments. Ovaries obtained during the second and third trimesters were assessed for the presence of AMH by immunohistochemistry. AMH (100-500 ng/ml) inhibited follicle activation in response to an activator (insulin) in ovarian cortical pieces from fetal ovaries in late gestation. Dose-dependent inhibitory effects on the diameters of primary follicles and their oocytes were also observed. These results were obtained only when AMH was added to cultures in advance of insulin (presumably because it penetrates tissue more slowly than insulin). Results of experiments with cortical pieces from fetal ovaries at mid-gestation, when follicles are forming, showed that AMH did not inhibit the formation of follicles. Immunohistochemical localization of AMH showed that it is not present in fetal ovaries until the third trimester, when it was localized to the granulosa cells of secondary and small antral follicles. The experiments were performed with fetal ovaries because follicles form and follicle activation begins during fetal life in cattle (as it does in humans), so fetal ovarian cortex of later gestation provides tissue rich in primordial follicles. We assume, but have no experimental evidence, that our findings also apply to post-natal ovaries. Although circulating AMH is used as an indication of the follicular reserve in women, little is known about AMH in human ovaries. Cattle are an excellent non-primate model for human ovarian follicular development and, hence, the findings suggest similar roles for AMH in human follicular development. Not applicable. This research was supported by National Research Initiative Competitive Grants no. 00-35203-9151, 2003-35203-13532, and 2008-35203-05989) from the U.S. Dept. of Agriculture's National Institute of Food and Agriculture to JEF and by an NIH National Research Service Award (F32 HD08264) to RAC. There are no conflicts of interest or competing interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Expression pattern of vascular endothelial growth factor in canine folliculogenesis and its effect on the growth and development of follicles after ovarian organ culture.

PubMed

Abdel-Ghani, M A; Shimizu, T; Suzuki, H

2014-10-01

In this study, the expressions of VEGF in dog follicles were detected by immunohistochemistry and the effects of VEGF treatment on the primordial to primary follicle transition and on subsequent follicle progression were examined using a dog ovary organ culture system. The frozen-thawed canine ovarian follicles within slices of ovarian cortical tissue were cultured for 7 and 14 days in presence or absence of VEGF. After culture, the ovaries were fixed, sectioned, stained and counted for morphologic analysis. The results showed that VEGF was expressed in the theca cells of antral follicles and in the granulosa cells nearest the oocyte in preantral follicle but not in granulosa cells of primordial and primary follicles; however, the VEGF protein was expressed in CL. After in vitro culture, VEGF caused a decrease in the number of primordial follicles and concomitant increase in the number of primary follicles that showed growth initiation and reached the secondary and preantral stages of development after 7 and 14 days. Follicular viability was also improved in the presence of VEGF after 7 and 14 days in culture. In conclusion, treatment with VEGF was found to promote the activation of primordial follicle development that could provide an alternative approach to stimulate early follicle development in dogs. © 2014 Blackwell Verlag GmbH.

Insulin improves in vitro survival of equine preantral follicles enclosed in ovarian tissue and reduces reactive oxygen species production after culture.

PubMed

Aguiar, F L N; Lunardi, F O; Lima, L F; Rocha, R M P; Bruno, J B; Magalhães-Padilha, D M; Cibin, F W S; Rodrigues, A P R; Gastal, M O; Gastal, E L; Figueiredo, J R

2016-04-01

This study investigated the effect of insulin concentration on the in vitro culture of equine preantral follicles enclosed in ovarian tissue. Ovarian tissue samples were immediately fixed (noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0 ng/mL, 10 ng/mL, or 10 μg/mL insulin. Ovarian tissues were processed and analyzed by classical histology. Culture medium samples were collected after 1 and 7 days of culture for steroid and reactive oxygen species (ROS) analyses. The percentage of morphologically normal follicles was greater (P < 0.001) in insulin-treated groups after 1 day of culture; likewise, more (P < 0.02) normal follicles were observed after 7 days of culture in medium supplemented with 10-ng/mL insulin. Furthermore, an increase (P < 0.01) in developing (transition, primary, and secondary) follicles between Days 1 and 7 of culture was observed only with the 10-ng/mL insulin treatment. ROS production after 1 or 7 days of culture was lower (P < 0.0001) in medium with 10-ng/mL insulin than the other treatments. Ovarian tissues containing preantral follicles were able to produce estradiol and progesterone after 1 and 7 days of culture; however, treatments did not differ in steroid production. In conclusion, the use of a physiological concentration (10 ng/mL) of insulin rather than the previously reported concentration (10 μg/mL) for in vitro culture of equine preantral follicles improved follicular survival and growth and lowered oxidative stress. Results from this study shed light on new perspectives for producing an appropriate medium to improve equine preantral follicle in vitro survival and growth. Copyright © 2016 Elsevier Inc. All rights reserved.

Melatonin and exposure to constant light/darkness affects ovarian follicular kinetics and estrous cycle in Indian desert gerbil Meriones hurrianae.

PubMed

Sinhasane, S V; Joshi, B N

1997-12-01

Melatonin mediates photoperiodic influence on reproduction and constant light and darkness affect pineal biosynthesis of melatonin. The present study was undertaken to assess the effects of melatonin and drastic photoperiodic changes on reproduction in a tropical desert species with a fossorial lifestyle. Ovarian follicular kinetics and estrous cycle were studied in the Indian desert gerbil Meriones hurrianae, after treatment with melatonin and exposure to constant light (LL) and darkness (DD) regimes. Melatonin treatment increased (P < 0.001) ovarian weights without changing the uterine weights. While exposure to LL decreased (P < 0.001) both ovarian and uterine weights, exposure to DD had no effect on these weights. Follicular kinetics of growing and regressing follicles revealed that ovaries of melatonin-treated and DD-exposed animals had significantly more growing follicles. Melatonin treatment increased all types of growing follicles, especially antral and Graafian follicles. Exposure to DD increased all types of growing follicles, with the medium sized antral and Graafian follicles being significant (P < 0.01). In contrast to stimulation of follicular growth by melatonin and DD, LL caused regression of all stages of follicular growth and also reduced the number of small preantral follicles. Melatonin treatment increased (P < 0.001) the length of estrous cycle (5.08 to 7.29 days). Gerbils treated with melatonin, exposed to LL and DD, had a longer (P < 0.001) metestrus. Animals held in LL, had the least number (P < 0.001) of estrous smears (1 in 30 days). The results suggest that melatonin is involved in growth of ovarian follicles in the Indian desert gerbil. Copyright 1997 Academic Press.

Human single follicle growth in vitro from cryopreserved ovarian tissue after slow freezing or vitrification.

PubMed

Wang, Tian-ren; Yan, Jie; Lu, Cui-ling; Xia, Xi; Yin, Tai-lang; Zhi, Xu; Zhu, Xiao-hui; Ding, Ting; Hu, Wei-hong; Guo, Hong-yan; Li, Rong; Yan, Li-ying; Qiao, Jie

2016-04-01

What is the effect of human ovarian tissue cryopreservation on single follicular development in vitro? Vitrification had a greater negative effect on growth and gene expression of human ovarian follicles when compared with fresh follicles. For human ovarian cortex cryopreservation, the conventional option is slow freezing while more recently vitrification has been demonstrated to maintain good quality and function of ovarian tissues. Ovarian tissues were collected from 11 patients. For every patient, the ovarian cortex was divided into three samples: Fresh, slow-rate freezing (Slow) and vitrification (Vit). Tissue histology was performed and follicles were isolated for single-cell mRNA analysis and in vitro culture (IVC) in 1% alginate for 8 days. Follicle morphology was assessed with hematoxylin-eosin analysis. Follicles were individually embedded in alginate (1% w/v) and cultured in vitro for 8 days. Follicle survival and growth were assessed by microscopy. Follicle viability was observed after Calcein-AM and ethidium homodimer-I (Ca-AM/EthD-I) staining. Expression of genes, including GDF9 (growth differentiation factor 9), BMP15 (bone morphogenetic protein 15) and ZP3 (zona pellucida glycoprotein 3) in oocytes and AMH (anti-Mullerian hormone), FSHR (FSH receptor), CYP11A (cholesterol side-chain cleavage cytochrome P450) and STAR (steroidogenic acute regulatory protein) in GCs, was evaluated by single-cell mRNA analysis. A total of 129 follicles were separated from ovarian cortex (Fresh n = 44; Slow n = 40; Vit n = 45). The percentage of damaged oocytes and granulosa cells was significantly higher in both the Slow and Vit groups, as compared with Fresh control (P< 0.05). The growth of follicles in vitro was significantly delayed in the Vit group compared with the Fresh group (P< 0.05). Both slow freezing (P< 0.05) and vitrification (P< 0.05) down-regulated the mRNA levels of ZP3 and CYP11A compared with Fresh group, while there was no significant difference between the Slow and Vit groups (P> 0.05). Vitrification also down-regulates AMH mRNA levels compared with Fresh group (P< 0.05). Only short-term IVC studies (8 days) are reported. Further study should be performed to examine and improve follicular development in a long-term culture system after cryopreservation. This is the first comparison of gene expression and growth of single human ovarian follicles in vitro after either slow freezing or vitrification. With the decreased gene expression and growth during IVC, damage by cryopreservation still exists and needs to be minimized during the long-term IVC of follicles in the future for eventual clinical application. This work was supported by the National Natural Science Foundation of China (31230047, 81571386, 81471508, 31429004 and 81501247), National Natural Science Foundation of Beijing (7142166) and Mega-projects of Science Research for the 12th five-year plan (2012ba132b05). There are no conflicts of interest to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Anti-Mullerian Hormone: A Marker of Ovarian Reserve and its Association with Polycystic Ovarian Syndrome.

PubMed

Verma, Anil Kumar; Rajbhar, Sarita; Mishra, Jyoti; Gupta, Mayank; Sharma, Mratunjai; Deshmukh, Geeta; Ali, Wahid

2016-12-01

Anti-Mullerian Hormone (AMH) is a useful endocrine marker for assessing the ovarian reserve. AMH serum level reflects the number of follicles that have made the transition from the primordial pool into the growing follicle pool, and it is not controlled by gonadotropins. The present study was conducted to correlate serum AMH levels with Polycystic Ovarian Syndrome (PCOS) and type of treatment protocol. Serum AMH levels were performed in the early follicular phase (on 2 nd day of menstrual cycle) both in infertile females including PCOS and control women. The results were analyzed in relation to age, Body Mass Index (BMI), ovarian volume, serum Follicle Stimulating Hormone (FSH) levels, Antral Follicle Count (AFC), type of treatment protocols and also in association with PCOS patients. The serum levels of AMH were measured in all the participants on 2 nd day of menstrual cycle using ultra sensitive Enzyme Linked Immunosorbent Assay (ELISA). The plasma AMH levels were significantly higher in women with polycystic ovarian syndrome. The significant association was seen between FSH and AFC with AMH. However, no significant association was observed between AMH levels with age, BMI, ovarian volume and type of treatment protocols. The serum AMH measurement was significantly higher in PCOS patients. No association with type of treatment protocol was obtained.

Xenotransplantation in immunodeficient mice to study ovarian follicular development in domestic animals.

PubMed

Bols, P E J; Aerts, J M J; Langbeen, A; Goovaerts, I G F; Leroy, J L M R

2010-04-01

Nowadays, in vitro study of follicular dynamics of primordial and primary follicular stages is limited because in vitro culture systems for these follicles are lacking, both in domestic animal species and in human. Therefore, additional insights might be generated by grafting ovarian tissue into immunodeficient mice to study activation and maturation of early follicular stages. A considerable amount of data has already been gathered in laboratory animals and through clinical application of human assisted reproduction technologies where live births were reported recently after the use of (cryopreserved) ovarian grafts. However, given that human preantral follicles are difficult to obtain and that there are many similarities between the bovine and human species with regard to ovarian physiology, the bovine model offers exciting additional prospects and is therefore discussed in more detail. This review will focus on recent developments related to preantral follicle and (repeated) ovarian tissue retrieval and xenotransplantation of (bovine) ovarian tissue strips to immunodeficient mice as a model to study preantral follicular dynamics. Different grafting strategies will be discussed as well as the consequences of this procedure on the viability and dynamic behavior of the grafted tissue and follicles. 2010 Elsevier Inc. All rights reserved.

Ontogeny of the ovary in polycystic ovary syndrome

PubMed Central

Dumesic, Daniel A.; Richards, JoAnne S.

2015-01-01

Activation of primordial follicles into the growing pool, selection of the dominant follicle, and its eventual ovulation require complex endocrine and metabolic interactions as well as intraovarian paracrine signals to coordinate granulosa cell proliferation, theca cell differentiation, and oocyte maturation. Early preantral follicle development relies mostly upon mesenchymal-epithelial cell interactions, intraovarian paracrine signals, and oocyte-secreted factors, whereas development of the antral follicle depends on circulating gonadotropins as well as locally derived regulators. In women with polycystic ovary syndrome (PCOS), ovarian hyperandrogenism, hyperinsulinemia from insulin resistance, and altered intrafollicular paracrine signaling perturb the activation, survival, growth, and selection of follicles, causing accumulation of small antral follicles within the periphery of the ovary, giving it a polycystic morphology. Altered adipocyte-ovarian interactions further compound these adverse events on follicle development and also can harm the oocyte, particularly in the presence of increased adiposity. Finally, endocrine antecedents of PCOS occur in female infants born to mothers with PCOS, which suggests that interactions between genes and the maternal-fetal hormonal environment may program ovarian function after birth. PMID:23472949

FSH supplementation to culture medium is beneficial for activation and survival of preantral follicles enclosed in equine ovarian tissue.

PubMed

Aguiar, F L N; Lunardi, F O; Lima, L F; Rocha, R M P; Bruno, J B; Magalhães-Padilha, D M; Cibin, F W S; Nunes-Pinheiro, D C S; Gastal, M O; Rodrigues, A P R; Apgar, G A; Gastal, E L; Figueiredo, J R

2016-04-01

This study investigated the effect of adding different concentrations of bovine recombinant follicle-stimulating hormone on the IVC of equine preantral follicles enclosed in ovarian tissue fragments. Randomized ovarian fragments were fixed immediately (fresh noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0, 10, 50, and 100 ng/mL FSH and subsequently analyzed by classical histology. Culture media collected on Day 1 or Day 7 and were analyzed for steroids (estradiol and progesterone) and reactive oxygen species (ROS). After Day 1 and Day 7 of culture, 50-ng/mL FSH treatment had a greater (P < 0.05) percentage of morphologically normal follicles when compared to the other groups, except the 10-ng/mL FSH treatment at Day 1 of culture. The percentage of developing follicles (transition, primary, and secondary), and follicular and oocyte diameters were higher (P < 0.05) in the 50-ng/mL FSH treatment compared to the other groups after Day 7 of culture. Furthermore, estradiol secretion and ROS production were maintained (P > 0.05) throughout the culture in the 50-ng/mL FSH treatment. In conclusion, the addition of 50 ng/mL of FSH promoted activation of primordial follicles to developing follicles, improved survival of preantral follicles, and maintained estradiol and ROS production of equine ovarian tissue after 7 days of culture. Copyright © 2016 Elsevier Inc. All rights reserved.

The Steroid Metabolome in the Isolated Ovarian Follicle and Its Response to Androgen Exposure and Antagonism

PubMed Central

Lebbe, Marie; Taylor, Angela E.; Visser, Jenny A.; Kirkman-Brown, Jackson C.; Woodruff, Teresa K.

2017-01-01

The ovarian follicle is a major site of steroidogenesis, crucially required for normal ovarian function and female reproduction. Our understanding of androgen synthesis and metabolism in the developing follicle has been limited by the sensitivity and specificity issues of previously used assays. Here we used liquid chromatography–tandem mass spectrometry to map the stage-dependent endogenous steroid metabolome in an encapsulated in vitro follicle growth system, from murine secondary through antral follicles. Furthermore, follicles were cultured in the presence of androgen precursors, nonaromatizable active androgen, and androgen receptor (AR) antagonists to assess effects on steroidogenesis and follicle development. Cultured follicles showed a stage-dependent increase in endogenous androgen, estrogen, and progesterone production, and incubations with the sex steroid precursor dehydroepiandrosterone revealed the follicle as capable of active androgen synthesis at early developmental stages. Androgen exposure and antagonism demonstrated AR–mediated effects on follicle growth and antrum formation that followed a biphasic pattern, with low levels of androgens inducing more rapid follicle maturation and high doses inhibiting oocyte maturation and follicle growth. Crucially, our study provides evidence for an intrafollicular feedback circuit regulating steroidogenesis, with decreased follicle androgen synthesis after exogenous androgen exposure and increased androgen output after additional AR antagonist treatment. We propose that this feedback circuit helps maintain an equilibrium of androgen exposure in the developing follicle. The observed biphasic response of follicle growth and function in increasing androgen supplementations has implications for our understanding of polycystic ovary syndrome pathophysiology and the dose-dependent utility of androgens in in vitro fertilization settings. PMID:28323936

Short-Term PTEN Inhibition Improves In Vitro Activation of Primordial Follicles, Preserves Follicular Viability, and Restores AMH Levels in Cryopreserved Ovarian Tissue From Cancer Patients

PubMed Central

Novella-Maestre, Edurne; Herraiz, Sonia; Rodríguez-Iglesias, Beatriz; Díaz-García, César; Pellicer, Antonio

2015-01-01

Introduction In vitro activation and growth of primordial dormant follicles to produce fertilizable oocytes would provide a useful instrument for fertility preservation. The employment of Phosphatase and TENsin homolog (PTEN) inhibitors, in combination with Protein kinase B (Akt) stimulating molecules, has been previously employed to increase follicular activation through the stimulation of the PTEN-Akt pathway. Methods We aim to establish improved in vitro activation also for cancer patients whose ovarian tissue has already been cryopreserved. Fresh and previously cryopreserved human ovarian cortex were exposed to short-term, low-concentration and ovary-specific treatment with only a PTEN inhibitor. Results Our in vitro activation protocol enhances the activation mechanisms of primordial follicles in both fresh and cryopreserved samples, and enlarges growing populations without inducing apoptosis in either follicles or the surrounding stroma. Treatment augments estradiol secretion and restores the expression levels of the previously diminished Anti-Müllerian hormone by means of cryopreservation procedures. Genomic modulation of the relative expression of PTEN pathway genes was found in treated samples. Conclusion The in vitro activation protocol offers new alternatives for patients with cryopreserved tissue as it increases the pool of viable activated follicles available for in vitro growth procedures. The combination of ovarian tissue cryopreservation and in vitro activation of primordial follicles, the main ovarian reserve component, will be a major advancement in fertility preservation. PMID:26024525

First pregnancies, live birth, and in vitro fertilization outcomes after transplantation of frozen-banked ovarian tissue with a human extracellular matrix scaffold using robot-assisted minimally invasive surgery.

PubMed

Oktay, Kutluk; Bedoschi, Giuliano; Pacheco, Fernanda; Turan, Volkan; Emirdar, Volkan

2016-01-01

Ovarian tissue cryopreservation is an experimental fertility preservation method and the transplantation techniques are still evolving. We attempted to improve the technique with the utility of a human decellularized extracellular tissue matrix (ECTM) scaffold, robot-assisted minimally invasive surgery, and perioperative pharmacological support. We prospectively studied 2 subjects with hemophagocytic lymphohistiocytosis (patient A) and non-Hodgkin lymphoma (patient B) who underwent ovarian tissue cryopreservation at the age of 23 years, before receiving preconditioning chemotherapy for hematopoietic stem cell transplantation. Both experienced ovarian failure postchemotherapy and we transplanted ovarian cortical tissues to the contralateral menopausal ovary 7 and 12 years later, using a human ECTM scaffold and robotic assistance. The ECTM scaffold tissue compatibility was shown in preclinical studies. Patients also received estrogen supplementation and baby aspirin preoperatively to aid in the revascularization process. Ovarian follicle development was observed approximately 10 (patient A) and 8 (patient B) weeks after ovarian tissue transplantation. Following 8 and 7 cycles of in vitro fertilization, 9 and 10 day-3 embryos were cryopreserved (patients A and B, respectively). While the baseline follicle-stimulating hormone (range 3.6-15.4 mIU/mL) levels near normalized by 7 months and remained steady postovarian transplantation in patient A, patient B showed improved but elevated follicle-stimulating hormone levels throughout (range 21-31 mIU/mL). Highest follicle yield was achieved 14 (8 follicles; patient A) and 11 (6 follicles; patient B) months postintervention. Patient A experienced a chemical pregnancy after the third frozen embryo transfer attempt. She then conceived following her first fresh in vitro fertilization embryo transfer and the pregnancy is currently ongoing. Patient B conceived after the first frozen embryo transfer attempt and delivered a healthy girl at term. We report the first pregnancies after the minimally invasive transplantation of previously cryopreserved ovarian tissue with an ECTM scaffold. This approach seems to be associated with steady ovarian function after a follow-up of up to 2 years. Copyright © 2016 Elsevier Inc. All rights reserved.

Survival and growth of isolated pre-antral follicles from human ovarian medulla tissue during long-term 3D culture.

PubMed

Yin, H; Kristensen, S G; Jiang, H; Rasmussen, A; Andersen, C Yding

2016-07-01

Can human pre-antral follicles isolated enzymatically from surplus medulla tissue survive and grow in vitro during long-term 3D culture? Secondary human follicles can develop to small antral follicles and remain hormonally active in an alginate-encapsulation culture system for more than 30 days. Ovarian tissue cryopreservation followed by transplantation is a promising fertility preservation approach for cancer patients. However, transplantation of cryopreserved tissue to patients may carry the risk of re-implanting malignant cells. Grafting of follicles enzymatically isolated from ovarian tissue or developing a method for follicular culture and maturation in vitro may provide fertility to such patients without the risk of reintroducing the malignancy. However, the growth of pre-antral follicles isolated by enzymatic digestion from medulla tissue during long-term culture has received only little attention. Two to ten human pre-antral follicles were encapsulated together within an alginate bead and cultured with or without ovarian interstitial tissue for either 7 days or >30 days. Follicles were cultured in either 20% oxygen or 5% oxygen or encapsulated in a lower concentration of alginate together with a lower concentration of FSH in high oxygen. A total of 395 pre-antral follicles from 16 cancer patients, aged 9-37 years, were co-cultured for either 7 days or >30 days. A proportion of follicle (64) were removed from culture on Day 7 and assessed for viability using confocal fluorescence microscopy following calcein-AM and ethidium homodimer-1 staining or histology. The remaining follicles (331) were continued in culture for >30 days then assessed for survival and growth. Anti-Müllerian hormone (AMH) and estradiol levels were quantified in the medium. An optimized protocol for isolation of intact healthy pre-antral follicles from ovarian medulla was developed. After 7 days of culture, secondary follicles had a significantly higher survival rates compared with primary and primordial follicles (70 versus <38%). Primordial and primary follicles did not develop into the antral follicle stage. In contrast, secondary follicles continued to develop in all culture conditions examined. Based on growth rate and morphology, four distinct cohorts of surviving follicles, 'fast growth', 'slow growth', 'no growth' and 'extruded oocyte' were identified. From Day 1 to Day 30, the mean diameter of follicles increased from 184 ± 35 to 661 ± 120 μm (significant from Day 18), 145 ± 19 to 318 ± 68 μm and 136 ± 15 to 162 ± 25 μm (mean ± SD) in the 'fast growth', 'slow growth' and 'no growth' patterns, respectively. The fast growth follicles also contained a larger diameter oocyte than other follicle groups. From the pre-antral follicle to antral stage, follicles became steroidogenically active and secretion of AMH and estradiol increased. No significant difference between the follicles cultured with or without ovarian interstitial tissue was observed. The number of surviving follicles at the end of study was low in each of the culture conditions therefore whether there is a benefit with any of the conditions is difficult to ascertain. Multiple pre-antral follicles were cultured within the same alginate bead which may affect the in vitro development of the secondary follicles. These findings show that pre-antral follicles, isolated enzymatically from surplus medulla tissue that is normally discarded, possess a developmental potential which may be used to devise safer fertility preservation methods for patients who are at high risk of malignant contamination of their ovarian tissue. The Child Cancer Foundation in Denmark (2012-26) and the EU interregional project ReproHigh are thanked for having funded this study; and the Key Program of Medical Science and Technology Innovation of Nanjing Military Area Command in China (14ZX06; 11Z010). They had no role in the study design, collection and analysis of data, data interpretation or in writing the report. The authors have no conflicts of interest to disclose. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Very low doses of heavy oxygen ion radiation induce premature ovarian failure.

PubMed

Mishra, Birendra; Ripperdan, Ryan; Ortiz, Laura; Luderer, Ulrike

2017-08-01

Astronauts are exposed to charged particles during space travel, and charged particles are also used for cancer radiotherapy. Premature ovarian failure is a well-known side effect of conventional, low linear energy transfer (LET) cancer radiotherapy, but little is known about the effects of high LET charged particles on the ovary. We hypothesized that lower LET (16.5 keV/µm) oxygen particles would be less damaging to the ovary than we previously found for iron (LET = 179 keV/µm). Adult female mice were irradiated with 0, 5, 30 or 50 cGy oxygen ions or 50 cGy oxygen plus dietary supplementation with the antioxidant alpha lipoic acid (ALA). Six-hour after irradiation, percentages of ovarian follicles immunopositive for γH2AX, a marker of DNA double strand breaks, 4-HNE, a marker of oxidative lipid damage and BBC3 (PUMA), a proapoptotic BCL-2 family protein, were dose dependently increased in irradiated mice compared to controls. One week after irradiation, numbers of primordial, primary and secondary follicles per ovary were dose dependently decreased, with complete absence of follicles in the 50 cGy groups. The ED 50 for primordial follicle destruction was 4.6 cGy for oxygen compared to 27.5 cGy for iron in our previous study. Serum FSH and LH concentrations were significantly elevated in 50 cGy groups at 8 week. Supplementation with ALA mitigated the early effects, but not the ultimate depletion of ovarian follicles. In conclusion, oxygen charged particles are even more potent inducers of ovarian follicle depletion than charged iron particles, raising concern for premature ovarian failure in astronauts exposed to both particles during space travel. © 2017 Society for Reproduction and Fertility.

Preservation of primordial follicles from lions by slow freezing and xenotransplantation of ovarian cortex into an immunodeficient mouse.

PubMed

Wiedemann, C; Hribal, R; Ringleb, J; Bertelsen, M F; Rasmusen, K; Andersen, C Y; Kristensen, S G; Jewgenow, K

2012-12-01

Assisted reproductive technology (ART) is considered an important tool in the conservation of endangered species, but often the most limiting factor of ART is the availability of mature oocytes. The aim of the present study was to investigate the feasibility of preserving female germ cells from ovaries of female lions (Panthera leo). Good quality cumulus-oocyte complexes (COCs) were isolated and subjected to in vitro maturation (IVM). In addition, ovarian cortex was obtained and cut into pieces for culture and cryopreservation by slow freezing. The survival of ovarian follicles was assessed by histology. Frozen-thawed samples of ovarian cortex samples were xenotransplanted under the skin of ovariectomized immunodeficient mouse for 28 days. Overall, 178 intact COCs were obtained from 13 lions, but only 28.1% were matured in vitro indicating insufficient IVM conditions. In contrast, almost all follicles within the ovarian cortex survived culture when the original sample was from a young healthy lion collected immediately after euthanasia. Within the xenotransplants, the number of primordial follicles decreased after 28 days by 20%, but the relation between primordial and growing follicles changed in favour of follicular growth. Female gamete rescue from valuable felids may be performed by slow freeze cryopreservation of ovarian cortex. Although the IVM protocol for lions is not yet optimized, mature oocytes may be obtained after long-term xenotransplantation and IVM and could potentially represent one way of salvage of endangered felid species in the future. © 2012 Blackwell Verlag GmbH.

Basic fibroblast growth factor promotes the development of human ovarian early follicles during growth in vitro.

PubMed

Wang, Tian-ren; Yan, Li-ying; Yan, Jie; Lu, Cui-ling; Xia, Xi; Yin, Tai-lang; Zhu, Xiao-hui; Gao, Jiang-man; Ding, Ting; Hu, Wei-hong; Guo, Hong-yan; Li, Rong; Qiao, Jie

2014-03-01

What is the effect of basic fibroblast growth factor (bFGF) on the growth of individual early human follicles in a three-dimensional (3D) culture system in vitro? The addition of 200 ng bFGF/ml improves human early follicle growth, survival and viability during growth in vitro. It has been demonstrated that bFGF enhances primordial follicle development in human ovarian tissue culture. However, the growth and survival of individual early follicles in encapsulated 3D culture have not been reported. The maturation in vitro of human ovarian follicles was investigated. Ovarian tissue (n= 11) was obtained from 11 women during laparoscopic surgery for gynecological disease, after obtaining written informed consent. One hundred and fifty-four early follicles were isolated by enzymic digestion and mechanical disruption. They were individually encapsulated into alginate (1% w/v) and randomly assigned to be cultured with 0, 100, 200 or 300 ng bFGF/ml for 8 days. Individual follicles were cultured in minimum essential medium α (αMEM) supplemented with bFGF. Follicle survival and growth were assessed by microscopy. Follicle viability was evaluated under confocal laser scanning microscope following Calcein-AM and Ethidium homodimer-I (Ca-AM/EthD-I) staining. After 8 days in culture, all 154 follicles had increased in size. The diameter and survival rate of the follicles and the percentage with good viability were significantly higher in the group cultured with 200 ng bFGF/ml than in the group without bFGF (P < 0.05). The percentage of follicles in the pre-antral stage was significantly higher in the 200 ng bFGF/ml group than in the group without bFGF (P < 0.05), while the percentages of primordial and primary follicles were significantly lower (P < 0.05). The study focuses on the effect of bFGF on the development of individual human early follicles in 3D culture in vitro and has limited ability to reveal the specific effect of bFGF at each different stage. The findings highlight the need to improve the acquisition and isolation of human ovarian follicles. The in vitro 3D culture of human follicles with appropriate dosage of bFGF offers an effective method to investigate their development. Moreover, it allows early follicles to be cultured to an advanced stage and therefore has the potential to become an important source of mature oocytes for assisted reproductive technology; particularly as an option for fertility preservation in women, including patients with cancer. This work was supported by the National Basic Research Program of China (2011|CB944504, 2011CB944503) and the National Natural Science Foundation of China (81200470, 81000275, 31230047, 8110197). There are no conflicts of interest to declare.

Primary Ovarian Insufficiency Induced by Fanconi Anemia E Mutation in a Mouse Model.

PubMed

Fu, Chun; Begum, Khurshida; Overbeek, Paul A

2016-01-01

In most cases of primary ovarian insufficiency (POI), the cause of the depletion of ovarian follicles is unknown. Fanconi anemia (FA) proteins are known to play important roles in follicular development. Using random insertional mutagenesis with a lentiviral transgene, we identified a family with reduced fertility in the homozygous transgenic mice. We identified the integration site and found that the lentivirus had integrated into intron 8 of the Fanconi E gene (Fance). By RT-PCR and in situ hybridization, we found that Fance transcript levels were significantly reduced. The Fance homozygous mutant mice were assayed for changes in ovarian development, follicle numbers and estrous cycle. Ovarian dysplasias and a severe lack of follicles were seen in the mutant mice. In addition, the estrous cycle was disrupted in adult females. Our results suggest that POI has been induced by the Fance mutation in this new mouse model.

Gonadotropin-dependent oocyte maturational competence requires activation of the protein kinase A pathway and synthesis of RNA and protein in ovarian follicles of Nibe, Nibea mitsukurii (Teleostei, Sciaenidae)

USGS Publications Warehouse

Yoshizaki, G.; Shusa, M.; Takeuchi, T.; Patino, R.

2002-01-01

Luteinizing hormone- (LH)-dependent ovarian follicle maturation has been recently described in two stages for teleost fishes. The oocyte's ability to respond to the steroidal maturation-inducing hormone (MIH), also known as oocyte maturational competence (OMC), is acquired during the first stage; whereas the MIH-dependent resumption of meiosis occurs during the second stage. However, studies directly addressing OMC have been performed with a limited number of species and therefore the general relevance of the two-stage model and its mechanisms remain uncertain. In this study, we examined the hormonal regulation of OMC and its basic transduction mechanisms in ovarian follicles of the sciaenid teleost, Nibe (Nibea mitsukurii). Exposure to MIH [17,20??-dihydroxy-4-pregnen-3-one or 17,20??,21-trihydroxy-4-pregnen-3-one] stimulated germinal vesicle breakdown (index of meiotic resumption) in full-grown follicles primed with human chorionic gonadotropin (HCG, an LH-like gonadotropin) but not in those pre-cultured in plain incubation medium. The induction of OMC by HCG was mimicked by protein kinase A (PKA) activators (forskolin and dibutyryl cyclic AMP), and blocked by specific inhibitors of PKA (H89 and H8) as well as inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Forskolin-induced OMC was also inhibited by actinomycin D and cycloheximide. A strong activator of protein kinase C, PMA, inhibited HCG-dependent OMC. In conclusion, OMC in Nibe ovarian follicles is gonadotropin-dependent and requires activation of the PKA pathway followed by gene transcription and translation events. These observations are consistent with the two-stage model of ovarian follicle maturation proposed for other teleosts, and suggest that Nibe can be used as new model species for mechanistic studies of ovarian follicle differentiation and maturation in fishes.

AMH/MIS as a contraceptive that protects the ovarian reserve during chemotherapy.

PubMed

Kano, Motohiro; Sosulski, Amanda E; Zhang, LiHua; Saatcioglu, Hatice D; Wang, Dan; Nagykery, Nicholas; Sabatini, Mary E; Gao, Guangping; Donahoe, Patricia K; Pépin, David

2017-02-28

The ovarian reserve represents the stock of quiescent primordial follicles in the ovary which is gradually depleted during a woman's reproductive lifespan, resulting in menopause. Müllerian inhibiting substance (MIS) (or anti-Müllerian hormone/AMH), which is produced by granulosa cells of growing follicles, has been proposed as a negative regulator of primordial follicle activation. Here we show that long-term parenteral administration of superphysiological doses of MIS, using either an adeno-associated virus serotype 9 (AAV9) gene therapy vector or recombinant protein, resulted in a complete arrest of folliculogenesis in mice. The ovaries of MIS-treated mice were smaller than those in controls and did not contain growing follicles but retained a normal ovarian reserve. When mice treated with AAV9/MIS were paired with male breeders, they exhibited complete and permanent contraception for their entire reproductive lifespan, disrupted vaginal cycling, and hypergonadotropic hypogonadism. However, when ovaries from AAV9-MIS-treated mice were transplanted orthotopically into normal recipient mice, or when treatment with the protein was discontinued, folliculogenesis resumed, suggesting reversibility. One of the important causes of primary ovarian insufficiency is chemotherapy-induced primordial follicle depletion, which has been proposed to be mediated in part by increased activation. To test the hypothesis that MIS could prevent chemotherapy-induced overactivation, mice were given carboplatin, doxorubicin, or cyclophosphamide and were cotreated with AAV9-MIS, recombinant MIS protein, or vehicle controls. We found significantly more primordial follicles in MIS-treated animals than in controls. Thus treatment with MIS may provide a method of contraception with the unique characteristic of blocking primordial follicle activation that could be exploited to prevent the primary ovarian insufficiency often associated with chemotherapy.

AMH/MIS as a contraceptive that protects the ovarian reserve during chemotherapy

PubMed Central

Kano, Motohiro; Sosulski, Amanda E.; Zhang, LiHua; Saatcioglu, Hatice D.; Wang, Dan; Nagykery, Nicholas; Sabatini, Mary E.; Gao, Guangping; Donahoe, Patricia K.; Pépin, David

2017-01-01

The ovarian reserve represents the stock of quiescent primordial follicles in the ovary which is gradually depleted during a woman’s reproductive lifespan, resulting in menopause. Müllerian inhibiting substance (MIS) (or anti-Müllerian hormone/AMH), which is produced by granulosa cells of growing follicles, has been proposed as a negative regulator of primordial follicle activation. Here we show that long-term parenteral administration of superphysiological doses of MIS, using either an adeno-associated virus serotype 9 (AAV9) gene therapy vector or recombinant protein, resulted in a complete arrest of folliculogenesis in mice. The ovaries of MIS-treated mice were smaller than those in controls and did not contain growing follicles but retained a normal ovarian reserve. When mice treated with AAV9/MIS were paired with male breeders, they exhibited complete and permanent contraception for their entire reproductive lifespan, disrupted vaginal cycling, and hypergonadotropic hypogonadism. However, when ovaries from AAV9-MIS–treated mice were transplanted orthotopically into normal recipient mice, or when treatment with the protein was discontinued, folliculogenesis resumed, suggesting reversibility. One of the important causes of primary ovarian insufficiency is chemotherapy-induced primordial follicle depletion, which has been proposed to be mediated in part by increased activation. To test the hypothesis that MIS could prevent chemotherapy-induced overactivation, mice were given carboplatin, doxorubicin, or cyclophosphamide and were cotreated with AAV9-MIS, recombinant MIS protein, or vehicle controls. We found significantly more primordial follicles in MIS-treated animals than in controls. Thus treatment with MIS may provide a method of contraception with the unique characteristic of blocking primordial follicle activation that could be exploited to prevent the primary ovarian insufficiency often associated with chemotherapy. PMID:28137855

Developmental programming: differential effects of prenatal testosterone and dihydrotestosterone on follicular recruitment, depletion of follicular reserve, and ovarian morphology in sheep.

PubMed

Smith, Peter; Steckler, Teresa L; Veiga-Lopez, Almudena; Padmanabhan, Vasantha

2009-04-01

Prenatal testosterone excess programs an array of adult reproductive disorders including luteinizing hormone excess, functional hyperandrogenism, neuroendocrine defects, polycystic ovarian morphology, and corpus luteum dysfunction, culminating in early reproductive failure. Polycystic ovarian morphology originates from enhanced follicular recruitment and follicular persistence. We tested to determine whether prenatal testosterone treatment, by its androgenic actions, enhances follicular recruitment, causes early depletion of follicular reserve, and disrupts the ovarian architecture. Pregnant sheep were given twice-weekly injections of testosterone or dihydrotestosterone (DHT), a nonaromatizable androgen, from Days 30 to 90 of gestation. Ovaries were obtained from Day-90 and Day-140 fetuses, and from 10-mo-old females during a synchronized follicular phase (n = 5-9 per treatment). Stereological techniques were used to quantify changes in ovarian follicle/germ cell populations. Results revealed no differences in numbers of oocytes and follicles between the three groups on Fetal Day 90. Greater numbers of early growing follicles were found in prenatal testosterone- and DHT-treated fetuses on Day 140. Increased numbers of growing follicles and reduced numbers of primordial follicles were found in 10-mo-old, prenatal testosterone-treated females, but not in those treated with DHT. Antral follicles of prenatal testosterone-treated females, but not those treated with DHT, manifested several abnormalities, which included the appearance of hemorrhagic and luteinized follicles and abnormal early antrum formation. Both treatment groups showed morphological differences in the rete ovarii. These findings suggest that increased follicular recruitment and morphologic changes in the rete ovarii of prenatal testosterone-treated females are facilitated by androgenic programming, but that postpubertal follicular growth, antral follicular disruptions, and follicular depletion largely occur through estrogenic programming.

Effect of non-ionizing electromagnetic field on the alteration of ovarian follicles in rats.

PubMed

Ahmadi, Seyed Shahin; Khaki, Amir Afshin; Ainehchi, Nava; Alihemmati, Alireza; Khatooni, Azam Asghari; Khaki, Arash; Asghari, Ali

2016-03-01

In recent years, there has been an increase in the attention paid to safety effects, environmental and society's health, extremely low frequency electromagnetic fields (ELF-EMF), and radio frequency electromagnetic fields (RF-EMF). The aim of this research was to determine the effect of EMF on the alteration of ovarian follicles. In this experimental study at Tabriz Medical University in 2015, we did EMF exposures and assessed the alteration of rats' ovarian follicles. Thirty three-month old rats were selected randomly from laboratory animals, and, after their ages and weights were determined, they were divided randomly into three groups. The control group consisted of 10 rats without any treatment, and they were kept in normal conditions. The second group of rats was influenced by a magnetic field of 50 Hz for eight weeks (three weeks intrauterine and five weeks ectopic). The third group of rats was influenced by a magnetic field of 50 Hz for 13 weeks (three weeks intrauterine and ten weeks ectopic). Samples were fixed in 10% buffered formaldehyde and cleared with Xylol and embedded in paraffin. After sectioning and staining, samples were studied by optic microscopy. Finally, SPSS version 17, were used for data analysis. EMF radiation increased the harmful effects on the formation of ovarian follicles and oocytes implantation. Studies on the effects of electromagnetic fields on ovarian follicles have shown that the nuclei of the oocytes become smaller and change shape. There were significant, harmful changes in the groups affected by electromagnetic waves. Atresia of ovarian follicles was significantly significant in both study groups compared to the control group (p < 0.05). Exposure to electromagnetic fields during embryonic development can cause morphological changes in oocytes and affect the differentiation of oocytes and folliculogenesis, resulting in decreased ovarian reserve leading to infertility or reduced fertility.

Matched-pairs analysis of ovarian suppression during oral versus vaginal hormonal contraceptive use

PubMed Central

Petrie, Kelsey A.; Torgal, Anu H.; Westhoff, Carolyn L.

2011-01-01

Background This study was conducted to compare ovarian suppression during oral versus vaginal hormonal contraceptive use. Secondary aims included comparison of endometrial thickness and bleeding patterns. Methods In two open-label trials assessing ovarian suppression, 33 compliant women completed both studies. They first used OCs [randomized to either 20 mcg ethinyl estradiol (EE)/100 mcg levonorgestrel (LNG) or 30 mcg EE/150 mcg LNG] and subsequently used contraceptive vaginal rings (CVR) (daily release of 15 mcg EE/120 mcg etonogestrel), all 21/7 day regimens. Participants had at least one run-in cycle using each contraceptive method prior to evaluation. During one cycle of each method, women underwent bi-weekly transvaginal sonography to measure ovarian follicular diameters and endometrial thickness. We also noted presence of a corpus luteum or a ruptured follicle as a marker of ovulation. Participants recorded bleeding days on paper calendars. We used matched pairs analyses as appropriate. Results During follow-up, we identified at least one ovarian follicle ≥8 mm in 20/33 (61%) OC users and 12/33 (36%) CVR users (matched pairs analysis, p=0.02). Similar trends were seen for larger follicles; however we had limited statistical power to evaluate these differences. Median follicular diameter among OC users was larger than median follicular diameter among CVR users (p=0.01). We did not observe a corpus luteum or ruptured follicle in any participant during either study. Endometrial thickness was similar during OC and CVR use (mean 4.1 ± 1.4 mm versus 4.1 ± 1.6 mm, p=0.9) as was the number of bleeding or spotting days (mean 2.1 ± 2.4 versus 1.9 ± 2.1, p=0.8). OC dose was unrelated to follicle diameter, endometrial thickness, or bleeding. Conclusions Ovarian follicles ≥8 mm were more common in 33 compliant women during OC use than during CVR use indicating CVR use results in greater ovarian suppression than OC use. PMID:22018131

Role of Insulin-like growth factors in initiation of follicle growth in normal and polycystic human ovaries.

PubMed

Stubbs, Sharron A; Webber, Lisa J; Stark, Jaroslav; Rice, Suman; Margara, Raul; Lavery, Stuart; Trew, Geoffrey H; Hardy, Kate; Franks, Stephen

2013-08-01

Polycystic ovary syndrome (PCOS), the commonest cause of anovulatory infertility, is characterized by disordered follicle development including increased activation and accelerated growth of preantral follicles. Data from experimental animals and preliminary results from studies of human ovarian tissue suggest that IGFs affect preantral follicle development. Our objectives were to investigate the expression of the type-1 IGF receptor (IGFR-1) in the human ovary and to determine whether IGFs are involved in stimulating the transition of follicles from primordial to primary stage in normal and polycystic ovaries. We used archived ovarian tissue for protein expression studies and small cortical biopsies for follicle isolation and for tissue culture. This was a laboratory-based study, using clinical tissue samples. A total of 54 women, 33 with normal ovaries and 21 with polycystic ovaries, were classified by reference to menstrual cycle history and ultrasonography. We evaluated expression of IGFR-1 mRNA in isolated preantral follicles and of IGFR-1 protein in archived ovarian tissue samples from normal and polycystic ovaries and effects of exogenous IGF-1 on preantral follicle development and survival in cultured fragments of normal and polycystic ovaries. IGFR-1 mRNA and protein was expressed in preantral follicles at all stages of development and enhanced expression was noted in PCOS follicles during early preantral development. IGF-1 stimulated initiation of follicle growth in normal tissue but had little effect on preantral follicle growth in polycystic ovaries in which, characteristically, there was a higher proportion of follicles that had entered the growing phase even before culture. IGFs are plausible candidates in regulation of initiation of human follicle growth, and accelerated preantral follicle growth in PCOS may be due to increased activity of endogenous IGFs.

Transvaginal ultrasound appearances of the ovary in normal women and hirsute women with oligomenorrhoea.

PubMed

Fox, R

1999-02-01

The transvaginal ultrasound appearances of the ovary were determined in women with clinical and endocrine features of polycystic ovarian disease (PCOD) and apparently normal women. At scan the number of small follicles were counted and ovarian volume was calculated. The maximum width of the ovarian cortex was also measured. Blood was sent for measurement of LH, FSH and testosterone. The women with oligomenorrhoea were scanned at random and the normal women were seen within the first 5 days of the start of menstruation. There were significant differences between median values for the 2 groups in terms of number of small follicles, ovarian volume and stromal width; the ovaries of the hirsute women had more follicles, were of larger volume, and had greater stromal width. The 2 ranges for number of follicles did overlap, however. Four hirsute oligomenorrhoeic women had a normal number of follicles; all 4 had the several clinical and endocrine features indicative of PCOD. These data suggest that the classical ultrasound features of PCOD are not consistently present and that the absence of increased follicularity at scan should not necessarily deter clinicians from making the functional diagnosis of PCOD.

Ovarian follicular dynamics, follicle deviation, and oocyte yield in Gyr breed (Bos indicus) cows undergoing repeated ovum pick-up.

PubMed

Viana, J H M; Palhao, M P; Siqueira, L G B; Fonseca, J F; Camargo, L S A

2010-04-15

The objective of this study was to evaluate ovarian follicular dynamics during intervals between successive ovum pick-up (OPU) and determine its effects on the number and quality of recovered cumulus-oocyte complexes (COCs) in Zebu cows (Bos indicus). Pluriparous nonlactating Gyr cows (Bos indicus; n=10) underwent four consecutive OPU sessions at 96-h intervals. The dynamics of ovarian follicular growth between OPU sessions was monitored by twice-daily ultrasonographic examinations. A single dominant follicle (DF) or two codominant (CDF) follicles (>9mm) were present in 63.3% (19 of 30) of intervals studied, with follicle deviation beginning when the future dominant follicle (F1) achieved a diameter of 6.2+/-0.3mm. The phenomenon of codominance was observed in four (13.3%) of the inter-OPU intervals. The remaining intervals (36.6%, 11 of 30) were characterized by a greater follicular population, lower rate of follicular growth, and a smaller diameter F1 (P<0.0001). There was a tendency (P=0.08) toward an increase in the number of recovered COCs when dominant follicles were not present (NDF). The quality of COCs was not affected by the presence of a single dominant follicle, but codominant follicles resulted in recovery of a lower proportion of viable embryos (40.0%, 62.1%, and 63.6%; P<0.05) and higher proportions of degenerate COCs (56.0%, 30.3%, and 28.6%; P<0.05) for CDF, NDF, and DF respectively. We concluded that, in Zebu cows, (a) repeated follicle aspirations altered ovarian follicular dynamics, perhaps by increasing follicular growth rate; (b) follicular dominance could be established in cows undergoing twice-a-week OPU; and (c) the presence of a dominant follicle during short inter-OPU intervals may not affect COC quality, except when a codominant follicle was present. Copyright 2010 Elsevier Inc. All rights reserved.

[Effects of electroacupuncture of "Guanyuan" (CV 4)-"Zhongji" (CV 3) on ovarian P450 arom and P450c 17alpha expression and relevant sex hormone levels in rats with polycystic ovary syndrome].

PubMed

Sun, Jie; Zhao, Ji-meng; Ji, Rong; Liu, Hui-rong; Shi, Yin; Jin, Chun-lan

2013-12-01

To observe the effect of electroacupuncture (EA) on ovarian P 450 arom and P 450 c 17 alpha (aromatases) expression and related sex hormone levels in polycystic ovary syndrome (PCOS) rats. Thirty SD rats were randomly divided into normal control group, model group and EA group (10 rats/group). PCOS model was made by intragastric administration of letrozole at 1 mg/kg per day for consecutive 21 days. "Guanyuan" (CV 4) and "Zhongji" (CV 3) acupoints were stimulated 20 min by EA (2 mA, 2 Hz), once daily for consecutive 14 days. The damp ovarian weight was weighed and the pathological changes of the ovarian tissue were observed after H. E. staining. Ultrastructural changes of the ovarian tissue were observed by transmission electron microscope. Immunohistochemical staining was adopted to detect ovarian follicle granulosa cell P 450 arom and follicle membrane cell P 450 c 17 alpha expression. The contents of estradiol (E 2), estrone (E 1), androstenedione (ASD), testosterone (T), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the ovarian tissue were measured by ELISA. Compared with the normal group, there was a significant increase in the damp weight of both left and right ovarian tissues in the model group (P < 0.01). After EA, the ovarian weight was remarkably reduced (P < 0.01). Pathological changes of the ovarian tissue such as thickening of the superficial albugineous coat of the ovary, thinning of the granular cell layer, and disappearance of the intraovular oocytes and coronaradiata under light microscope, and mitochondrion swelling, fracture or disappearance of mitochondrial cristae, and enlargement of the endoplasmic reticulum, etc. after modeling were obviously improved in the EA group. In comparison to the control group, the expression of the follicle granulosa cell P450 arom was significantly down-regulated and that of follicle membrane cell P 450 c 17 alpha was significantly upregulated in the model group (P < 0.01). After EA intervention these changes were obviously reversed (P < 0.05, P < 0.01). In the model group, there was a significant increase in the levels of ASD, T and LH in the ovarian tissues (P < 0.01) and a marked decrease in the contents of ovarian E 1 and E2 (P < 0.01) in comparison to the control group. After EA, the ovarian ASD, T and LH levels were notably decreased (P < 0.05, P < 0.01) and the E 1 and E 2 levels apparently increased (P < 0.01) compared with the model group. EA can improve letrozole-induced pathological changes of ovarian morphology and ultrastructure and obviously promote P 450 arom expression in the follicle granulosa cell layer and inhibit P 450 c 17 alpha expression in the follicle membrane cell layer, as well as regulate sex hormone levels in PCOS rats, facilitating the normal transformation of ovarian androgen to estrogen and restoring the local endocrine disorders.

Expression pattern of RAGE and IGF-1 in the human fetal ovary and ovarian serous carcinoma.

PubMed

Poljicanin, Ana; Filipovic, Natalija; Vukusic Pusic, Tanja; Soljic, Violeta; Caric, Ana; Saraga-Babic, Mirna; Vukojevic, Katarina

2015-01-01

The expression pattern of RAGE and IGF-1 proteins in different ovarian cell lineages was histologically analyzed in six fetal, nine adult human ovaries, and nine serous ovarian carcinomas (OSC) using immunohistochemical methods. Mild expression of IGF-1 in ovarian surface epithelium (Ose) and oocytes in the 15-week human ovaries increased to moderate or strong in the stromal cells, oocytes and follicular cells in week 22. Occasional mild RAGE expression was observed in Ose during week 15, while strong expression characterized primordial follicles in week 22. In the reproductive human ovary, IGF-1 was mildly to moderately expressed in all ovarian cell lineages except in theca cells of the tertiary follicle where IGF-1 was negative. RAGE was strongly positive in the granulosa cells and some theca cells of the tertiary follicle, while negative to mildly positive in all cells of the secondary follicle. In the postmenopausal human ovary IGF-1 and RAGE were mildly expressed in Ose and stroma. In OSC, cells were strongly positive to IGF-1 and RAGE, except for some negative stromal cells. Different levels of IGF-1 and RAGE co-expression characterized fetal ovarian cells during development. In reproductive ovaries, IGF-1 and RAGE were co-localized in the granulosa and theca interna cells of tertiary follicles, while in postmenopausal ovaries and OSC, IGF-1 and RAGE were co-localized in Ose and OSC cells respectively. Our results indicate that intracellular levels of IGF-1 and RAGE protein might regulate the final destiny of the ovarian cell populations prior and during folliculogenesis, possibly controlling the metastatic potential of OSC as well. Copyright © 2015. Published by Elsevier GmbH.

Association of Exon 10A and 10B inactivating mutation of follicle stimulating hormone receptor gene (FSHR) and Polycystic Ovarian Syndrome in Vellore cohort

NASA Astrophysics Data System (ADS)

Sekar, Nishu; Kulkarni, Rucha; Ozalkar, Sharvari; Prabhu, Yogamaya D.; Renu, Kaviyarasi; Ramgir, Shalaka S.; Abilash, V. G.

2017-11-01

Polycystic ovarian syndrome is the most common heterogenous endocrine disorder in women. Follicle stimulating hormone receptor is associated with normal development as well as maturation of follicles and triggers estrogen production in granulosa cells of the ovary. Inactivating mutation in FSHR gene correlated with reduction of ovarian function in women is due to damage to receptor function. This study aims to investigate whether inactivating mutations, in follicle stimulating hormone receptor gene is related to polycystic ovarian morphology in women with PCOS. Genomic DNA isolated from 15 subjects from Sandhya Hospital, Vellore (10 patients with PCOS and 5 healthy controls) was taken for this study. Patient data included a clinical report, hormonal levels, and ovarian morphological details. DNA isolation was followed by DNA amplification by polymerase chain reaction using Exon 10 A and Exon 10 B primers. The PCR-RFLP analysis was performed using Dde1 restriction enzyme. Here we discuss inactivating mutation found in Exon 10 of FSHR gene in patients with PCOS.The absence of inactivating mutation was observed through PCR-RFLP study on Exon 10A and Exon 10B.

DYNAMIC BEHAVIOR OF A DELAY-DIFFERENTIAL EQUATION MODEL FOR THE HORMONAL REGULATION OF THE MENSTRUAL CYCLE

EPA Science Inventory

During the menstrual cycle, pituitary hormones stimulate the growth and development of ovarian follicles and the release of an ovum to be fertilized. The ovarian follicles secrete hormones during the cycle that regulate the production of the pituitary hormones creating positi...

Maternal age influences folliculogenesis and gene networks in the ovaries of beef heifers

USDA-ARS?s Scientific Manuscript database

The size of the ovarian reserve is an important component of fertility and reproductive longevity in bovine females. Ultrasonographic determination of antral follicle count is the best method for estimating the size of the ovarian reserve in heifers and cows in a production setting. Antral follicl...

Short-term culture of ovarian cortex pieces to assess the cryopreservation outcome in wild felids for genome conservation

PubMed Central

2013-01-01

Background Cryopreservation of ovarian tissue has the potential to preserve female germ cells of endangered mammals. In the present study, a freezing protocol successfully used for human tissue, was adapted for preserving ovarian tissue of domestic and non-domestic felids. Ovaries from non-domestic felid species were obtained from seven freshly euthanized and two recently deceased wild felids kept in different European Zoos. In addition, ovaries from domestic cats were obtained after ovariectomy from local veterinary clinics for methological adaptations. Ovarian cortex was dissected and uniform sized pieces of 2 mm diameter were obtained. Using a slow freezing protocol (-0.3°C per min) in 1.5 mol/L ethylene glycol, 0.1 mol/L sucrose, the pieces were cultured for up to 14 days both before and after cryopreservation. The integrity of primordial follicles was assessed by histology, and the impact of different protein sources (FCS or BSA) and Vitamin C was determined during two weeks of culture. Results and conclusion During culture the number of primordial follicles decreased within the ovarian pieces (p < 0.05). This effect was less pronounced when FCS was used as the protein source instead of BSA. Supplementation with Vitamin C had a detrimental effect on follicle survival. Since the procedure of cryopreservation had no effect on the follicle survival after one week of culture we conclude that the freezing protocol was suitable for felids. This is the first report of preserving a huge amount of follicles within ovarian tissue by slow freezing performed in several wild feline species. PMID:23433001

Enhancement of Neoangiogenesis and Follicle Survival by Sphingosine-1-Phosphate in Human Ovarian Tissue Xenotransplants

PubMed Central

Oktay, Kutluk

2011-01-01

Ovarian transplantation is one of the key approaches to restoring fertility in women who became menopausal as a result of cancer treatments. A major limitation of human ovarian transplants is massive follicular loss during revascularization. Here we investigated whether sphingosine-1-phosphate or its receptor agonists could enhance neoangiogenesis and follicle survival in ovarian transplants in a xenograft model. Human ovarian tissue xenografts in severe-combined-immunodeficient mice were treated with sphingosine-1-phosphate, its analogs, or vehicle for 1–10 days. We found that sphingosine-1-phosphate treatment increased vascular density in ovarian transplants significantly whereas FTY720 and SEW2871 had the opposite effect. In addition, sphingosine-1-phosphate accelerated the angiogenic process compared to vehicle-treated controls. Furthermore, sphingosine-1-phosphate treatment was associated with a significant proliferation of ovarian stromal cell as well as reduced necrosis and tissue hypoxia compared to the vehicle-treated controls. This resulted in a significantly lower percentage of apoptotic follicles in sphingosine-1-phosphate-treated transplants. We conclude that while sphingosine-1-phosphate promotes neoangiogenesis in ovarian transplants and reduces ischemic reperfusion injury, sphingosine-1-phosphate receptor agonists appear to functionally antagonize this process. Sphingosine-1-phosphate holds great promise to clinically enhance the survival and longevity of human autologous ovarian transplants. PMID:21559342

Age-related decline in ovarian follicle stocks differ between chimpanzees (Pan troglodytes) and humans.

PubMed

Cloutier, Christina T; Coxworth, James E; Hawkes, Kristen

2015-02-01

Similarity in oldest parturitions in humans and great apes suggests that we maintain ancestral rates of ovarian aging. Consistent with that hypothesis, previous counts of primordial follicles in postmortem ovarian sections from chimpanzees (Pan troglodytes) showed follicle stock decline at the same rate that human stocks decline across the same ages. Here, we correct that finding with a chimpanzee sample more than three times larger than the previous one, which also allows comparison into older ages. Analyses show depletion rates similar until about age 35, but after 35, the human counts continue to fall with age, while the change is much less steep in chimpanzees. This difference implicates likely effects on ovarian dynamics from other physiological systems that are senescing at different rates, and, potentially, different perimenopausal experience for chimpanzees and humans.

Sex steroid receptors and apoptosis-related proteins are differentially expressed in polycystic ovaries of adult dogs.

PubMed

Chuffa, Luiz Gustavo de Almeida; Lupi Júnior, Luiz Antonio; da Maia Lima, Alfredo Feio

2016-02-01

In Polycystic Ovaries (PCOs), the dynamics of sex hormone receptors and follicle-related apoptotic signaling remain unknown. In this study, we investigated the expression of androgen receptors (AR), estrogen receptors (ERα and ERβ), and apoptosis-related molecules (BAX, active caspase-3, Bcl-2 and Survivin) on different follicular stages of PCOs in adult dogs. Clinical evidences of high estradiol and testosterone levels, persistent estrus and vaginal discharge were observed. Inhibin B immunolabeling was increased in primary and 2 to 5-mm follicles, and a marked epithelial hyperplasia was common in the ovarian surface. Ovarian epithelia and primary follicles showed low expression of AR, ERα, and ERβ, whereas a moderate immunoexpression of AR was found in theca cells of secondary follicles and cysts. In PCOs, growing follicles displayed ERα expression, and secondary follicles exhibited higher ERβ expression. In addition, while few ERα-positive cells were found in the cysts, ERβ was moderately expressed in growing follicles and cysts. BAX was upregulated in the ovarian epithelium, primary follicles, and in the wall of follicular cysts. Active caspase-3 was significantly downregulated in the epithelium, primary follicles, and follicular cysts, whereas growing follicles had a strong immunoexpression in the granulosa cells. Bcl-2 and survivin were increased in the epithelium and primary follicles, and only survivin was upregulated in secondary and growing follicles. While Bcl-2 had a diffuse immunexpression in the follicular cysts, survivin was overexpressed by these cells. We concluded that sex steroid receptors and apoptotic proteins are differentially expressed in the follicles of adult dogs with PCOs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Feeling the ovaries prior to insemination. Clinical implications for improving the fertility of the dairy cow.

PubMed

López-Gatius, F

2011-07-01

During the periovulatory period in dairy cattle, the largest ovarian follicle can be felt by palpation per rectum as a firm/soft follicle (young preovulatory follicle), a very soft follicle separating it from the remainder of the ovary (mature preovulatory follicle), or an evacuated follicle (follicle associated with ovulation). Because any one of these three follicle types may be present at the time of artificial insemination, the objective of this study was to identify possible differences between the effects of a firm/soft, very soft, or evacuated ovarian follicle on fertility. Out of a study sample of 2365 inseminations, very soft, firm/soft, and evacuated follicles were recorded in 1689 (71%), 593 (25%), and 83 (3.5%) inseminations, respectively. Logistic regression analysis indicated no significant effects of largest follicle type, vaginal discharge, season, days in milk, parity, synchronized or natural estrus, and semen-providing bull on the pregnancy rate. The only variable included in the final logistic regression model was the interaction season-follicle type. This interaction determined that the likelihood of pregnancy decreased significantly by factors of 0.86 or 0.82 in cows with a firm/soft follicle inseminated during the cool or warm period, respectively, and by a factor of 0.09 in cows with evacuated follicles inseminated during the warm period, using as reference cows with a very soft follicle inseminated during the cool period (yielding the highest pregnancy rate). As an overall conclusion, the state of the periovulatory follicle at insemination was clearly related to fertility and masked the effects of factors commonly affecting fertility such as parity, days in milk at AI and inseminating bull. More importantly they suggest that by including ovarian follicle checks in artificial insemination routines, the success of this procedure could be improved. Copyright © 2011 Elsevier Inc. All rights reserved.

Synergistic effect of melatonin and ghrelin in preventing cisplatin-induced ovarian damage via regulation of FOXO3a phosphorylation and binding to the p27Kip1 promoter in primordial follicles.

PubMed

Jang, Hoon; Na, Younghwa; Hong, Kwonho; Lee, Sangho; Moon, Sohyeon; Cho, Minha; Park, Miseon; Lee, Ok-Hee; Chang, Eun Mi; Lee, Dong Ryul; Ko, Jung Jae; Lee, Woo Sik; Choi, Youngsok

2017-10-01

Premature ovarian failure during chemotherapy is a serious problem for young women with cancer. To preserve the fertility of these patients, approaches to prevent chemotherapy-induced ovarian failure are needed. In a previous study, we reported that melatonin treatment prevents the depletion of the dormant follicle pool via repression of the simultaneous activation of dormant primordial follicles by cisplatin. However, melatonin's protective effect was only partial and thus insufficient. In this study, we found that the hormone ghrelin enhances the protective effect of melatonin against cisplatin-induced ovarian failure in mouse model. Co-administration of melatonin and ghrelin more effectively prevented cisplatin-induced follicle disruption. Simultaneous treatment with melatonin and ghrelin almost restored the number of primordial follicles and the corpus luteum in cisplatin-treated ovaries, compared with single administration. We found melatonin and ghrelin receptors on the cell membrane of premature oocytes of primordial follicles. In addition, melatonin and ghrelin co-administration inhibited the cisplatin-induced phosphorylation of PTEN and FOXO3a that induces cytoplasmic translocation of FOXO3a. Inhibition of FOXO3a phosphorylation by melatonin and ghrelin increased the binding affinity of FOXO3a for the p27 Kip1 promoter in primordial follicles. Co-administration of melatonin and ghrelin in cisplatin-treated ovaries restored the expression of p27 Kip1 , which is critical for retention of the dormant status of primordial follicles. In conclusion, these findings suggest that melatonin and ghrelin co-administration is suitable for use as a fertoprotective adjuvant therapy during cisplatin chemotherapy in young female cancer patients. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Pax2.1 is required for the development of thyroid follicles in zebrafish.

PubMed

Wendl, Thomas; Lun, Klaus; Mione, Marina; Favor, Jack; Brand, Michael; Wilson, Stephen W; Rohr, Klaus B

2002-08-01

The thyroid gland is an organ primarily composed of endoderm-derived follicular cells. Although disturbed embryonic development of the thyroid gland leads to congenital hypothyroidism in humans and mammals, the underlying principles of thyroid organogenesis are largely unknown. In this study, we introduce zebrafish as a model to investigate the molecular and genetic mechanisms that control thyroid development. Marker gene expression suggests that the molecular pathways of early thyroid development are essentially conserved between fish and mammals. However during larval stages, we find both conserved and divergent features of development compared with mammals. A major difference is that in fish, we find evidence for hormone production not only in thyroid follicular cells, but also in an anterior non-follicular group of cells. We show that pax2.1 and pax8, members of the zebrafish pax2/5/8 paralogue group, are expressed in the thyroid primordium. Whereas in mice, only Pax8 has a function during thyroid development, analysis of the zebrafish pax2.1 mutant no isthmus (noi(-/-)) demonstrates that pax2.1 has a role comparable with mouse Pax8 in differentiation of the thyroid follicular cells. Early steps of thyroid development are normal in noi(-/-), but later expression of molecular markers is lost and the formation of follicles fails. Interestingly, the anterior non-follicular site of thyroid hormone production is not affected in noi(-/-). Thus, in zebrafish, some remaining thyroid hormone synthesis takes place independent of the pathway leading to thyroid follicle formation. We suggest that the noi(-/-) mutant serves as a new zebrafish model for hypothyroidism.

In vitro culture of early secondary preantral follicles in hanging drop of ovarian cell-conditioned medium to obtain MII oocytes from outbred deer mice.

PubMed

Choi, Jung Kyu; Agarwal, Pranay; He, Xiaoming

2013-12-01

The ovarian follicle (each contains a single oocyte) is the fundamental functional tissue unit of mammalian ovaries. In humans, it has been long held true that females are born with a maximum number of follicles (or oocytes) that are not only nonrenewable, but also undergoing degeneration with time with a sharply decreased oocyte quality after the age of ∼35. Therefore, it is of importance to isolate and bank ovarian follicles for in vitro culture to obtain fertilizable oocytes later, to preserve the fertility of professional women who may want to delay childbearing, young and unmarried women who may lose gonadal function because of exposure to environmental/occupational hazards or aggressive medical treatments, such as radiation and chemotherapy, and even endangered species and breeds. Although they contributed significantly to the understanding of follicle science and biology, most studies reported to date on this topic were done using the man-made, unnatural inbred animal species. It was found in this study that the conventional two-dimensional microliter drop and three-dimensional hanging drop (HD) methods, reported to be effective for in vitro culture of preantral follicles from inbred mice, are not directly transferrable to outbred deer mice. Therefore, a modified HD method was developed in this study to achieve a much higher (>5 times compared to the best conventional methods) percentage of developing early secondary preantral follicles from the outbred mice to the antral stage, for which, the use of an ovarian cell-conditioned medium and multiple follicles per HD were identified to be crucial. It was further found that the method for in vitro maturation of oocytes in antral follicles obtained by in vitro culture of preantral follicles could be very different from that for oocytes in antral follicles obtained by hormone stimulation in vivo. Therefore, this study should provide important guidance for establishing effective protocols of in vitro follicle culture to preserve the fertility of wildlife and humans outbred by nature.

In Vitro Culture of Early Secondary Preantral Follicles in Hanging Drop of Ovarian Cell-Conditioned Medium to Obtain MII Oocytes from Outbred Deer Mice

PubMed Central

Choi, Jung Kyu; Agarwal, Pranay

2013-01-01

The ovarian follicle (each contains a single oocyte) is the fundamental functional tissue unit of mammalian ovaries. In humans, it has been long held true that females are born with a maximum number of follicles (or oocytes) that are not only nonrenewable, but also undergoing degeneration with time with a sharply decreased oocyte quality after the age of ∼35. Therefore, it is of importance to isolate and bank ovarian follicles for in vitro culture to obtain fertilizable oocytes later, to preserve the fertility of professional women who may want to delay childbearing, young and unmarried women who may lose gonadal function because of exposure to environmental/occupational hazards or aggressive medical treatments, such as radiation and chemotherapy, and even endangered species and breeds. Although they contributed significantly to the understanding of follicle science and biology, most studies reported to date on this topic were done using the man-made, unnatural inbred animal species. It was found in this study that the conventional two-dimensional microliter drop and three-dimensional hanging drop (HD) methods, reported to be effective for in vitro culture of preantral follicles from inbred mice, are not directly transferrable to outbred deer mice. Therefore, a modified HD method was developed in this study to achieve a much higher (>5 times compared to the best conventional methods) percentage of developing early secondary preantral follicles from the outbred mice to the antral stage, for which, the use of an ovarian cell-conditioned medium and multiple follicles per HD were identified to be crucial. It was further found that the method for in vitro maturation of oocytes in antral follicles obtained by in vitro culture of preantral follicles could be very different from that for oocytes in antral follicles obtained by hormone stimulation in vivo. Therefore, this study should provide important guidance for establishing effective protocols of in vitro follicle culture to preserve the fertility of wildlife and humans outbred by nature. PMID:23789595

Amygdala Kindling Alters Estrus Cycle and Ovarian Morphology in the Rat.

PubMed

Pan, Juan; Zhang, Lingwu; Wang, Feng; Liu, Dan; Li, P Andy; Sun, Tao

2013-11-01

The objective of this study is to explore the effects of amygdala kindling on estrus cycle and ovarian morphology. Thirty-five female rats at the age of 8 weeks were randomly designated to electrode kindled, sham-kindled, and normal controls. Kindled rats were implanted with kindling electrodes in the left basolateral amygdala and kindled by brief suprathreshold stimulations with a bipolar electrode. Estrous cycles were daily monitored through vaginal smears. Electrographic and behavioral seizures were recorded and ovarian morphology was evaluated by light and electron microscopies. Our results showed that the kindled rats lost their ovarian periodicity displayed significant ovarian enlargement. H&E staining revealed increased number of growing follicles and total follicles, as well as polycysts in the ovaries of the kindled animals compared to sham and control animals. Ultrastructural study detected numerous apoptotic granulosa cells in growing follicles and thecal cell hyperplasia with secretary granules in the thecal cells in the kindled rats. The results suggest that amygdala kindling is a risk factor for the development of polycystic ovary syndrome.

Effect of medroxyprogesterone acetate (Provera) on ovarian radiosensitivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jarrell, J.; YoungLai, E.V.; McMahon, A.

1989-04-01

Medroxyprogesterone acetate (Provera) is a drug that is commonly given to young women with cancer during chemotherapy and radiation to control heavy bleeding associated with anovulation. Because hypothalamic-pituitary-ovarian suppression has been associated with ovarian protection from the effects of chemotherapy and medroxyprogesterone acetate has been identified as a radiosensitizing agent, we explored the effects of medroxyprogesterone acetate on a rat model with known radiation injury characteristics. Sprague-Dawley rats were treated with medroxyprogesterone acetate or vehicle from day 22 to day 37 of life and were either irradiated or sham-irradiated on day 30 of life and then killed on day 44.more » Radiation with medroxyprogesterone acetate administration produced a greater loss in preantral and healthy control follicles than in control follicles. No suppression of luteinizing hormone or follicle-stimulating hormone had occurred by day 30 but ovarian glutathione content was reduced. These findings indicate that the administration of medroxyprogesterone acetate with radiotherapy may enhance ovarian injury.« less

Reference values in ovarian response to controlled ovarian stimulation throughout the reproductive period.

PubMed

La Marca, Antonio; Grisendi, Valentina; Spada, Elena; Argento, Cindy; Milani, Silvano; Plebani, Maddalena; Seracchioli, Renato; Volpe, Annibale

2014-01-01

Abstract The age-related decline in ovarian response to gonadotropins has been well known since the beginning of ovarian stimulation in IVF cycles and has been considered secondary to the age-related decline in ovarian reserve. The objective of this study was to establish reference values and to construct nomograms of ovarian response for any specific age to gonadotropins in IVF/ICSI cycles. We analyzed our database containing information on IVF cycles. According to inclusion and exclusion criteria, a total of 703 patients were selected. Among inclusion criteria, there were regular menstrual cycle, treatment with a long GnRH agonist protocol and starting follicle-stimulating hormone (FSH) dose of at least 200 IU per day. To estimate the reference values of ovarian response, the CG-LMS method was used. A linear decline in the parameters of ovarian response with age was observed: the median number of oocytes decreases approximately by one every three years, and the median number of follicles >16 mm by one every eight years. The number of oocytes and growing follicles corresponding to the 5th, 25th, 50th, 75th and 95th centiles has been calculated. This study confirmed the well known negative relationship between ovarian response to FSH and female ageing and permitted the construction of nomograms of ovarian response.

Influence of ovarian reserves in beef heifers on uterine morphometry and function

USDA-ARS?s Scientific Manuscript database

The size of the reproductive tract and the number of follicles in the ovaries are positively associated with fertility and early conception in beef heifers. Heifers with greater numbers of ovarian follicles have larger uteri that secrete a greater amount of protein on day 16 after estrus. Therefor...

Activation of dormant ovarian follicles to generate mature eggs.

PubMed

Li, Jing; Kawamura, Kazuhiro; Cheng, Yuan; Liu, Shuang; Klein, Cynthia; Liu, Shu; Duan, En-Kui; Hsueh, Aaron J W

2010-06-01

Although multiple follicles are present in mammalian ovaries, most of them remain dormant for years or decades. During reproductive life, some follicles are activated for development. Genetically modified mouse models with oocyte-specific deletion of genes in the PTEN-PI3K-Akt-Foxo3 pathway exhibited premature activation of all dormant follicles. Using an inhibitor of the Phosphatase with TENsin homology deleted in chromosome 10 (PTEN) phosphatase and a PI3K activating peptide, we found that short-term treatment of neonatal mouse ovaries increased nuclear exclusion of Foxo3 in primordial oocytes. After transplantation under kidney capsules of ovariectomized hosts, treated follicles developed to the preovulatory stage with mature eggs displaying normal epigenetic changes of imprinted genes. After in vitro fertilization and embryo transfer, healthy progeny with proven fertility were delivered. Human ovarian cortical fragments from cancer patients were also treated with the PTEN inhibitor. After xeno-transplantation to immune-deficient mice for 6 months, primordial follicles developed to the preovulatory stage with oocytes capable of undergoing nuclear maturation. Major differences between male and female mammals are unlimited number of sperm and paucity of mature oocytes. Thus, short-term in vitro activation of dormant ovarian follicles after stimulation of the PI3K-Akt pathway allows the generation of a large supply of mature female germ cells for future treatment of infertile women with a diminishing ovarian reserve and for cancer patients with cryo-preserved ovaries. Generation of a large number of human oocytes also facilitates future derivation of embryonic stem cells for regenerative medicine.

Development of the ovarian follicular epithelium.

PubMed

Rodgers, R J; Lavranos, T C; van Wezel, I L; Irving-Rodgers, H F

1999-05-25

A lot is known about the endocrine control of the development of ovarian follicles, but a key question now facing researchers is which molecular and cellular processes take part in control of follicular growth and development. The growth and development of ovarian follicles occurs postnatally and throughout adult life. In this review, we focus on the follicular epithelium (membrana granulosa) and its basal lamina. We discuss a model of how granulosa cells arise from a population of stem cells and then enter different lineages before differentiation. The structure of the epithelium at the antral stage of development is presented, and the effects that follicle growth has on the behavior of the granulosa cells are discussed. Finally, we discuss the evidence that during follicle development the follicular basal lamina changes in composition. This would be expected if the behavior of the granulosa cells changes, or if the permeability of the basal lamina changes. It will be evident that the follicular epithelium has similarities to other epithelia in the body, but that it is more dynamic, as gross changes occur during the course of follicle development. This basic information will be important for the development of future reproductive technologies in both humans and animals, and possibly for understanding polycystic ovarian syndrome in women.

Phosphoinositide 3-Kinase p110δ Mediates Estrogen- and FSH-Stimulated Ovarian Follicle Growth

PubMed Central

Li, Qian; He, Hui; Zhang, Yin-Li; Li, Xiao-Meng; Guo, Xuejiang; Huo, Ran; Bi, Ye; Li, Jing

2013-01-01

In the mammalian ovary, primordial follicles are generated early in life and remain dormant for prolonged periods. Their growth resumes via primordial follicle activation, and they continue to grow until the preovulatory stage under the regulation of hormones and growth factors, such as estrogen, FSH, and IGF-1. Both FSH and IGF-1 activate the phosphatidylinositol-3 kinase (PI3K)/Akt (acute transforming retrovirus thymoma protein kinase) signaling pathway in granulosa cells (GCs), yet it remains inconclusive whether the PI3K pathway is crucial for follicle growth. In this study, we investigated the p110δ isoform (encoded by the Pik3cd gene) of PI3K catalytic subunit expression in the mouse ovary and its function in fertility. Pik3cd-null females were subfertile, exhibited fewer growing follicles and more atretic antral follicles in the ovary, and responded poorly to exogenous gonadotropins compared with controls. Ovary transplantation showed that Pik3cd-null ovaries responded poorly to FSH stimulation in vitro; this confirmed that the follicle growth defect was intrinsically ovarian. In addition, estradiol (E2)-stimulated follicle growth and GC proliferation in preantral follicles was impaired in Pik3cd-null ovaries. FSH and E2 substantially activated the PI3K/Akt pathway in GCs of control mice but not in those of Pik3cd-null mice. However, primordial follicle activation and oocyte meiotic maturation were not affected by Pik3cd knockout. Taken together, our findings indicate that the p110δ isoform of the PI3K catalytic subunit is a key component of the PI3K pathway for both FSH and E2-stimulated follicle growth in ovarian GCs; however, it is not required for primordial follicle activation and oocyte development. PMID:23820902

Assessment of follicle viability using fluorescence microscopy before and after ovarian thawing.

PubMed

Sofoudis, C; Zervomanolakis, I; Galani, I; Grigoriou, V; Botsis, D; Vlahos, N

2017-01-01

The incidence of young women diagnosed with cancer has been globally increasing. In many cases the surgical approach followed by chemotherapy, radiotherapy or hormonal therapy could lead to infertility or premature ovarian failure. Several options are available in order to preserve fertility and increase the future gestation rate. Among embryo cryopreservation and oocyte cryopreservation, ovarian tissue cryopreservation represents an ideal option, especially for premenopausal women and for those who cannot delay the start of chemotherapy. The purpose of this study was to examine the follicle viability using fluorescence microscope before and after ovarian thawing.

A novel fibrin-based artificial ovary prototype resembling human ovarian tissue in terms of architecture and rigidity.

PubMed

Chiti, Maria Costanza; Dolmans, Marie-Madeleine; Mortiaux, Lucie; Zhuge, Flanco; Ouni, Emna; Shahri, Parinaz Asiabi Kohneh; Van Ruymbeke, Evelyne; Champagne, Sophie-Demoustier; Donnez, Jacques; Amorim, Christiani Andrade

2018-01-01

The aim of this study is to optimize fibrin matrix composition in order to mimic human ovarian tissue architecture for human ovarian follicle encapsulation and grafting. Ultrastructure of fresh human ovarian cortex in age-related women (n = 3) and different fibrin formulations (F12.5/T1, F30/T50, F50/T50, F75/T75), rheology of fibrin matrices and histology of isolated and encapsulated human ovarian follicles in these matrices. Fresh human ovarian cortex showed a highly fibrous and structurally inhomogeneous architecture in three age-related patients, but the mean ± SD of fiber thickness (61.3 to 72.4 nm) was comparable between patients. When the fiber thickness of four different fibrin formulations was compared with human ovarian cortex, F50/T50 and F75/T75 showed similar fiber diameters to native tissue, while F12.5/T1 was significantly different (p value < 0.01). In addition, increased concentrations of fibrin exhibited enhanced storage modulus with F50/T50, resembling physiological ovarian rigidity. Excluding F12.5/T1 from further analysis, only three remaining fibrin matrices (F30/T50, F50/T50, F75/T75) were histologically investigated. For this, frozen-thawed fragments of human ovarian tissue collected from 22 patients were used to isolate ovarian follicles and encapsulate them in the three fibrin formulations. All three yielded similar follicle recovery and loss rates soon after encapsulation. Therefore, based on fiber thickness, porosity, and rigidity, we selected F50/T50 as the fibrin formulation that best mimics native tissue. Of all the different fibrin matrix concentrations tested, F50/T50 emerged as the combination of choice in terms of ultrastructure and rigidity, most closely resembling human ovarian cortex.

Culture of domestic cat ovarian tissue in vitro and in the chick embryo chorioallantoic membrane.

PubMed

Vilela, J M V; Leonel, E C R; D'Oliveira, L; Paiva, R E G; Miranda-Vilela, A L; Amorim, C A; Pic-Taylor, A; Lucci, C M

2016-10-15

In vitro culture and transplantation procedures are essential protocols employed in the evaluation of ovarian follicle survival and development. Culture in the chorioallantoic membrane (CAM) of chick embryos is an intermediate method that provides important follicle development information and has not been tested for cat ovaries to date. The aim of this study was to investigate if in vitro and CAM culture could be used as short-term systems to study cat ovarian tissue development. The ovaries of eight cats were dissected into 3-mm(3) cubes, cultured in vitro and in CAM for up to 5 days, and stained with hematoxylin-eosin and Gomori trichrome. Cell proliferation was analyzed using anti-Ki67. Possible differences among groups were investigated by analysis of variance or the Kruskal-Wallis test followed by Bonferroni correction. The T-test or Wilcoxon test was used to verify differences between the CAM and IVC. Results revealed that 87.5% of all follicles were primordial during culture. The percentage of primordial follicles in the morphologically normal follicles (MNF) pool was always higher than 80%, with the exception of Day 3 of CAM culture, but the number of MNF reduced significantly from Day 0 (600 out of 777 follicles) to Day 5 in the CAM (91 out of 171) and IVC (296 out of 686). The number of primordial follicles in 1 mm(3) in Days 2, 3, and 5 in the CAM was significantly lower than that in the control (Day 0). No cellular proliferation was observed in culture. Vascularization occurred in the CAM culture, but with no association to follicular viability. In addition, both methods showed an increase in connective tissue during culture. Although no significant differences were observed in the percentage of MNF, there was a reduction in the total number of follicles, both for IVC and CAM-cultured ovarian tissue. Furthermore, anti-Ki67 did not stain any follicle after Day 0 in IVC or in CAM culture. Neither system was capable of promoting follicle growth and/or development. The results show that the CAM is not a suitable system for feline ovarian tissue and highlight the necessity to improve IVC systems in cats. Copyright © 2016 Elsevier Inc. All rights reserved.

Impaired insulin signaling pathway in ovarian follicles of cows with cystic ovarian disease.

PubMed

Hein, G J; Panzani, C G; Rodríguez, F M; Salvetti, N R; Díaz, P U; Gareis, N C; Benítez, G A; Ortega, H H; Rey, F

2015-05-01

Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. Follicular cell steroidogenesis and proliferation in ovulatory follicles is stimulated by hormones such as insulin and its necessary post-receptor response. The aim of this study was to determine the expression of insulin receptor (IR), IR substrate-1 (IRS1) and phosphatidylinositol 3-kinase (PI3K), key intermediates in the insulin pathway, in control cows and cows with spontaneous COD and ACTH-induced COD. IR and IRS1 mRNA levels were greater in granulosa cells and lower in follicular cysts than in control tertiary follicles. PI3K mRNA levels were similar in all follicles evaluated, whereas the expression of IR, IRS1 and PI3K was similar in theca cells. Protein expression of IR was higher in control tertiary follicles than in the same structures in animals with COD and with cysts. IRS1 and PI3K protein expression showed the same pattern in tertiary and cystic follicles. However, the protein expression of subunit alpha p85 of PI3K was greater in theca cells from tertiary follicles than in cystic follicles. These results provide new insights into the insulin response in cows with COD. The lower gene and protein expressions of some insulin downstream effectors at an early stage of the signaling pathway could negatively influence the functionality of ovaries and contribute to follicle persistence. Copyright © 2015 Elsevier B.V. All rights reserved.

Variation in ovarian follicle density during human fetal development.

PubMed

Geber, Selmo; Megale, Rodrigo; Vale, Fabiene; Lanna, Ana Maria Arruda; Cabral, Antônio Carlos Vieira

2012-09-01

To obtain a precise estimate of ovarian follicle density and variation in the number of follicles at several gestational ages during human fetal development. Twelve necropsied ovaries from 9 fetuses (gestational age: 24 to 36 weeks) and 3 neonates (who died within the first hours of life) were studied. Ovaries were fixed with 4 % formaldehyde and embedded in paraffin. Serial, 7 mm thick sections of the ovaries were cut and evaluated at every 50 cuts. Follicles were counted in 10 regions (each measuring 625 μm(2)) of the ovarian cortex and the number of follicles per mm³ was calculated. The number of follicles per 0.25 mm² ranged from 10.9 (± 4.8) in a neonate to 34.7 (± 10.6) also in a neonate. Among fetuses, follicle density was lowest at 36 weeks of gestation (11.1 ± 6.2) and highest at 26 weeks (32 ± 8.9). The total number of follicles ranged from 500,000 at the age of 22 weeks to > 1,000,000 at the age of 39 weeks. Our results show a peak in the number of follicles during intrauterine life at approximately 26 weeks, followed by a rapid reduction in this number before birth, providing a step forward towards the understanding of primordial follicular assembly in humans and, ultimately, the identification of the determinants of reproductive capacity.

An imbalance between apoptosis and proliferation contributes to follicular persistence in polycystic ovaries in rats

PubMed Central

Salvetti, Natalia R; Panzani, Carolina G; Gimeno, Eduardo J; Neme, Leandro G; Alfaro, Natalia S; Ortega, Hugo H

2009-01-01

Background Cystic ovarian disease is an important cause of infertility that affects bovine, ovine, caprine and porcine species and even human beings. Alterations in the ovarian micro-environment of females with follicular cysts could alter the normal processes of proliferation and programmed cell death in ovarian cells. Thus, our objective was to evaluate apoptosis and proliferation in ovarian cystic follicles in rats in order to investigate the cause of cystic follicle formation and persistence. Methods We compared the number of in situ apoptotic cells by TUNEL assay, expression of active caspase-3 and members of Bcl-2 family by immunohistochemistry; and cell proliferation by the expression of the proliferation markers: PCNA and Ki-67. Results The proliferation index was low in granulosa of tertiary and cystic follicles of light exposed rats when compared with tertiary follicles of control animals, while in theca interna only cystic follicles presented low proliferation index when compared with tertiary follicles (p < 0.05). The granulosa of cysts exhibited a similar cell DNA fragmentation to early atretic follicles. In the granulosa and theca interna, active caspase-3 shown similar immunostaining levels in tertiary and cystic follicles (p < 0.05). The granulosa cells presented high expression of Bcl-2, Bcl-xL and Bcl-w in the tertiary and cystic follicles with diminishing intensity in the atretic follicles, except with Bcl-w where the intensity was maintained in the atretic follicles (p < 0.05). The expression of Bax was weak in the healthy and cystic follicles. In the theca interna, Bcl-2 expression was the same as the pattern found in the granulosa; no differences were found between tertiary and cystic follicles from both groups for Bcl-xL and Bcl-w. The expression of Bax in this layer was higher in the tertiary follicles of the treated animals (p < 0.05) while the values for cystic follicles were similar to those in the tertiary follicles of controls. The theca externa showed low expression of the pro and anti-apoptotic proteins. Conclusion These results show that the combination of weak proliferation indices and low apoptosis observed in follicular cysts, could explain the cause of the slow growth of cystic follicles and the maintenance of a static condition without degeneration, which leads to their persistence. These alterations may be due to structural and functional modifications that take place in these cells and could be related to hormonal changes in animals with this condition. PMID:19570211

How to personalize ovarian stimulation in clinical practice.

PubMed

Sighinolfi, Giovanna; Grisendi, Valentina; La Marca, Antonio

2017-09-01

Controlled ovarian stimulation (COS) in in vitro fertilization (IVF) cycles is the starting point from which couple's prognosis depends. Individualization in follicle-stimulating hormone (FSH) starting dose and protocol used is based on ovarian response prediction, which depends on ovarian reserve. Anti-Müllerian hormone levels and the antral follicle count are considered the most accurate and reliable markers of ovarian reserve. A literature search was performed for studies that addressed the ability of ovarian reserve markers to predict poor and high ovarian response in assisted reproductive technology cycles. According to the predicted response to ovarian stimulation (poor- normal- or high- response), it is possible to counsel couples before treatment about the prognosis, and also to individualize ovarian stimulation protocols, choosing among GnRH-agonists or antagonists for endogenous FSH suppression, and the FSH starting dose in order to decrease the risk of cycle cancellation and ovarian hyperstimulation syndrome. In this review we discuss how to choose the best COS therapy, based on ovarian reserve markers, in order to enhance chances in IVF.

Gonadotropin and intra-ovarian signals regulating follicle development and atresia: the delicate balance between life and death.

PubMed

Craig, Jesse; Orisaka, Makoto; Wang, Hongmei; Orisaka, Sanae; Thompson, Winston; Zhu, Cheng; Kotsuji, Fumikazu; Tsang, Benjamin K

2007-05-01

Regulation of mammalian follicular development is tightly regulated by both cell death and survival signals, including endocrine (e.g. gonadotropin) and intra-ovarian regulators (e.g. Nodal and GDF9). The destiny of the individual follicle (growth/ovulation or atresia) is dependent on a delicate balance in the expression and action of factors promoting follicular cell proliferation, growth and differentiation, and of those promoting programmed cell death (apoptosis). Development of the follicle from the primordial to preantral stage is regulated by oocyte-derived factors including GDF9 and BMP15, and is not dependent on gonadotropin support (gonadotropin-independent stage). As the follicle transits into the early antral stage it becomes responsive to gonadotropin (gonadotropin-responsive stages) and further development renders the follicle completely dependent on the presence of gonadotropin while modulated by intra-ovarian regulators (gonadotropin-dependent). Follicle fate is also regulated by pro-apoptotic factors such as the intraovarian regulator Nodal, which is secreted by the theca and promotes apoptosis of differentiated granulosa cells through a mechanism involving Smad2 signaling and suppression of the PI3K/Akt pathway. The intracellular protein prohibitin (PHB) appears to have a dual role during folliculogenesis; acting as a cell survival factor in undifferentiated cells, and as a pro-apoptotic factor following differentiation. Further investigations of the interplay between these endocrine and ovarian regulators will lead to a better understanding into the regulation of follicular development and atresia, allowing development of new techniques for assisted reproduction.

In vitro effects of diethylstilbestrol, genistein, 4-tert-butylphenol, and 4-tert-octylphenol on steroidogenic activity of isolated immature rat ovarian follicles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myllymaeki, Sari; Haavisto, Tapio; Vainio, Minna

2005-04-01

Isolated rat ovarian follicles grow and produce steroid hormones in vitro and so provide a good model for studying the effects of hormonally active compounds on follicular steroidogenesis. We have evaluated the effects of diethylstilbestrol (DES), genistein (GEN) and two alkylphenols, 4-tert-butylphenol (BP) and 4-tert-octylphenol (OP) on the growth, survival, and steroid hormone and cAMP production by isolated 14-day-old rat (Sprague-Dawley) ovarian follicles. During a 5-day culture, FSH was obligatory for follicle growth and increased estradiol and testosterone secretion in a dose-dependent manner. DES (10{sup -6} M) caused the strongest decline in estradiol and testosterone levels but did not havemore » detectable effects on either cAMP production or aromatase enzyme activity. GEN caused a prominent decrease in cAMP and testosterone levels without significant changes in secreted estradiol. The latter, apparently, was due to a dose-dependent stimulation of aromatase enzyme activity in the presence of genistein. Both BP and OP decreased estradiol and testosterone secretion in a dose-dependent manner while no effect on aromatase activity was observed. OP, unlike BP, decreased forskolin-induced cAMP levels. Xenoestrogens at the used concentrations did not interfere with the growth and survival of the follicles. The results indicate that isolated ovarian follicles representing intact morphological and functional units offer a sensitive model system for elucidating the female-specific reproductive effects of environmental chemicals.« less

There is a Positive Correlation Between Socioeconomic Status and Ovarian Reserve in Women of Reproductive Age.

PubMed

Barut, Mert Ulas; Agacayak, Elif; Bozkurt, Murat; Aksu, Tarık; Gul, Talip

2016-11-16

BACKGROUND The purpose of this study was to investigate the potential association between socioeconomic status and ovarian reserve, anti-Mullerian hormone level, antral follicle count, and follicle stimulating hormone level in women of reproductive age. MATERIAL AND METHODS A total of 101 married women between 20-35 years of age who presented to the Department of Obstetrics and Gynecology, Health Research System In Vitro Fertilization (HRS IVF) Center between October 2014 and November 2015 and met the inclusion criteria were included in this study. The participants were divided into three socioeconomic groups using Kuppuswamy's socioeconomic status scale. Thirty-one participants were assigned to the low socioeconomic status group, 37 to the middle socioeconomic status group, and 33 to the high socioeconomic status group. On days 3-6 of the menstrual cycle, 10 mL of blood was collected from the participants for follicle stimulating hormone and anti-Mullerian hormone measurements. Transvaginal ultrasonography was performed for both ovaries for the purpose of counting antral follicles measuring 2-10 mm in diameter. RESULTS Both ovarian reserve parameters, namely anti-Mullerian hormone level and antral follicle count, exhibited a significant association with socioeconomic status (p=0.000 and p=0.000, respectively). The association between follicle stimulating hormone level and socioeconomic status was also significant (p=0.000). CONCLUSIONS A low socioeconomic status aggravated by sources of stress such as undernutrition and financial hardships affects ovarian reserve, which should be remembered in approaching infertile patients.

Does the Ovarian Stimulation Phase Length Predict In vitro Fertilization Outcomes?

PubMed Central

Alport, Brie; Case, Allison; Lim, Hyun; Baerwald, Angela

2011-01-01

Background Bi-directional communication between the follicle and oocyte is necessary to regulate follicle and oocyte development. Currently, it is not practical to monitor the serial growth of individual follicles during assisted reproduction. The ovarian stimulation phase length (SPL) is an indirect measure of mean follicular growth rate. The objective of this study was to test the hypothesis that a short or long SPL would be associated with suboptimal outcomes in women undergoing in vitro fertilization (IVF). Materials and Methods A retrospective cohort study was conducted in 140 women who underwent IVF. Follicle development was monitored every 2-3 days during ovarian stimulation using transvaginal ultrasonography. Once > 3 follicles reached ≥ 17 mm, human chorionic gonadotropin (hCG) was administered. Oocyte retrieval was performed approximately 35 hours after hCG. Oocytes underwent IVF on the day of collection and were evaluated daily thereafter. Embryos were transferred on days 3 or 5, depending on the number and quality of embryos available. Associations between SPL, age, follicle, oocyte, embryo and pregnancy outcomes were evaluated (SPSS version 17.0; SPSS Inc., Chicago, IL, USA). Results A SPL of 11 days was associated with an optimal number of follicles that developed to ≥ 6 mm, ≥ 10 mm and ≥ 15 mm; serum estradiol concentrations; and number of oocytes collected (p<0.05). Gradual reductions in the number of developing follicles, serum estradiol concentrations and number of oocytes collected occurred with SPL less than or greater than 11 days (p<0.05). The SPL did not influence endometrial, embryo or pregnancy outcomes (p>0.05). Associations between SPL and outcomes were not influenced by age (p>0.05). Conclusion The ovarian SPL can be used to predict the number of follicles that develop, oocytes collected and serum estradiol concentrations, but not embryo or pregnancy outcomes. PMID:25101156

Effects of ammonium perchlorate on the reproductive performance and thyroid follicle histology of zebrafish

USGS Publications Warehouse

Patino, R.; Wainscott, M.R.; Cruz-Li, E. I.; Balakrishnan, S.; McMurry, C.; Blazer, V.S.; Anderson, T.A.

2003-01-01

Adult zebrafish were reared up to eight weeks in control water or in water containing ammonium perchlorate (AP) at measured perchlorate concentrations of 18 (environmentally relevant, high) and 677 ppm. Groups of eight females were paired with four males on a weekly basis to assess AP effects on spawned egg volume, an index of reproductive performance. All treatments were applied to four to five spawning replicates. At 677 ppm, spawn volume was reduced within one week and became negligible after four weeks. At 18 ppm, spawn volume was unaffected even after eight weeks. Also, perchlorate at 18 ppm did not affect percentage egg fertilization. Fish were collected at the end of the exposures (677 ppm, four weeks; control and 18 ppm, eight weeks) for whole-body perchlorate content and thyroid histopathological analysis. Fish perchlorate levels were about one-hundredth of those of treatment water levels, indicating that waterborne perchlorate does not accumulate in whole fish. At 677 ppm for four weeks, perchlorate caused thyroid follicle cell (nuclear) hypertrophy and angiogenesis, whereas at 18 ppm for eight weeks, its effects were more pronounced and included hypertrophy, angiogenesis, hyperplasia, and colloid depletion. In conclusion, an eight-week exposure of adult zebrafish to 18 ppm perchlorate (high environmentally relevant concentrations) affected the histological condition of their thyroid follicles but not their reproductive performance. The effect of 677 ppm perchlorate on reproduction may be due to extrathyroidal toxicity. Further research is needed to determine if AP at lower environmentally relevant concentrations also affects the thyroid follicles of zebrafish.

Neurokinin B Exerts Direct Effects on the Ovary to Stimulate Estradiol Production.

PubMed

Qi, Xin; Salem, Mohamed; Zhou, Wenyi; Sato-Shimizu, Miwa; Ye, Gang; Smitz, Johan; Peng, Chun

2016-09-01

Neurokinin B (NKB) and its receptor, NK3R, play critical roles in reproduction by regulating the secretion of the hypothalamic GnRH. NKB and NK3R genes are also expressed in the ovary; however, their physiological roles within the ovary are unknown. The aim of this study was to determine whether NKB acts directly on the ovary to regulate reproduction. Injection of NKB into zebrafish accelerated follicle development, increased the mRNA levels of cyp11a1 and cyp19a1, and enhanced estradiol production. Similarly, NKB induced cyp11a1 and cyp19a1 expression in primary cultures of zebrafish follicular cells and stimulated estradiol production from cultured follicles. Furthermore, NKB activates cAMP response element-binding protein and ERK, and ERK inhibitors abolished the effect of NKB on cyp11a1, whereas protein kinase A and calmodulin-dependent protein kinase II inhibitors that blocked the activation of cAMP response element-binding protein, attenuated the effect of NKB on cyp19a1 expression. In a human granulosa cell line, COV434, a NKB agonist, senktide, also increased CYP11A1 and CYP19A1 mRNA levels and enhanced aromatase protein levels and activities. Small interfering RNA-mediated knockdown of NK3R reduced senktide-induced CYP11A1 and CYP19A1 mRNA levels. Finally, we found that NK3R mRNA was strongly down-regulated in granulosa cells obtained from polycystic ovary syndrome (PCOS) patients when compared with non-PCOS subjects. Taken together, our findings establish a direct action of NKB to induce ovarian estrogen production and raise the possibility that defective signaling of this pathway may contribute to the development of PCOS.

Effects of maturation-inducing hormone on heterologous gap junctional coupling in ovarian follicles of Atlantic croaker

USGS Publications Warehouse

Yoshizaki, G.; Patino, R.; Thomas, P.; Bolamba, D.; Chang, Xiaotian

2001-01-01

A previous ultrastructural study of heterologous (granulosa cell-oocyte) gap junction (GJ) contacts in ovarian follicles of Atlantic croaker suggested that these contacts disappear late during the process of resumption of oocyte meiosis. This observation suggested that, unlike scenarios proposed for a number of other species, uncoupling of GJ is not necessary for the onset of meiotic resumption in croaker follicles. However, the functionality of heterologous GJ contacts and the temporal association between maturation-inducing hormone (MIH)-induced changes in heterologous coupling and resumption of oocyte meiosis have not been examined in Atlantic croaker. These questions were addressed with a cell-cell coupling assay that is based on the transfer of a GJ marker, Lucifer Yellow, from oocytes to granulosa cells. Follicle-enclosed oocytes injected with Lucifer Yellow allowed transfer of the dye into the follicle cell layer, thus confirming that there is functional heterologous coupling between the oocyte and the granulosa cells. Dye transfer was observed in vitellogenic, full-grown/maturation-incompetent, and full-grown /maturation-competent follicles. Treatment of maturation-competent follicles with MIH caused a time-dependent decline in the number of follicles transferring dye. However, although GJ uncoupling in some of the follicles was observed before germinal vesicle breakdown (GVBD, index of meiotic resumption), about 50% of the follicles maintained the ability to transfer dye even after GVBD had occurred. Further, a known GJ inhibitor (phorbol 12-myristate 13-acetate) blocked heterologous GJ within a time frame similar to that seen with MIH but without inducing any of the morphological changes (including GVBD) associated with follicular maturation. In conclusion, uncoupling of heterologous GJ seems insufficient and unnecessary for the onset of meiotic resumption in ovarian follicles of Atlantic croaker. ?? 2001 Elsevier Science.

Ovarian Stem Cell Nests in Reproduction and Ovarian Aging.

PubMed

Ye, Haifeng; Zheng, Tuochen; Li, Wei; Li, Xiaoyan; Fu, Xinxin; Huang, Yaoqi; Hu, Chuan; Li, Jia; Huang, Jian; Liu, Zhengyv; Zheng, Liping; Zheng, Yuehui

2017-01-01

The fixed primordial follicles pool theory, which monopolized reproductive medicine for more than one hundred years, has been broken by the discovery, successful isolation and establishment of ovarian stem cells. It has brought more hope than ever of increasing the size of primordial follicle pool, improving ovarian function and delaying ovarian consenescence. Traditional view holds that stem cell aging contributes to the senility of body and organs. However, in the process of ovarian aging, the main factor leading to the decline of the reproductive function is the aging and degradation of ovarian stem cell nests, rather than the senescence of ovarian germ cells themselves. Recent studies have found that the immune system and circulatory system are involved in the formation of ovarian germline stem cell niches, as well as regulating the proliferation and differentiation of ovarian germline stem cells through cellular and hormonal signals. Therefore, we can improve ovarian function and delay ovarian aging by improving the immune system and circulatory system, which will provide an updated program for the treatment of premature ovarian failure (POF) and infertility. © 2017 The Author(s). Published by S. Karger AG, Basel.

Multidetector computed tomographic morphology of ovaries in cynomolgus macaques (Macaca fascicularis).

PubMed

Jones, Jeryl C; Appt, Susan E; Bourland, J Daniel; Hoyer, Patricia B; Clarkson, Thomas B; Kaplan, Jay R

2007-09-01

Macaques are important models for menopause and associated diseases in women. A sensitive, noninvasive technique for quantifying changes in ovarian morphology would facilitate longitudinal studies focused on the health-related sequelae of naturally occurring or experimentally induced alterations in ovarian structure and function. Multidetector computed tomography (MDCT) is a fast, non-invasive imaging technique that uses X-rays, multiple rows of detectors, and computers to generate detailed slice images of structures. The purpose of this study was to describe the utility of MDCT for reliably characterizing ovarian morphology in macaques. Five macaques were scanned using contrast-enhanced MDCT. The following characteristics were described: 1) appearance of ovaries and adjacent landmarks, 2) effects of varying technical protocols on ovarian image quality, 3) radiation doses delivered to the pelvic region during scanning, and 4) MDCT estimates of ovarian volume and antral follicle counts versus those measured directly in ovarian tissue. Ovaries were distinguishable in all MDCT scans and exhibited heterogeneous contrast enhancement. Antral follicles appeared as focal areas of nonenhancement. Ovarian image quality with 5 pediatric scanning protocols was sufficient for discriminating ovarian margins. Pelvic region radiation doses ranged from 0.5 to 0.7 rad. Antral follicles counted using MDCT ranged from 3 to 5 compared with 3 to 4 counted using histology. Ovarian volumes measured using MDCT ranged from 0.41 to 0.67 ml compared with 0.40 to 0.65 ml by water displacement. MDCT is a promising technique for measuring longitudinal changes in macaque ovarian morphology reliably and noninvasively.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Daud, A.I.; Bumpus, F.M.; Husain, A.

Ovarian angiotensin I (Ang I)-converting enzyme (ACE), estimated by the specific binding of the ACE inhibitor (125I)iodo-MK-351A, is localized on multiple ovarian structures, including follicular granulosa cells, corpora lutea, terminal epithelium, and ovarian blood vessels, but total ovarian ACE does not display a cyclic pattern of variation during the rat estrous cycle. We have previously shown that ACE is localized on the granulosa cell layer of a subpopulation of rat ovarian follicles. Our present study shows that ovarian granulosa cells contain high affinity (binding site affinity (Kd), approximately 90 pM) and low capacity (binding site density (Bmax), approximately 12 fmol/2.5more » X 10(5) cells) (125I)iodo-MK-351A-binding sites and convert (125I)iodo-Ang I to (125I)iodo-Ang II (greater than 85% of this conversion was inhibited by the ACE inhibitor captopril). Throughout the rat estrous cycle, 94-100% of developing follicles and 89-96% of atretic follicles contained high levels of ACE; however, ACE was either not observed or its levels were very low in preovulatory follicles. These findings indicate the presence of high levels of biologically active ACE on the surface of granulosa cells and suggest a potential role for follicular ACE in early stages of follicular maturation and atresia. Although ACE is known to process a variety of peptides found within the ovary, and these peptides may have opposing effects on follicular maturation, we attempted to define the cumulative effect of ACE inhibition on follicular maturation.« less

Expression of Folliculogenesis-Related Genes in Vitrified Human Ovarian Tissue after Two Weeks In Vitro Culture.

PubMed

Shams Mofarahe, Zahra; Salehnia, Mojdeh; Ghaffari Novin, Marefat; Ghorbanmehr, Nassim; Fesharaki, Mohammad Gholami

2017-01-01

This study was designed to evaluate the effects of vitrification and in vitro culture of human ovarian tissue on the expression of oocytic and follicular cell-related genes. In this experimental study, ovarian tissue samples were obtained from eight transsexual women. Samples were cut into small fragments and were then assigned to vitrified and non-vitrified groups. In each group, some tissue fragments were divided into un-cultured and cultured (in α-MEM medium for 2 weeks) subgroups. The normality of follicles was assessed by morphological observation under a light microscope using hematoxylin and eosin (H&E) staining. Expression levels of factor in the germ line alpha ( FIGLA ), KIT ligand ( KL ), growth differentiation factor 9 ( GDF-9 ) and follicle stimulating hormone receptor ( FSHR ) genes were quantified in both groups by real-time reverse transcriptase polymerase chain reaction (RT-PCR) at the beginning and the end of culture. The percentage of normal follicles was similar between non-cultured vitrified and non-vitrified groups (P>0.05), however, cultured tissues had significantly fewer normal follicles than non-cultured tissues in both vitrified and non-vitrified groups (P<0.05). In both cultured groups the rate of primary and secondary follicles was significantly higher than non-cultured tissues (P<0.05). The expression of all examined genes was not significantly altered in both non-cultured groups. Whiles, in comparison with cultured tissues non-cultured tissues, the expression of FIGLA gene was significantly decreased, KL gene was not changed, GDF-9 and FSHR genes was significantly increased (P<0.05). Human ovarian vitrification following in vitro culture has no impairing effects on follicle normality and development and expression of related-genes. However, in vitro culture condition has deleterious effects on normality of follicles.

Maternally Contributed Folate Receptor 1 Is Expressed in Ovarian Follicles and Contributes to Preimplantation Development

PubMed Central

Strandgaard, Trine; Foder, Solveig; Heuck, Anders; Ernst, Erik; Nielsen, Morten S.; Lykke-Hartmann, Karin

2017-01-01

Folates have been shown to play a crucial role for proper development of the embryo as folate deficiency has been associated with reduced developmental capacity such as increased risk of fetal neural tube defects and spontanous abortion. Transcripts encoding the reduced folate carrier RFC1 (SLC19A1 protein) and the high-affinity folate receptor FOLR1 are expressed in oocytes and preimplantation embryos, respectively. In this study, we observed maternally contributed FOLR1 protein during mouse and human ovarian follicle development, and 2-cell mouse embryos. In mice, FOLR1 was highly enriched in oocytes from primary, secondary and tertiary follicles, and in the surrounding granulosa cells. Interestingly, during human follicle development, we noted a high and specific presence of FOLR1 in oocytes from primary and intermediate follicles, but not in the granulosa cells. The distribution of FOLR1 in follicles was noted as membrane-enriched but also seen in the cytoplasm in oocytes and granulosa cells. In 2-cell embryos, FOLR1-eGFP fusion protein was detected as cytoplasmic and membrane-associated dense structures, resembling the distribution pattern observed in ovarian follicle development. Knock-down of Folr1 mRNA function was accomplished by microinjection of short interference (si)RNA targeting Folr1, into mouse pronuclear zygotes. This revealed a reduced capacity of Folr1 siRNA-treated embryos to develop to blastocyst compared to the siRNA-scrambled control group, indicating that maternally contributed protein and zygotic transcripts sustain embryonic development combined. In summary, maternally contributed FOLR1 protein appears to maintain ovarian functions, and contribute to preimplantation development combined with embryonically synthesized FOLR1. PMID:29034232

Ovarian function and reproductive senescence in the rat: role of ovarian sympathetic innervation.

PubMed

Cruz, Gonzalo; Fernandois, Daniela; Paredes, Alfonso H

2017-02-01

Successful reproduction is the result of a myriad interactions in which the ovary and the ovarian follicular reserve play a fundamental role. At present, women who delay maternity until after 30 years of age have a decreased fertility rate due to various causes, including damaged follicles and a reduction in the reserve pool of follicles. Therefore, the period just prior to menopause, also known as the subfertile period, is important. The possibility of modulating the follicular pool and the health of follicles during this period to improve fertility is worth exploring. We have developed an animal model to study the ovarian ageing process during this subfertile period to understand the mechanisms responsible for reproductive senescence. In the rat model, we have shown that the sympathetic nervous system participates in regulating the follicular development during ovarian ageing. This article reviews the existing evidence on the presence and functional role of sympathetic nerve activity in regulating the follicular development during ovarian ageing, with a focus on the subfertile period.Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/153/2/R61/suppl/DC1. © 2017 Society for Reproduction and Fertility.

Evaluation of results obtained with corifollitropin alfa after poor ovarian response in previous cycle using recombinant follicular stimulating hormone in the long-term protocol.

PubMed

Salgueiro, Lister L; Rolim, Juliana R; Moura, Bernardo R L; Machado, Suelen P P; Haddad, Carolina

2016-08-01

This study evaluated the use of Corifollitropin alfa in patients with previous poor response to recombinant follicle stimulating hormone in long-term protocols using gonadotropin-releasing hormone. Twenty-seven poor responders to previous treatment with the long term protocol using the recombinant follicle stimulating hormone (Group 1) were selected and then submitted to a second attempt using the same long term protocol with Corifollitropin alfa instead of the recombinant follicle stimulating hormone (Group 2).Ovarian down-regulation was achieved using subcutaneous administration of Leuprolide Acetate. Ovarian stimulation was performed with recombinant follicle stimulating hormone until the administration of human chorionic gonadotropin, followed by follicular aspiration (Group 1). Group 2 was submitted to this same protocol using Corifollitropin alfa instead of recombinant follicle stimulating hormone. There were significant differences in the number of aspirated oocytes, percentage of mature oocytes, amount of injected oocytes and transferred embryos - with all of these parameters being increased in the Corifollitropin alfa group. In addition, the rates of pregnancy and ongoing pregnancy were also significantly higher in the Corifollitropin alfa group. The present study demonstrated that the use of Corifollitropin alfa in the long-term protocol could be a highly effective alternative for patients with poor ovarian response, who were unsuccessful in a previous treatment with In Vitro Fertilization - Intracytoplasmic Sperm Injection.

Increased abundance of aromatase and follicle stimulating hormone receptor mRNA and decreased insulin-like growth factor-2 receptor mRNA in small ovarian follicles of cattle selected for twin births

USDA-ARS?s Scientific Manuscript database

Cattle genetically selected for twin ovulations and births (Twinner) exhibit increased ovarian follicular development, increased ovulation rate, and greater blood and follicular fluid IGF 1 concentrations compared with contemporary cattle not selected for twins (Control). Experimental objectives wer...

Dysregulation of follicle development in a mouse model of premature ovarian insufficiency

PubMed Central

Grasa, P; Sheikh, S; Krzys, N; Millar, K; Janjua, S; Nawaggi, P

2016-01-01

Premature ovarian insufficiency (POI) occurs in 1% of reproductive-age women. The ovarian manifestation ranges from the presence of a variable population of follicles (follicular) to the absence of follicles (afollicular), and in the majority of cases the cause is unknown. A transgenic mouse model of follicular POI, the Double Mutant (DM), arises from oocyte-specific deletion of Mgat1 and C1galt1 required for the generation of O- and N-glycans. DM females are subfertile at 6 weeks, infertile by 9 weeks and exhibit POI by 12 weeks of age. In this study we investigate the cause of the reduced fertility at 6 weeks and infertility at 9 weeks of DM females. Ovary sections were used to analyse follicle and corpora lutea (CL) numbers, apoptosis, and levels of laminin and 3β-hydroxysteroid dehydrogenase using immunohistochemistry. After POI, DM females unexpectedly remained sexually receptive. At both 6 and 9 weeks, DM ovaries contained more primary follicles, however, at 9 weeks DM follicles were proportionally healthier, revealed by TUNEL analysis compared with Controls. In 9 week DM ovaries (collected post-mating), secondary follicles had theca and basal lamina structure abnormalities, whilst preovulatory follicles failed to ovulate resulting in the presence of numerous luteinised unruptured follicles, indicative of ovulation failure. Finally, DM ovaries contained more regressing CL with decreased luteal cell apoptosis indicative of a defect in CL regression. Identifying these follicular modifications have provided insight into the aetiology of a model of POI and highlight targets to investigate with the hope of developing new fertility treatments. PMID:27581083

Bisphenol A and Ovarian Reserve among Infertile Women with Polycystic Ovarian Syndrome.

PubMed

Zhou, Wei; Fang, Fang; Zhu, Wenting; Chen, Zi-Jiang; Du, Yanzhi; Zhang, Jun

2016-12-27

To better understand possible effects of bisphenol A (BPA) exposure on ovarian reserve in women with polycystic ovary syndrome (PCOS), we measured creatinine adjusted urinary BPA (BPA_Cre) concentrations and used regression models to evaluate the association between urinary BPA level and antral follicle count (AFC), antimullerian hormone (AMH), day-3 follicle stimulating hormone levels (FSH) and inhibin B (INHB) in 268 infertile women diagnosed with PCOS. BPA was detected in all women with a median concentration of 2.35 ng/mL (the 25th and 75th percentiles of 1.47 ng/mL and 3.95 ng/mL). A unit increase in BPA_Cre was associated with a significant decrease of 0.34 in AFC (β = -0.34, 95% CI = -0.60, -0.08; p = 0.01). Likewise, BPA was negatively associated with AMH and day-3 FSH levels, but neither of them reached statistical significance. No association was observed between BPA and INHB. Our results suggest that in women with PCOS, BPA may affect ovarian follicles and, therefore, reduce ovarian reserve.

Amygdala Kindling Alters Estrus Cycle and Ovarian Morphology in the Rat

PubMed Central

Pan, Juan; Zhang, Lingwu; Wang, Feng; Liu, Dan

2014-01-01

The objective of this study is to explore the effects of amygdala kindling on estrus cycle and ovarian morphology. Thirty-five female rats at the age of 8 weeks were randomly designated to electrode kindled, sham-kindled, and normal controls. Kindled rats were implanted with kindling electrodes in the left basolateral amygdala and kindled by brief suprathreshold stimulations with a bipolar electrode. Estrous cycles were daily monitored through vaginal smears. Electrographic and behavioral seizures were recorded and ovarian morphology was evaluated by light and electron microscopies. Our results showed that the kindled rats lost their ovarian periodicity displayed significant ovarian enlargement. H&E staining revealed increased number of growing follicles and total follicles, as well as polycysts in the ovaries of the kindled animals compared to sham and control animals. Ultrastructural study detected numerous apoptotic granulosa cells in growing follicles and thecal cell hyperplasia with secretary granules in the thecal cells in the kindled rats. The results suggest that amygdala kindling is a risk factor for the development of polycystic ovary syndrome. PMID:25285307

Polycystic ovary morphology: age-based ultrasound criteria.

PubMed

Kim, Hyun-Jun; Adams, Judith M; Gudmundsson, Jens A; Arason, Gudmundur; Pau, Cindy T; Welt, Corrine K

2017-09-01

To determine age-based criteria for polycystic ovary morphology. Cross-sectional, case-control design. Outpatient setting. Women with polycystic ovary syndrome (PCOS) defined by hyperandrogenism and irregular menses (n = 544) and controls with regular menses and no evidence of hyperandrogenism (n = 666) participated. Parameters were tested in a second cohort of women with PCOS (n = 105) and controls (n = 32) meeting the same criteria. Subjects underwent a pelvic ultrasound documenting ovarian volume and maximum follicle number in a single plane. A receiver operating characteristic curve was constructed to determine the ovarian volume and follicle number with the best sensitivity and specificity to define PCOS for age groups at approximately 5-year intervals from age 18 to >44 years. The best sensitivity and specificity were obtained using a threshold volume of 12 mL and 13 follicles for ages ≤24 years, 10 mL and 14 follicles for ages 25-29 years, 9 mL and 10 follicles for ages 30-34 years, 8 mL and 10 follicles for ages 35-39 years, 10 mL and 9 follicles for ages 40-44 years, and 6 mL and 7 follicles for ages >44 years. Data from a second cohort confirmed the need to decrease volume and follicle number with increasing age to diagnose PCOS. Polycystic ovary morphology was most accurate at predicting the PCOS diagnosis for women ages 30-39 years. The ovarian volume and follicle number threshold to define polycystic ovary morphology should be lowered starting at age 30. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Detection of ovarian matrix metalloproteinase mRNAs by in situ hybridization.

PubMed

Rosewell, Katherine L; Curry, Thomas E

2009-01-01

In situ hybridization represents a powerful technique to localize DNA or RNA of interest at the chromosomal or cellular level. In endocrine tissues composed of diverse and varied cell types, in situ hybridization has allowed the identification of specific cells responsible for the expression of genes controlling the function of the tissue. Our laboratory has routinely used this approach to understand the cellular expression of genes associated with the growth of the ovarian follicle, rupture of the follicle, and transformation of the ruptured follicle into the corpus luteum. The current study outlines the procedural details of in situ detection of mRNA in tissues and illustrates the utility of this approach in identifying the ovarian cells expressing the matrix metalloproteinases and their endogenous inhibitors, the TIMPs, in the human ovary.

Targeting of follicle stimulating hormone peptide-conjugated dendrimers to ovarian cancer cells

NASA Astrophysics Data System (ADS)

Modi, Dimple A.; Sunoqrot, Suhair; Bugno, Jason; Lantvit, Daniel D.; Hong, Seungpyo; Burdette, Joanna E.

2014-02-01

Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side effects. To address these issues, we have designed poly(amidoamine) (PAMAM) dendrimers to selectively target the follicle stimulating hormone receptor (FSHR), which is overexpressed by tumorigenic ovarian cancer cells but not by immature primordial follicles and other non-tumorigenic cells. Fluorescein-labeled generation 5 (G5) PAMAM dendrimers were conjugated with the binding peptide domain of FSH (FSH33) that has a high affinity to FSHR. The targeted dendrimers exhibited high receptor selectivity to FSHR-expressing OVCAR-3 cells, resulting in significant uptake and downregulation of an anti-apoptotic protein survivin, while showing minimal interactions with SKOV-3 cells that do not express FSHR. The selectivity of the FSH33-targeted dendrimers was further validated in 3D organ cultures of normal mouse ovaries. Immunostaining of the conjugates revealed their selective binding and uptake by ovarian surface epithelium (OSE) cells that express FSHR, while sparing the immature primordial follicles. In addition, an in vivo study monitoring tissue accumulation following a single intraperitoneal (i.p.) injection of the conjugates showed significantly higher accumulation of FSH33-targeted dendrimers in the ovary and oviduct compared to the non-targeted conjugates. These proof-of-concept findings highlight the potential of these FSH33-targeted dendrimers to serve as a delivery platform for anti-ovarian cancer drugs, while reducing their systemic side effects by preventing nonspecific uptake by the primordial follicles.Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side effects. To address these issues, we have designed poly(amidoamine) (PAMAM) dendrimers to selectively target the follicle stimulating hormone receptor (FSHR), which is overexpressed by tumorigenic ovarian cancer cells but not by immature primordial follicles and other non-tumorigenic cells. Fluorescein-labeled generation 5 (G5) PAMAM dendrimers were conjugated with the binding peptide domain of FSH (FSH33) that has a high affinity to FSHR. The targeted dendrimers exhibited high receptor selectivity to FSHR-expressing OVCAR-3 cells, resulting in significant uptake and downregulation of an anti-apoptotic protein survivin, while showing minimal interactions with SKOV-3 cells that do not express FSHR. The selectivity of the FSH33-targeted dendrimers was further validated in 3D organ cultures of normal mouse ovaries. Immunostaining of the conjugates revealed their selective binding and uptake by ovarian surface epithelium (OSE) cells that express FSHR, while sparing the immature primordial follicles. In addition, an in vivo study monitoring tissue accumulation following a single intraperitoneal (i.p.) injection of the conjugates showed significantly higher accumulation of FSH33-targeted dendrimers in the ovary and oviduct compared to the non-targeted conjugates. These proof-of-concept findings highlight the potential of these FSH33-targeted dendrimers to serve as a delivery platform for anti-ovarian cancer drugs, while reducing their systemic side effects by preventing nonspecific uptake by the primordial follicles. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr05042d

Gonadotropin binding sites in human ovarian follicles and corpora lutea during the menstrual cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shima, K.; Kitayama, S.; Nakano, R.

Gonadotropin binding sites were localized by autoradiography after incubation of human ovarian sections with /sup 125/I-labeled gonadotropins. The binding sites for /sup 125/I-labeled human follicle-stimulating hormone (/sup 125/I-hFSH) were identified in the granulosa cells and in the newly formed corpora lutea. The /sup 125/I-labeled human luteinizing hormone (/sup 125/I-hLH) binding to the thecal cells increased during follicular maturation, and a dramatic increase was preferentially observed in the granulosa cells of the large preovulatory follicle. In the corpora lutea, the binding of /sup 125/I-hLH increased from the early luteal phase and decreased toward the late luteal phase. The changes in 3more » beta-hydroxysteroid dehydrogenase activity in the corpora lutea corresponded to the /sup 125/I-hLH binding. Thus, the changes in gonadotropin binding sites in the follicles and corpora lutea during the menstrual cycle may help in some important way to regulate human ovarian function.« less

Left-right differences in ovarian volume and antral follicle count in 1423 women of reproductive age.

PubMed

Korsholm, Anne-Sofie; Hvidman, Helene Westring; Bentzen, Janne Gasseholm; Nyboe Andersen, Anders; Birch Petersen, Kathrine

2017-04-01

The aim of this cross-sectional study was to investigate side differences in antral follicle count (AFC) and ovarian volume in left versus right ovaries in relation to chronological and "biological" age, the latter estimated by anti-Müllerian hormone (AMH) levels. The cohort comprised 1423 women: 1014 fertile and 409 infertile. All were examined by transvaginal sonography and serum AMH. Overall the right ovary contained 8.1% more antral follicles (p = 0.002) and had 10.7% larger volume compared with the left (p < 0.001). In all AMH quartiles, the right ovarian volume was larger than the left (p ≤ 0.003). AFC was significantly higher in the right compared to the left ovary in the three upper AMH quartiles (p ≤ 0.005). The findings were similar when stratified in age quartiles. More than half (54.8%) had polycystic ovarian (PCO) morphology in at least one ovary. Of these women, 46.3% (n = 361) had PCO morphology unilateral - most frequently on the right side (27.6%) compared to the left (18.7%, p < 0.001). The consistent difference in AFC and ovarian volume found in AMH and age quartiles may be explained by presence of a larger pool of primordial follicles in the right ovary established during fetal life.

Effects of thermal regime on ovarian maturation and plasma sex steroids in farmed white sturgeon, Acipenser transmontanus

USGS Publications Warehouse

Webb, M.A.H.; Van Eenennaam, J. P.; Feist, G.W.; Linares-Casenave, J.; Fitzpatrick, M.S.; Schreck, C.B.; Doroshov, S.I.

2001-01-01

Recently, commercial aquaculture farms in Northern California have exposed gravid, cultured white sturgeon females to cold water (12 ?? 1??C) throughout the late phase of vitellogenesis and ovarian follicle maturation resulting in improved ovulation rates and egg quality. However, the optimum timing for transfer of broodfish to the cold water and the capacity of transferred broodfish to maintain reproductive competence over an extended time in cold water had not been evaluated. Gravid white sturgeon females that have been raised at water temperatures of 16-20??C were transported to either cold water (12 ?? 1??C; Group 1) in November 1997 or maintained in ambient water temperatures (10-19??C; Group 2) until early spring. In March 1998, half of the fish in Group 2 had regressed ovaries, but the remaining females had intact ovarian follicles and were transported to the cold water. Ovarian follicles and blood were collected from females until they reached the stage of spawning readiness (determined by germinal vesicle position and an oocyte maturation assay) or underwent ovarian regression. Exposure of gravid sturgeon females to ambient water temperatures (14.5 ?? 2.3??C, mean ?? S.D.) from October to March led to a decrease in plasma sex steroids and a high incidence of ovarian regression in fish with a more advanced stage of oocyte development. Transfer of females with intact ovarian follicles to cold water (12 ?? 1??C) in the fall or early spring resulted in normal ovarian development in the majority of females. Holding females in cold water does not seem to override their endogenous reproductive rhythms but extends their capacity to maintain oocyte maturational competence over a longer period of time. A temperature-sensitive phase in ovarian development may occur during the transition from vitellogenic growth to oocyte maturation, and the degree and timing of sensitivity to environmental temperature are dependent on the female's endogenous reproductive rhythm. ?? 2001 Elsevier Science B.V. All Rights reserved.

Disruption of the ovarian follicle reservoir of prepubertal rats following prenatal exposure to a continuous 900-MHz electromagnetic field.

PubMed

Türedi, Sibel; Hancı, Hatice; Çolakoğlu, Serdar; Kaya, Haydar; Odacı, Ersan

2016-06-01

The effects on human health of electromagnetic field (EMF) have begun to be seriously questioned with the entry into daily life of devices establishing EMF, such as cell phones, wireless fidelity, and masts. Recent studies have reported that exposure to EMF, particularly during pregnancy, affects the developing embryo/fetus. The aim of this study was therefore to examine the effects of exposure to continuous 900-Megahertz (MHz) EMF applied in the prenatal period on ovarian follicle development and oocyte differentiation. Six pregnant Sprague Dawley rats were divided equally into a non-exposed control group (CNGr) and a group (EMFGr) exposed to continuous 900-MHz EMF for 1 h daily, at the same time every day, on days 13-21 of pregnancy. New groups were established from pups obtained from both groups after birth. One group consisting of female pups from CNGr rats was adopted as newborn CNGr (New-CNGr, n = 6), and another group consisting of female pups from EMFGr rats was adopted as newborn EMFGr (New-EMFGr, n = 6). No procedure was performed on New-CNGr or New-EMFGr rats. All rat pups were sacrificed on the postnatal 34th day, and their ovarian tissues were removed. Follicle count, histological injury scoring and morphological assessment with apoptotic index criteria were performed with sections obtained following routine histological tissue preparation. Follicle count results revealed a statistically significant decrease in primordial and tertiary follicle numbers in New-EMFGr compared to New-CNGr (p < 0.05), while atretic follicle numbers and apoptotic index levels increased significantly (p < 0.05). Histopathological examination revealed severe follicle degeneration, vasocongestion, a low level of increased stromal fibrotic tissue and cytoplasmic vacuolization in granulosa cell in New-EMFGr. Prenatal exposure to continuous 900-MHz EMF for 1 h each day from days 13-21 led to a decrease in ovarian follicle reservoirs in female rat pups at the beginning of the prepubertal period.

Confirmation of ovarian homogeneity in post-vitellogenic cultured white sturgeon, Acipenser transmontanus.

PubMed

Talbott, Mariah J; Servid, Sarah A; Cavinato, Anna G; Van Eenennaam, Joel P; Doroshov, Serge I; Struffenegger, Peter; Webb, Molly A H

2014-02-01

Assessing stage of oocyte maturity in female sturgeon by calculating oocyte polarization index (PI) is a necessary tool for both conservation propagation managers and caviar producers to know when to hormonally induce spawning. We tested the assumption that sampling ovarian follicles from one section of one ovary is sufficient for calculating an oocyte PI representative of oocyte maturity for an individual animal. Short-wavelength near-infrared spectroscopy (SW-NIR) scans were performed on three positions per ovary for five fish prior to caviar harvest. Samples of ovarian follicles were subsequently taken from the exact location of the SW-NIR scans for calculation of oocyte PI and follicle diameter. Oocyte PI was statistically different though not biologically relevant within an ovary and between ovaries in four of five fish. Follicle diameter was statistically different but not biologically relevant within an ovary in three of five fish. There were no differences in follicle diameter between ovaries. No statistical differences were observed between SW-NIR spectra collected at different locations within an ovary or between ovaries. These results emphasize the importance of utilizing both oocyte PI measurement and progesterone-induced oocyte maturation assays while deciding when to hormonally induce spawning in sturgeon females.

Female reproductive impacts of dietary methylmercury in yellow perch (Perca flavescens) and zebrafish (Danio rerio).

PubMed

DeBofsky, Abigail R; Klingler, Rebekah H; Mora-Zamorano, Francisco X; Walz, Marcus; Shepherd, Brian; Larson, Jeremy K; Anderson, David; Yang, Luobin; Goetz, Frederick; Basu, Niladri; Head, Jessica; Tonellato, Peter; Armstrong, Brandon M; Murphy, Cheryl; Carvan, Michael J

2018-03-01

The purpose of this study was to evaluate the effects of environmentally relevant dietary MeHg exposures on adult female yellow perch (Perca flavescens) and female zebrafish (Danio rerio) ovarian development and reproduction. Yellow perch were used in the study for their socioeconomic and ecological importance within the Great Lakes basin, and the use of zebrafish allowed for a detailed analysis of the molecular effects of MeHg following a whole life-cycle exposure. Chronic whole life dietary exposure of F 1 zebrafish to MeHg mimics realistic wildlife exposure scenarios, and the twenty-week adult yellow perch exposure (where whole life-cycle exposures are difficult) captures early seasonal ovarian development. For both species, target dietary accumulation values were achieved prior to analyses. In zebrafish, several genes involved in reproductive processes were shown to be dysregulated by RNA-sequencing and quantitative real-time polymerase chain reaction (QPCR), but no significant phenotypic changes were observed regarding ovarian staging, fecundity, or embryo mortality. Yellow perch were exposed to dietary MeHg for 12, 16, or 20 weeks. In this species, a set of eight genes were assessed by QPCR in the pituitary, liver, and ovary, and no exposure-related changes were observed. The lack of genomic resources in yellow perch hinders the characterization of subtle molecular impacts. The ovarian somatic index, circulating estradiol and testosterone, and ovarian staging were not significantly altered by MeHg exposure in yellow perch. These results suggest that environmentally relevant MeHg exposures do not drastically reduce the reproductively important endpoints in these fish, but to capture realistic exposure scenarios, whole life-cycle yellow perch exposures are needed. Copyright © 2017 Elsevier Ltd. All rights reserved.

The numbers of 2- 5 and 6-9 mm ovarian follicles are inversely correlated in both normal women and in polycystic ovary syndrome patients: what is the missing link?

PubMed

Peigné, M; Catteau-Jonard, S; Robin, G; Dumont, A; Pigny, P; Dewailly, D

2018-04-01

Is the negative correlation between the numbers of 2-5 and 6-9 mm follicles influenced by ovarian and/or metabolic parameter(s) in young control women and in patients with polycystic ovarian syndrome (PCOS)? Our study confirmed that the negative correlation between numbers of follicles sized 2-5 and 6-9 mm was stronger in PCOS than in young control women and was not linked to any ovarian or metabolic parameter. Previous reports described a direct negative correlation between the number of small antral follicles (2-5 mm) and large antral follicle (6-9 mm) during the early follicular phase (cycle Days 2-5) in normal and PCOS women. Numerous factors, that could be either intrinsic to the ovary or secondary to metabolic influence and/or gonadotropin regulation, might account for this. Six hundred and thirty-nine patients with PCOS according to Rotterdam Criteria and 157 control women were recruited in this retrospective cross-sectional study from January 2009 to January 2016. Data were obtained from a database of clinical, hormonal and ultrasound (U/S) features recorded consecutively in a single reproductive medicine centre. Univariate correlations between the various parameters were analysed by the Spearman's correlation test. All variables significantly related to the 2-5 and/or 6-9 mm follicle numbers were included in a principal component analysis (PCA) in order to structure the data and to obtain collections of uncorrelated variables, called principal components (PC), which are linear combinations of the original variables. By univariate analysis, the 2-5 and 6-9 mm follicle numbers were strongly but negatively correlated in both populations. Many other variables were correlated to the 2-5 and/or 6-9 mm follicle numbers and to each other. By PCA, these relationships were gathered into four independent PCs in each population. In both groups, the 2-5 and 6-9 mm follicle numbers correlated strongly and inversely to a specific PC. Among the other variables tested, only serum oestradiol level correlated weakly to this PC in the control group. Two other uncorrelated PCs gathered relationships between variables linked to the metabolic status and the gonadotropin regulation both in control and PCOS women. Lastly, a fourth PC included relationships which linked to ovarian ageing in controls and to follicle dysregulation in patients with PCOS. Our controls did not represent the general population since they were recruited in an ART centre; we used a modified Rotterdam classification for PCOS using follicle count and/or serum AMH level with in-house thresholds to define the follicle excess; the AMH assay used is no longer commercially available. Factor(s) regulating specifically the equilibrium between the 2-5 and 6-9 mm follicle numbers still need(s) to be identified. More attention should be paid to the oocyte. None.

Bisphenol A Exposure, Ovarian Follicle Numbers, and Female Sex Steroid Hormone Levels: Results From a CLARITY-BPA Study

PubMed Central

Patel, Shreya; Brehm, Emily; Gao, Liying; Rattan, Saniya; Ziv-Gal, Ayelet

2017-01-01

Bisphenol A (BPA) is an industrial chemical found in thermal receipts and food and beverage containers. Previous studies have shown that BPA can affect the numbers and health of ovarian follicles and the production of sex steroid hormones, but they often did not include a wide range of doses of BPA, used a small sample size, focused on relatively short-term exposures to BPA, and/or did not examine the consequences of chronic BPA exposure on the ovaries or steroid levels. Thus, this study was designed to examine the effects of a wide range of doses of BPA on ovarian morphology and sex steroid hormone production. Specifically, this study tested the hypothesis that prenatal and continuous BPA exposure reduces ovarian follicle numbers and sex steroid hormone levels. To test this hypothesis, rats were dosed with vehicle, ethinyl estradiol (0.05 and 0.5 μg/kg body weight/d), or BPA (2.5, 25, 250, 2500, and 25,000 μg/kg body weight/d) from gestation day 6 until 1 year as part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA). Ovaries and sera were collected on postnatal days 1, 21, and 90, and at 6 months and 1 year. The ovaries were subjected to histological evaluation of follicle numbers and the sera were subjected to measurements of estradiol and progesterone. Collectively, these data indicate that BPA exposure at some doses and time points affects ovarian follicle numbers and sex steroid levels, but these effects are different than those observed with ethinyl estradiol exposure and some previous studies on BPA. PMID:28324068

Bisphenol A Exposure, Ovarian Follicle Numbers, and Female Sex Steroid Hormone Levels: Results From a CLARITY-BPA Study.

PubMed

Patel, Shreya; Brehm, Emily; Gao, Liying; Rattan, Saniya; Ziv-Gal, Ayelet; Flaws, Jodi A

2017-06-01

Bisphenol A (BPA) is an industrial chemical found in thermal receipts and food and beverage containers. Previous studies have shown that BPA can affect the numbers and health of ovarian follicles and the production of sex steroid hormones, but they often did not include a wide range of doses of BPA, used a small sample size, focused on relatively short-term exposures to BPA, and/or did not examine the consequences of chronic BPA exposure on the ovaries or steroid levels. Thus, this study was designed to examine the effects of a wide range of doses of BPA on ovarian morphology and sex steroid hormone production. Specifically, this study tested the hypothesis that prenatal and continuous BPA exposure reduces ovarian follicle numbers and sex steroid hormone levels. To test this hypothesis, rats were dosed with vehicle, ethinyl estradiol (0.05 and 0.5 μg/kg body weight/d), or BPA (2.5, 25, 250, 2500, and 25,000 μg/kg body weight/d) from gestation day 6 until 1 year as part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA). Ovaries and sera were collected on postnatal days 1, 21, and 90, and at 6 months and 1 year. The ovaries were subjected to histological evaluation of follicle numbers and the sera were subjected to measurements of estradiol and progesterone. Collectively, these data indicate that BPA exposure at some doses and time points affects ovarian follicle numbers and sex steroid levels, but these effects are different than those observed with ethinyl estradiol exposure and some previous studies on BPA. Copyright © 2017 Endocrine Society.

Short photoperiod-induced ovarian regression is mediated by apoptosis in Siberian hamsters (Phodopus sungorus)

PubMed Central

Moffatt-Blue, C S; Sury, J J; Young, Kelly A

2009-01-01

Siberian hamster reproduction is mediated by photoperiod-induced changes in gonadal activity. However, little is known about how photoperiod induces cellular changes in ovarian function. We hypothesized that exposing female hamsters to short (inhibitory) as opposed to long (control) photoperiods would induce an apoptosis-mediated disruption of ovarian function. Ovaries and plasma from hamsters exposed to either long (LD, 16 h light:8 h darkness) or short (SD, 8 h light:16 h darkness) days were collected during diestrus II after 3, 6, 9 and 12 weeks and processed for histology or RIA respectively. Apoptosis was assessed by in situ TUNEL and active caspase-3 protein immunolabeling. No significant differences were observed among LD hamsters for any parameter; therefore, these control data were pooled. SD exposure induced a decline in preantral follicles (P < 0.05), early antral/antral follicles (P < 0.01) and corpora lutea (P < 0.01) by week 12 as compared with LD. Terminal atretic follicles appeared by SD week 9; by week 12, these had become the predominant ovarian structures. Estradiol concentrations decreased by weeks 9 and 12 SD when compared with both LD and week-3 SD hamsters (P < 0.05); however, no changes were observed for progesterone. TUNEL-positive follicles in SD ovaries increased at week 3 and subsequently declined by week 12 as compared with LD ovaries (P < 0.01). Active capsase-3 protein immunostaining peaked at SD week 3 as compared with all other groups (P < 0.01). TUNEL and capsase-3 immunolabeling were localized to granulosa cells of late-preantral and early-antral/antral follicles. These data indicate that SD exposure rapidly induces follicular apoptosis in Siberian hamsters, which ultimately disrupts both estradiol secretion and folliculogenesis, resulting in the seasonal loss of ovarian function. PMID:16595728

The ovarian DNA damage repair response is induced prior to phosphoramide mustard-induced follicle depletion, and ataxia telangiectasia mutated inhibition prevents PM-induced follicle depletion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

Phosphoramide mustard (PM) is an ovotoxic metabolite of cyclophosphamide and destroys primordial and primary follicles potentially by DNA damage induction. The temporal pattern by which PM induces DNA damage and initiation of the ovarian response to DNA damage has not yet been well characterized. This study investigated DNA damage initiation, the DNA repair response, as well as induction of follicular demise using a neonatal rat ovarian culture system. Additionally, to delineate specific mechanisms involved in the ovarian response to PM exposure, utility was made of PKC delta (PKCδ) deficient mice as well as an ATM inhibitor (KU 55933; AI). Fishermore » 344 PND4 rat ovaries were cultured for 12, 24, 48 or 96 h in medium containing DMSO ± 60 μM PM or KU 55933 (48 h; 10 nM). PM-induced activation of DNA damage repair genes was observed as early as 12 h post-exposure. ATM, PARP1, E2F7, P73 and CASP3 abundance were increased but RAD51 and BCL2 protein decreased after 96 h of PM exposure. PKCδ deficiency reduced numbers of all follicular stages, but did not have an additive impact on PM-induced ovotoxicity. ATM inhibition protected all follicle stages from PM-induced depletion. In conclusion, the ovarian DNA damage repair response is active post-PM exposure, supporting that DNA damage contributes to PM-induced ovotoxicity. - Highlights: • PM exposure induces DNA damage repair gene expression. • Inhibition of ATM prevented PM-induced follicle depletion. • PKCδ deficiency did not impact PM-induced ovotoxicity.« less

Individualization of controlled ovarian stimulation in vitro fertilization using ovarian reserve markers.

PubMed

Grisendi, Valentina; La Marca, Antonio

2017-06-01

In assisted reproduction technologies (ART) the controlled ovarian stimulation (COS) therapy is the starting point from which a good oocytes retrieval depends. Treatment individualization is based on ovarian response prediction, which largely depends on a woman's ovarian reserve. Anti-Müllerian hormone (AMH) and antral follicle count (AFC) are considered the most accurate and reliable markers of ovarian reserve. A literature search was carried out for studies that addressed the ability of AMH and AFC to predict poor and/or excessive ovarian response in IVF cycles. According to the predicted response to ovarian stimulation (poor- normal- or high-response) is today possible not only to personalize pre-treatment counseling with the couple, but also to individualize the ovarian stimulation protocol, choosing among GnRH-agonists or antagonists for endogenous follicle-stimulating hormone (FSH) suppression and formulating the FSH starting dose most adequate for the single patients. In this review we discuss how to choose the best COS therapy for the single patient, on the basis of the markers-guided ovarian response prediction.

Current understanding of factors influencing antral follicle count and applications to reproductive management in cattle

USDA-ARS?s Scientific Manuscript database

In recent years, an increasing amount of research has focused on variation in numbers of antral follicles in the bovine ovary. This measurement is used as a proxy for the number of microscopic primordial follicles in the ovaries (e.g., the ovarian reserve), because antral follicles can be visualize...

Optical coherence tomography imaging and fluorescence spectroscopy of a novel rat model of ovarian cancer

NASA Astrophysics Data System (ADS)

Kanter, Elizabeth; Walker, Ross; Marion, Sam; Hoyer, Patricia; Barton, Jennifer K.

2005-08-01

Ovarian cancer is relatively rare but is the fifth leading cause of death from cancer in women. Little is known about the precursors and early stages of ovarian cancer partially due to the lack of a realistic animal model. A cohesive model that incorporates ovarian cancer induction into a menopausal rodent would be well suited for comprehensive studies of ovarian cancer, and non-destructive imaging would allow carcinogenesis to be followed. Optical Coherence Tomography (OCT) and Light-Induced Fluorescence (LIF) are minimally invasive optical modalities that allow both structural and biochemical changes to be noted. Rat ovaries were exposed to 4-vinylcyclohexene diepoxide (VCD) for 20 days in order to destroy the primordial follicles. Sutures coated with 7,12-dimethylbenz(a)anthracene (DMBA) were implanted in the right ovary, in order to produce epithelial based ovarian cancers. Rats were sacrificed at 1, 3, and 5 months and ovaries were harvested and imaged with a combined OCT/LIF system. Histology was preformed on the harvested ovaries and any pathology determined. OCT was able to visualize follicle loss and DMBA-induced abnormalities. LIF spectra were also different between cycling, follicle deplete, and DMBA-exposed ovaries. Overall this pilot study demonstrated the feasibility of both the animal model and optical imaging.

Effects of mating behaviour and the ovarian follicular state of female alpacas on conception.

PubMed

Vaughan, J L; Macmillan, K L; Anderson, G A; D'Occhio, M J

2003-01-01

To determine relationships between mating behaviour, ovarian follicular state and successful conception in receptive female alpacas. Seventy pen matings were observed at a commercial alpaca stud in south-western Victoria. The behaviours observed included time taken to assume sternal recumbency, mating duration, and evidence of nonreceptive behaviour such as spitting, kicking and vocalisation. Ovarian follicular state was determined by ultrasonography, which was complemented by measuring plasma concentrations of oestradiol and progesterone. Pregnancies were confirmed by transabdominal ultrasonography between days 45 and 80 after mating. There were no significant differences between receptive females that conceived and those that failed to conceive in the time taken to adopt the copulation position of sternal recumbency, mating duration, or maximum follicle diameter. There was no significant relationship between time taken to assume sternal recumbency (log10) and maximum follicle diameter or plasma oestradiol (log10). However, there was a significant quadratic relationship between plasma oestradiol concentration (log10) and follicle diameter, and the probability of pregnancy increased as the plasma concentration of oestradiol (log10) at the time of mating increased. Females were sexually receptive most of the time in the absence of a corpus luteum, and regardless of size of the largest follicle or plasma concentration of oestradiol. Breed (Huacaya vs Suri), site of the dominant follicle (left or right ovary), lactation state, number of matings by the male (1 or 2), or interval between parturition and mating, did not affect pregnancy outcome. Follicles with a diameter less than 7 mm were able to ovulate in response to mating. This was smaller than previously reported. Thirty-four pregnancies (49% pregnancy rate) resulted in 30 (88%) births with a gestation length of 343 days (SEM +/- 2, range 316-367 days). There were 4 (12%) abortions between days 45 and 80 of gestation and full term. It was not possible to correlate mating behaviour and ovarian state with conception. To optimise pregnancy rates in receptive alpacas, matings need to occur in the presence of an oestrogenic follicle that is capable of ovulation in response to mating. A simple method of detecting alpacas with follicles in this state is not currently available and treatments that control ovarian follicular growth should therefore be investigated.

Platelet-Derived Growth Factor in the Ovarian Follicle Attracts the Stromal Cells of the Fallopian Tube Fimbriae

PubMed Central

Chen, Chiu-Hua; Hsu, Che-Fang; Huang, Rui-Len; Ding, Dah-Ching; Chu, Tang-Yuan

2016-01-01

During human ovulation, the fallopian tube fimbriae must move to the ovulation site to catch the oocyte. As the tissue-of-origin of the majority of ovarian high-grade serous carcinoma (HGSC), the fallopian tube fimbriae carrying a precursor cancer lesion may also approach the ovulatory site for metastasis. We hypothesize that platelet-derived growth factor (PDGF) in mature follicle fluid (FF) attracts the migration of PDGFR-expressing fimbriae toward the ovulating follicle. We observed that more PDGFR-β was expressed in the distal part than in the proximal parts of the fallopian tube, particularly in stromal cells in the lamina propria. The stromal cells, but not the epithelial cells, from normal fimbriae and fallopian tube HGSC were highly chemotactic to mature FF. The chemotactic activities were positively correlated with PDGF-BB and estradiol levels in FF and were abolished by a blocking antibody of PDGFR-β and by tyrosine kinase inhibitor imatinib. When PDGF-BB/AB was depleted from the FF, more than 80% of chemotaxis activities were diminished. This study suggests an ovarian follicle-directed and PDGF-dependent attraction of fallopian tube fimbriae before ovulation. The same mechanism may also be crucial for the ovarian homing of HGSC, which largely originates in the fimbriae. PMID:27379403

In vivo blockade of acetylcholinesterase increases intraovarian acetylcholine and enhances follicular development and fertility in the rat.

PubMed

Urra, Javier; Blohberger, Jan; Tiszavari, Michelle; Mayerhofer, Artur; Lara, Hernan E

2016-07-21

Growth and differentiation of ovarian follicles are regulated by systemic and local factors, which may include acetylcholine (ACh). Granulosa cells (GCs) of growing follicles and luteal cells produce ACh and in cultured GCs it exerts trophic actions via muscarinic receptors. However, such actions were not studied in vivo. After having established that rat ovarian GCs and luteal cells express the ACh-metabolizing enzyme ACh esterase (AChE), we examined the consequences of local application of an AChE inhibitor, huperzine A (HupA), by osmotic minipump delivery into the ovarian bursa of hemiovariectomized rats. Saline was used in the control group. Local delivery of HupA for 4 weeks increased ovarian ACh content. Estrus cyclicity was not changed indicating a locally restricted range of HupA action. The number of primordial and primary follicles was unaffected, but small secondary follicles significantly increased in the HupA group. Furthermore, a significant increase in the number of corpora lutea suggested increased ovulatory events. In support, as shown upon mating, HupA-treated females had significantly increased implantation sites and more pups. Thus the data are in support of a trophic role of ACh in follicular development and ovulation and point to an important role of ACh in female fertility.

In vivo blockade of acetylcholinesterase increases intraovarian acetylcholine and enhances follicular development and fertility in the rat

PubMed Central

Blohberger, Jan; Tiszavari, Michelle; Mayerhofer, Artur; Lara, Hernan E.

2016-01-01

Growth and differentiation of ovarian follicles are regulated by systemic and local factors, which may include acetylcholine (ACh). Granulosa cells (GCs) of growing follicles and luteal cells produce ACh and in cultured GCs it exerts trophic actions via muscarinic receptors. However, such actions were not studied in vivo. After having established that rat ovarian GCs and luteal cells express the ACh-metabolizing enzyme ACh esterase (AChE), we examined the consequences of local application of an AChE inhibitor, huperzine A (HupA), by osmotic minipump delivery into the ovarian bursa of hemiovariectomized rats. Saline was used in the control group. Local delivery of HupA for 4 weeks increased ovarian ACh content. Estrus cyclicity was not changed indicating a locally restricted range of HupA action. The number of primordial and primary follicles was unaffected, but small secondary follicles significantly increased in the HupA group. Furthermore, a significant increase in the number of corpora lutea suggested increased ovulatory events. In support, as shown upon mating, HupA-treated females had significantly increased implantation sites and more pups. Thus the data are in support of a trophic role of ACh in follicular development and ovulation and point to an important role of ACh in female fertility. PMID:27440195

MALDI Mass Spectrometry Imaging of Lipids and Gene Expression Reveals Differences in Fatty Acid Metabolism between Follicular Compartments in Porcine Ovaries

PubMed Central

Uzbekova, Svetlana; Elis, Sebastien; Teixeira-Gomes, Ana-Paula; Desmarchais, Alice; Maillard, Virginie; Labas, Valerie

2015-01-01

In mammals, oocytes develop inside the ovarian follicles; this process is strongly supported by the surrounding follicular environment consisting of cumulus, granulosa and theca cells, and follicular fluid. In the antral follicle, the final stages of oogenesis require large amounts of energy that is produced by follicular cells from substrates including glucose, amino acids and fatty acids (FAs). Since lipid metabolism plays an important role in acquiring oocyte developmental competence, the aim of this study was to investigate site-specificity of lipid metabolism in ovaries by comparing lipid profiles and expression of FA metabolism-related genes in different ovarian compartments. Using MALDI Mass Spectrometry Imaging, images of porcine ovary sections were reconstructed from lipid ion signals for the first time. Cluster analysis of ion spectra revealed differences in spatial distribution of lipid species among ovarian compartments, notably between the follicles and interstitial tissue. Inside the follicles analysis differentiated follicular fluid, granulosa, theca and the oocyte-cumulus complex. Moreover, by transcript quantification using real time PCR, we showed that expression of five key genes in FA metabolism significantly varied between somatic follicular cells (theca, granulosa and cumulus) and the oocyte. In conclusion, lipid metabolism differs between ovarian and follicular compartments. PMID:25756245

Synthetic polymers improve vitrification outcomes of macaque ovarian tissue as assessed by histological integrity and the in vitro development of secondary follicles☆

PubMed Central

Ting, Alison Y.; Yeoman, Richard R.; Lawson, Maralee S.; Zelinski, Mary B.

2013-01-01

Ovarian tissue cryopreservation is the only proven option for fertility preservation in female cancer patients who are prepubertal or require immediate treatment. However it remains unclear which cryopreservation protocol is best in cases where the tissue may contain cancerous cells, as these should be matured in vitro rather than autografted. This study evaluated different cryoprotectant exposure times and whether the addition of synthetic polymers (Supercool X-1000, Z-1000 and polyvinylpyrrolidone [PVP K-12]) to the vitrification solution is beneficial to tissue morphology, cellular proliferation and subsequent in vitro function of secondary follicles. Pieces of macaque (n = 4) ovarian cortex were exposed to vitrification solution containing glycerol (25%, v/v) and ethylene glycol (25%, v/v) for 3 or 8 min, without (V3, V8) or with (VP3, VP8) polymers (0.2% [v/v] X-1000, 0.4% Z-1000 and 0.2% PVP). Fresh and vitrified tissues were fixed for histology and phosphohistone H3 (PPH3) analysis, or used for secondary follicle isolation followed by encapsulated 3D culture. Five-week follicle survival and growth, as well as steroid hormones (estradiol [E2], progesterone, androstenedione) were measured weekly. Morphology of the stroma and preantral follicles as well as PPH3 expression, was preserved in all vitrified tissues. Vitrification with polymers and shorter incubation time (VP3) increased in vitro follicle survival and E2 production compared to other vitrified groups. Thus, a short exposure of macaque ovarian tissue to a vitrification solution containing synthetic polymers preserves morphology and improves in vitro function of secondary follicles. PMID:22569078

Pretreatment with oral contraceptives in infertile anovulatory patients with polycystic ovary syndrome who receive gonadotropins for controlled ovarian stimulation.

PubMed

Palomba, Stefano; Falbo, Angela; Orio, Francesco; Russo, Tiziana; Tolino, Achille; Zullo, Fulvio

2008-06-01

This study was intended to assess the effects of oral contraceptives given before treatment in infertile anovulatory patients with polycystic ovary syndrome who receive gonadotropins for controlled ovarian stimulation. Pretreatment with oral contraceptives increased the mono-ovulatory cycles, the duration of stimulation for noncanceled cycles, and the number of vials of gonadotropins used. It also reduced the number of dominant follicles and the peak E(2) levels for dominant follicles. No effect was observed in rates of cycle cancellation, pregnancy, abortion, live birth, multiple pregnancies, and ovarian hyperstimulation syndrome.

Relationships between ovarian blood flow and ovarian response to eCG-treatment of dairy cows.

PubMed

Honnens, A; Niemann, H; Herzog, K; Paul, V; Meyer, H H D; Bollwein, H

2009-07-01

The goal of the present study was to investigate ovarian blood flow and ovarian response in cows undergoing a gonadotropin treatment to induce a superovulatory response, using transrectal colour Doppler sonography. Forty-two cows including 19 cross-bred, 14 German Holstein and 9 German Black Pied cows were examined sonographically before hormonal stimulation on Day 10 of the oestrous cycle, three days after administration of eCG (Day 13) and seven days after artificial insemination (Day 7(p.i.)). After each Doppler examination, blood was collected for determination of total oestrogens (E) and progesterone (P4) in peripheral plasma. The blood flow volume (BFV) and pulsatility index (PI), which is a measure for blood flow resistance, were determined in the ovarian artery, and B-mode sonography was used to count dominant follicles and corpora lutea. Important criteria to assess the ovarian response following the hormonal treatment were the number of follicles >5mm in diameter on Day 13 and the number of corpora lutea on Day 7(p.i.) per cow. The number of follicles ranged from 2 to 61 (mean+/-S.E.M.: 17.5+/-1.7) and corpora lutea from 0 to 50 (mean+/-S.E.M.: 17.0+/-1.6). The BFV increased from 28.4 to 45.0 ml/min between Days 10 and 13 and reached a maximum of 108.5 ml/min on Day 7(p.i.) The PI decreased from 6.25 on Day 10 to 4.70 on Day 13 and to 2.10 on Day 7(p.i.) The BFV and PI on Day 13 did not correlate with the number of follicles (P>0.05). However, on Day 7(p.i.) the number of corpora lutea correlated positively with the BFV (r=0.64; P<0.0001), and an inverse relationship was found for the PI (r=-0.51; P=0.0005). There were no correlations (P>0.05) between the BFV and PI on Day 10 and the number of follicles on Day 13 or the number of corpora lutea on Day 7(p.i.) Results of the present study show that in cows, a hormonal treatment to induce a superovulatory response yielded a marked increase in BFV and a marked decrease in PI in the ovarian artery. However, there was no correlation between BFV and PI in the ovarian arteries before hormonal stimulation and the number of follicles and corpora lutea that developed after stimulation. Thus BFV and PI measured in the ovarian arteries have limited diagnostic value to predict the outcome of a gonadotropin treatment.

High levels of testosterone inhibit ovarian follicle development by repressing the FSH signaling pathway.

PubMed

Liu, Tao; Cui, Yu-qian; Zhao, Han; Liu, Hong-bin; Zhao, Shi-dou; Gao, Yuan; Mu, Xiao-li; Gao, Fei; Chen, Zi-jiang

2015-10-01

The effect of high concentrations of testosterone on ovarian follicle development was investigated. Primary follicles and granulosa cells were cultured in vitro in media supplemented with a testosterone concentration gradient. The combined effects of testosterone and follicle-stimulating hormone (FSH) on follicular growth and granulosa cell gonadotropin receptor mRNA expression were also investigated. Follicle growth in the presence of high testosterone concentrations was promoted at early stages (days 1-7), but inhibited at later stage (days 7-14) of in vitro culture. Interestingly, testosterone-induced follicle development arrest was rescued by treatment with high concentrations of FSH (400 mIU/mL). In addition, in cultured granulosa cells, high testosterone concentrations induced cell proliferation, and increased the mRNA expression level of FSH receptor (FSHR), and luteinized hormone/choriogonadotropin receptor. It was concluded that high concentrations of testosterone inhibited follicle development, most likely through regulation of the FSH signaling pathway, although independently from FSHR downregulation. These findings are an important step in further understanding the pathogenesis of polycystic ovary syndrome.

Characterization of ovarian aging and reproductive senescence in vervet monkeys (Chlorocebus aethiops sabaeus).

PubMed

Atkins, Hannah M; Willson, Cynthia J; Silverstein, Marnie; Jorgensen, Matthew; Floyd, Edison; Kaplan, Jay R; Appt, Susan E

2014-02-01

Female vervet monkeys (Chlorocebus aethiops sabaeus) are used as an experimental model for chronic diseases relevant to women's health. However, reproductive senescence (menopause) has not yet been characterized for vervet monkeys. Here we describe the histologic, hormonal, and menstrual markers of reproductive senescence in vervet monkeys from the Wake Forest Vervet Research Colony. Ovaries from monkeys (age, 0 to 27 y) were serially sectioned (5 μm), stained, and photographed. In every 100th section, the numbers of primordial, primary, and secondary follicles were determined, and triplicate measurements were used to calculate mean numbers of follicles per ovary. Antimüllerian hormone (AMH), follicle stimulating hormone, and menstrual cycle length were measured in additional monkeys. Primordial follicles and AMH decreased significantly with age, and significant correlations between numbers of primordial and primary follicles and between numbers of primary and secondary follicles were noted. Histologic evaluation revealed that ovaries from 4 aged monkeys (older than 23 y) were senescent. One aged monkey transitioned to menopause, experiencing cycle irregularity over 4 y, eventual cessation of menses, and plasma AMH below the level of detection. Finally, with increasing age, the percentage of female vervets with offspring declined significantly. The present study provides insight into ovarian aging and reproductive senescence in vervet monkeys. Results highlight the importance of considering this nonhuman primate as a model to investigate the relationships between ovarian aging and chronic disease risk.

Anogenital distance is related to ovarian follicular number in young Spanish women: a cross-sectional study

PubMed Central

2012-01-01

Background In animals, anogenital distance (AGD) at birth reflects androgen levels during pregnancy and predicts adult AGD. Little is known about AGD in relation to female reproductive characteristics in humans, a question this study was designed to explore. Methods We used multiple linear and logistic regression analyses to model the relationships between adult female reproductive system characteristics (e.g. ovarian morphology, menstrual cycle) and two measures of AGD [anus-fourchette (AGDAF) and anus-clitoris (AGDAC)] in 100 college-age volunteers in Spain. Ovarian morphology was classified as having < 6 or ≥ 6 follicles per ovary. Results Both AGD measures were positively associated with ovarian follicle number, with AGDAF being more strongly associated. Women in the upper tertile of the AGDAF and AGDAC distributions were more likely to have ≥ 6 ovarian follicles [OR: 6.0 (95% CI 2.0, 17.6) and 3.0 (95% CI 1.1, 8.6), respectively] compared to women in the lowest tertile. Conclusions Increased follicular recruitment has been related to excess androgen exposure in utero in toxicological studies. Our results suggest that the androgenic environment during early fetal life may influence reproductive system development, including AGD, in human females. PMID:23217457

Oocyte cryopreservation for fertility preservation in postpubertal female children at risk for premature ovarian failure due to accelerated follicle loss in Turner syndrome or cancer treatments.

PubMed

Oktay, K; Bedoschi, G

2014-12-01

To preliminarily study the feasibility of oocyte cryopreservation in postpubertal girls aged between 13 and 15 years who were at risk for premature ovarian failure due to the accelerated follicle loss associated with Turner syndrome or cancer treatments. Retrospective cohort and review of literature. Academic fertility preservation unit. Three girls diagnosed with Turner syndrome, 1 girl diagnosed with germ-cell tumor. and 1 girl diagnosed with lymphoblastic leukemia. Assessment of ovarian reserve, ovarian stimulation, oocyte retrieval, in vitro maturation, and mature oocyte cryopreservation. Response to ovarian stimulation, number of mature oocytes cryopreserved and complications, if any. Mean anti-müllerian hormone, baseline follical stimulating hormone, estradiol, and antral follicle counts were 1.30 ± 0.39, 6.08 ± 2.63, 41.39 ± 24.68, 8.0 ± 3.2; respectively. In Turner girls the ovarian reserve assessment indicated already diminished ovarian reserve. Ovarian stimulation and oocyte cryopreservation was successfully performed in all female children referred for fertility preservation. A range of 4-11 mature oocytes (mean 8.1 ± 3.4) was cryopreserved without any complications. All girls tolerated the procedure well. Oocyte cryopreservation is a feasible technique in selected female children at risk for premature ovarian failure. Further studies would be beneficial to test the success of oocyte cryopreservation in young girls. Copyright © 2014 North American Society for Pediatric and Adolescent Gynecology. Published by Elsevier Inc. All rights reserved.

Ovarian fragment sizes affect viability and morphology of preantral follicles during storage at 4°C

USDA-ARS?s Scientific Manuscript database

The efficient transportation of ovarian tissues is affected b various factors compromising their viability. We tested various ovarian sample sizes (whole ovary, biopsy, and transplantation size) during various transportation times....

In vitro culture thawed human ovarian tissue: NIV versus slow freezing method.

PubMed

Xiao, Zhun; Wang, Yan; Li, Ling-Ling; Li, Shang-wei

2013-01-01

The aim of this study was to determine if the needle immersed vitrification method (NIV) can improve the growth potential of thawed ovarian tissue in vitro culture. Human ovarian cortical tissues were cryopreserved using NIV and slow freezing method. After 14 days of culture, the preservation outcomes of NIV and slow freezing groups were analyzed histologically using light microscope and apoptosis was assessed by TUNEL assay. The result showed that the percentage of morphologically abnormal primordial follicles was lower in NIV group than in slow freezing group (P < 0.05). The incidence of TUNEL-positive primordial follicles was lower in NIV group than in slow freezing group (P < 0.05). The study showed that cryopreservation of human ovarian tissue with NIV was effective in improving the growth potential of frozen-thawed ovarian tissue in vitro culture.

Impact of environmental exposures on ovarian function and role of xenobiotic metabolism during ovotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharya, Poulomi; Keating, Aileen F., E-mail: akeating@iastate.edu

2012-06-15

The mammalian ovary is a heterogeneous organ and contains oocyte-containing follicles at varying stages of development. The most immature follicular stage, the primordial follicle, comprises the ovarian reserve and is a finite number, defined at the time of birth. Depletion of all follicles within the ovary leads to reproductive senescence, known as menopause. A number of chemical classes can destroy follicles, thus hastening entry into the menopausal state. The ovarian response to chemical exposure can determine the extent of ovotoxicity that occurs. Enzymes capable of bioactivating as well as detoxifying xenobiotics are expressed in the ovary and their impact onmore » ovotoxicity has been partially characterized for trichloroethylene, 7,12-dimethylbenz[a]anthracene, and 4-vinylcyclohexene. This review will discuss those studies, as well as illustrate where knowledge gaps remain for chemicals that have also been established as ovotoxicants. -- Highlights: ► Summary of ovotoxicant action during ovotoxicity. ► Discussion of impact of biotransformation on chemical toxicity. ► Identification of knowledge gaps in chemical metabolism.« less

Di (2-ethylhexyl) phthalate inhibits growth of mouse ovarian antral follicles through an oxidative stress pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei, E-mail: weiwang2@illinois.edu; Craig, Zelieann R., E-mail: zelieann@illinois.edu; Basavarajappa, Mallikarjuna S., E-mail: mbasava2@illinois.edu

2012-01-15

Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer that has been shown to inhibit growth of mouse antral follicles, however, little is known about the mechanisms by which DEHP does so. Oxidative stress has been linked to follicle growth inhibition as well as phthalate-induced toxicity in non-ovarian tissues. Thus, we hypothesized that DEHP causes oxidative stress and that this leads to inhibition of the growth of antral follicles. To test this hypothesis, antral follicles isolated from CD-1 mice (age 31–35 days) were cultured with vehicle control (dimethylsulfoxide [DMSO]) or DEHP (1–100 μg/ml) ± N-acetyl cysteine (NAC, an antioxidant at 0.25–1 mM).more » During culture, follicles were measured daily. At the end of culture, follicles were collected and processed for in vitro reactive oxygen species (ROS) assays to measure the presence of free radicals or for measurement of the expression and activity of various key antioxidant enzymes: Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPX) and catalase (CAT). The results indicate that DEHP inhibits the growth of follicles compared to DMSO control and that NAC (0.25–1 mM) blocks the ability of DEHP to inhibit follicle growth. Furthermore, DEHP (10 μg/ml) significantly increases ROS levels and reduces the expression and activity of SOD1 compared to DMSO controls, whereas NAC (0.5 mM) rescues the effects of DEHP on ROS levels and SOD1. However, the expression and activity of GPX and CAT were not affected by DEHP treatment. Collectively, these data suggest that DEHP inhibits follicle growth by inducing production of ROS and by decreasing the expression and activity of SOD1. -- Highlights: ► DEHP inhibits growth and increases reactive oxygen species in ovarian antral follicles in vitro. ► NAC rescues the effects of DEHP on the growth and reactive oxygen species levels in follicles. ► DEHP decreases the expression and activity of Cu/Zn superoxide dismutase, which can be rescued by NAC, in antral follicles.« less

Luteal activity and effect of dietary energy restriction on follicular development in lactating cows.

PubMed

Comin, A; Peric, T; Montillo, M; Cappa, A; Marchi, V; Veronesi, M C; Prandi, A

2017-08-01

The aim of this research has been to evaluate the presence of anomalies in the ovarian cycle activity during postpartum and to verify whether 72-hr dietary fasting during the dominance phase, the phase before ovulation, might modify the ovarian follicle population. The presence of anomalies in ovarian cycle activity has been evaluated in 30 Italian Friesian cows starting from 20 days postpartum until 211 days of lactation. Long oestrus and brief dioestrus or scarce luteal activity have been the main anomalies found through measuring progesterone concentrations in the whey. Until 100 days of lactation, the BCS values of the problematic animals have been significantly lower than those in animals with normal ovarian activity. After 100 days of lactation, the ovarian anomalies continued to appear despite the fact that all the animals have reached comparable BCS values. Starting from the results of this trial, the effect of 72-hr dietary fasting on dominant follicles has been studied in six cows. Ultrasonography revealed that the diameter of the follicles at 71 days postpartum has been significantly lower than at 181 days. A 72-hr dietary restriction at 101 and 211 days postpartum did not affect the size of the dominant follicle. However, at 101 days postpartum, half of the animals presented follicular cysts. The effect of fasting differed if the animal has been in early postpartum or 211 days of lactation. Further researches are necessary to understand how different metabolic conditions can modify the follicular population but on the other hand the study shows the utility for farmers and field veterinarians of monitoring the resumption of the ovarian cycle postpartum through the whey progesterone concentrations. © 2017 Blackwell Verlag GmbH.

Equine ovarian tissue viability after cryopreservation and in vitro culture.

PubMed

Gastal, G D A; Aguiar, F L N; Alves, B G; Alves, K A; de Tarso, S G S; Ishak, G M; Cavinder, C A; Feugang, J M; Gastal, E L

2017-07-15

Ovarian tissue cryopreservation allows the preservation of the female fertility potential for an undetermined period. The objectives of this study were to compare the efficiency of cryoprotective agents (CPAs; dimethyl sulfoxide, DMSO; ethylene glycol, EG; and propylene glycol, PROH) using slow-freezing and vitrification methods, and evaluate the viability of cryopreserved equine ovarian tissue after 7 days of culture. Fresh and cryopreserved ovarian fragments were evaluated for preantral follicle morphology, stromal cell density, EGFR, Ki-67, Bax, and Bcl-2 protein expression, and DNA fragmentation. Vitrification with EG had the highest rate of morphologically normal preantral follicles, while DMSO had the lowest (76.1 ± 6.1% and 40.9 ± 14.8%, respectively; P < 0.05). In slow-freezing, despite that DMSO had the highest percentage of morphologically normal follicles (77.7 ± 5.8%), no difference among the CPAs was observed. Fluorescence intensity of EGFR and Ki-67 was greater when vitrification with EG was used. Regardless of the cryopreservation treatment, DMSO had the highest (P < 0.05) Bax/Bcl-2 ratio; however, DNA fragmentation was similar (P > 0.05) among treatments after thawing. After in vitro culture, the percentage of normal follicles was similar (P > 0.05) between slow-freezing and vitrification methods; however, vitrification had greater (P < 0.05) stromal cell density than slow-freezing. In summary, equine ovarian tissue was successfully cryopreserved, increasing the viability of the cells in the ovarian tissue after thawing when using DMSO and EG for slow-freezing and vitrification methods, respectively. Therefore, these results are relevant for fertility preservation programs. Copyright © 2017 Elsevier Inc. All rights reserved.

A monogamous pipefish has the same type of ovary as observed in monogamous seahorses.

PubMed

Sogabe, Atsushi; Matsumoto, Koji; Ohashi, Mizuki; Watanabe, Aki; Takata, Hiromi; Murakami, Yasunori; Omori, Koji; Yanagisawa, Yasunobu

2008-08-23

Syngnathid fish (pipefish and seahorses) are unique among teleost fish in that their ovary consists of a rolled sheet with germinal ridge(s) on the dorsal side running along the entire length of the sheet. A distinct difference is seen in the ovarian structure between polygamous Syngnathus pipefish and monogamous seahorses (Hippocampus spp.), the former having one germinal ridge and the latter with two ridges. This study examined the ovarian structure and the mode of egg production in a monogamous pipefish Corythoichthys haematopterus. The ovary of C. haematopterus had two germinal ridges like that observed in monogamous seahorses. There were two distinct groups of follicles in the ovary, one being a cohort of extremely small follicles and the other a cohort of follicles developing and increasing in size with the passage of time. We suggest that the ovarian structure and the mode of egg production in this pipefish are adaptations to monogamy.

Luteinizing hormone receptors in human ovarian follicles and corpora lutea during the menstrual cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamoto, M.; Nakano, R.; Iwasaki, M.

The binding of /sup 125/I-labeled human luteinizing hormone (hLH) to the 2000-g fraction of human ovarian follicles and corpora lutea during the entire menstrual cycle was examined. Specific high affinity, low capacity receptors for hLH were demonstrated in the 2000-g fraction of both follicles and corpora lutea. Specific binding of /sup 125/I-labeled hLH to follicular tissue increased from the early follicular phase to the ovulatory phase. Specific binding of /sup 125/I-labeled hLH to luteal tissue increased from the early luteal phase to the midluteal phase and decreased towards the late luteal phase. The results of the present study indicate thatmore » the increase and decrease in receptors for hLH during the menstrual cycle might play an important role in the regulation of the ovarian cycle.« less

ATP-binding cassette (ABC) transporters in caprine preantral follicles: gene and protein expression.

PubMed

Guerreiro, Denise Damasceno; de Lima, Laritza Ferreira; Mbemya, Gildas Tetaping; Maside, Carolina Mielgo; Miranda, André Marrocos; Tavares, Kaio César Simiano; Alves, Benner Geraldo; Faustino, Luciana Rocha; Smitz, Johan; de Figueiredo, José Ricardo; Rodrigues, Ana Paula Ribeiro

2018-06-01

The multidrug resistance proteins ABCB1, ABCC2 and ABCG2 are an energy-dependent efflux pump that functions in systemic detoxification processes. Physiologically expressed in a variety of tissues, most abundantly in the liver and intestinal epithelia, placenta, blood-brain barrier and various stem cells, until now, these pumps were not identified in goat ovarian tissue. Therefore, the aim of this study is to analyze ABCB1, ABCC2, and ABCG2 mRNA and protein expression in goat preantral follicles. Fragments (3 × 3 × 1 mm) from five pairs of ovary (n = 10) obtained from five goat were collected and immediately submitted to qPCR, Western blot, and immunofluorescence assay for mRNA detection and identification and localization of the ABC transporters, respectively. mRNA for ABCB1, ABCC2, and ABCG2 and the presence of their proteins were observed on ovarian tissue samples. Positive marks were observed for the three transport proteins in all follicular categories studied. However, the marks were primarily localized in the oocyte of primordial, transition and primary follicle categories. In conclusion, goat ovarian tissue expresses mRNA for the ABCB1, ABCC2 and ABCG2 transporters and the expression of these proteins in the preantral follicles is a follicle-dependent stage.

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) pre-exposure ensures follicle integrity during in vitro culture of ovarian tissue but not during cryopreservation in the domestic cat model.

PubMed

Tanpradit, Nae; Chatdarong, Kaywalee; Comizzoli, Pierre

2016-12-01

Temporary and reversible downregulation of metabolism may improve the survival of tissues exposed to non-physiological conditions during transport, in vitro culture, and cryopreservation. The objectives of the study were to (1) optimize the concentration and duration of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP-a mitochondrial uncoupling agent) exposures for biopsies of domestic cat ovarian tissue and (2) examine the effects of FCCP pre-exposures on follicle integrity after tissue culture and/or cryopreservation. Biopsies of cat ovarian tissue were first treated with various concentrations of FCCP (0, 10, 40, or 200 nM) for 10 or 120 min to determine the most suitable pre-exposure conditions. Based on these results, tissues were pre-exposed to 200 nM FCCP for 120 min for the subsequent studies on culture and cryopreservation. In all experiments and for each treatment group, tissue activity and integrity were measured by mitochondrial membrane potential (relative optical density of rhodamine 123 fluorescence), follicular viability (calcein assay), follicular morphology (histology), granulosa cell proliferation (Ki-67 immunostaining), and follicular density. Ovarian tissues incubated with 200 nM FCCP for 120 min led to the lowest mitochondrial activity (1.17 ± 0.09; P < 0.05) compared to control group (0 nM; 1.30 ± 0.12) while maintaining a constant percentage of viable follicles (75.3 ± 7.8 %) similar to the control group (71.8 ± 11.7 %; P > 0.05). After 2 days of in vitro culture, percentage of viable follicles (78.8 ± 8.9 %) in similar pre-exposure conditions was higher (P < 0.05) than in the absence of FCCP (61.2 ± 12.0 %) with percentages of morphologically normal follicles (57.6 ± 17.3 %) not different from the fresh tissue (70.2 ± 7.1 %; P > 0.05). Interestingly, percentages of cellular proliferation and follicular density were unaltered by the FCCP exposures. Based on the indicators mentioned above, the FCCP-treated tissue fragments did not have a better follicle integrity after freezing and thawing. Pre-exposure to 200 nM FCCP during 120 min protects and enhances the follicle integrity in cat ovarian tissue during short-term in vitro culture. However, FCCP does not appear to exert a beneficial or detrimental effect during ovarian tissue cryopreservation.

White spotting variant mouse as an experimental model for ovarian aging and menopausal biology.

PubMed

Smith, Elizabeth R; Yeasky, Toni; Wei, Jain Qin; Miki, Roberto A; Cai, Kathy Q; Smedberg, Jennifer L; Yang, Wan-Lin; Xu, Xiang-Xi

2012-05-01

Menopause is a unique phenomenon in modern women, as most mammalian species possess a reproductive period comparable with their life span. Menopause is caused by the depletion of germ cell-containing ovarian follicles and in laboratory studies is usually modeled in animals in which the ovarian function is removed through ovariectomy or chemical poisoning of the germ cells. Our objective was to explore and characterize the white spotting variant (Wv) mice that have reduced ovarian germ cell abundance, a result of a point mutation in the c-kit gene that decreases kinase activity, as a genetic model for use in menopause studies. Physiological and morphological features associated with menopause were determined in female Wv/Wv mice compared with age-matched wildtype controls. Immunohistochemistry was used to evaluate the presence and number of follicles in paraffin-embedded ovaries. Bone density and body composition were evaluated using the PIXImus x-ray densitometer, and lipids, calcium, and hormone levels were determined in serum using antigen-specific enzyme immunoassays. Heart and body weight were measured, and cardiac function was evaluated using transthoracic echocardiography. The ovaries of the Wv/Wv females have a greatly reduced number of normal germ cells at birth compared with wildtype mice. The remaining follicles are depleted by around 2 months, and the ovaries develop benign epithelial lesions that resemble morphological changes that occur during ovarian aging, whereas a normal mouse ovary has numerous follicles at all stages of development and retains some follicles even in advanced age. Wv mice have elevated plasma gonadotropins and reduced estrogen and progesterone levels, a significant reduction in bone mass density, and elevated serum cholesterol and lipoprotein levels. Moreover, the Wv female mice have enlarged hearts and reduced cardiac function. The reduction of c-kit activity in Wv mice leads to a substantially diminished follicular endowment in newborn mice and premature depletion of follicles in young mice, although mutant females have a normal life span after cessation of ovarian function. The Wv female mice exhibit consistent physiological changes that resemble common features of postmenopausal women. These alterations include follicle depletion, morphological aging of the ovary, altered serum levels of cholesterol, gonadotropins and steroid hormones, decreased bone density, and reduced cardiac function. These changes were not observed in male mice, either age-matched male Wv/Wv or wildtype mice, and are improbably caused by global loss of c-kit function. The Wv mouse may be a genetic, intact-ovary model that mimics closely the phenotypes of human menopause to be used for further studies to understand the mechanisms of menopausal biology.

Changes in the transcriptome of bovine ovarian cortex during follicle activation in vitro

PubMed Central

Yang, M. Y.

2015-01-01

The signals that regulate activation, a key transition in ovarian follicular development, are still not well understood, especially in nonrodent species. To gain insight into the regulation of this transition in cattle, we combined a microarray approach with an in vitro system in which ovarian cortical pieces cultured in control medium are enriched for primordial follicles, whereas pieces cultured with insulin are enriched for primary follicles. Total RNA was extracted from cultured cortical pieces, and then transcripts were identified and analyzed using the Affymetrix Bovine Genome GeneChip array. Around 65% of the transcripts in the bovine GeneChip were detected in cultured cortical pieces. Comparison between pieces cultured with or without insulin generated 158 differentially expressed transcripts. Compared with controls, 90 transcripts were upregulated and 68 were downregulated by insulin. These transcripts are involved in many biological processes and functions, but most are associated with cellular growth or cell cycle/cell death. The transcript encoding ubiquitin-conjugating enzyme E2C (UBE2C) was significantly upregulated during follicle activation, and Ingenuity Pathways Analysis revealed that UBE2C can interact with the tumor suppressor phosphatase and tensin homolog (PTEN). Both PTEN mRNA and protein were lower in cortical pieces cultured with insulin than in controls. In addition, FOXO3a, a downstream effector of PTEN signaling, underwent nuclear-cytoplasmic shuttling during primordial to primary follicle development in bovine fetal ovaries, further suggesting the involvement of the PTEN pathway in follicle activation in cattle. Genes and pathways identified in this study provide interesting candidates for further investigation of mechanisms underlying follicle activation. PMID:26443523

Characterization of folliculogenesis and the occurrence of apoptosis in the development of the bovine fetal ovary.

PubMed

Santos, S S D; Ferreira, M A P; Pinto, J A; Sampaio, R V; Carvalho, A C; Silva, T V G; Costa, N N; Cordeiro, M S; Miranda, M S; Ribeiro, H F L; Ohashi, O M

2013-01-15

The aim of this research was to perform in situ quantification, morphometry evaluation, and apoptosis analysis of ovarian follicular wall cells in mechanically isolated follicles obtained from ovaries of bovine fetuses (Bos taurus indicus) between 3 and 9 months of age. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The number of isolated follicles increased from 3 months onward (102.5 ± 141.1, mean ± SEM), peaked at 6 months (12855.0 ± 9030.1), and then decreased by 7 months (3208.7 ± 3249.5), consistent with atresia occurring at these stages. Follicular density was greatest at 4 months, consistent with a sudden boost in follicular activity independent of a corresponding increase in ovarian size. Antral follicles were first observed at 5 months. As fetal age increased, there was a tendency for the percentage of primordial and primary follicles to decrease, and the percentage of secondary follicles to increase. However, the high variability (P < 0.05) for all follicle populations up to 5 months of age precluded further interpretation of these results. Oocyte diameter increased from the primordial (23.6 ± 4.4 μm) to the secondary follicular stages (38.0 ± 14.9 μm). Apoptosis was observed in ovaries from all fetal ages analyzed. We concluded that preantral follicles could be isolated from bovine fetuses by 3 months of age, with apoptosis affecting ovarian follicular dynamics throughout fetal life. Copyright © 2013 Elsevier Inc. All rights reserved.

Di(2-ethylhexyl) phthalate inhibits antral follicle growth, induces atresia, and inhibits steroid hormone production in cultured mouse antral follicles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hannon, Patrick R., E-mail: phannon2@illinois.edu; Brannick, Katherine E., E-mail: kbran@illinois.edu; Wang, Wei, E-mail: Wei.Wang2@covance.com

Di(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental toxicant found in consumer products that causes ovarian toxicity. Antral follicles are the functional ovarian units and must undergo growth, survival from atresia, and proper regulation of steroidogenesis to ovulate and produce hormones. Previous studies have determined that DEHP inhibits antral follicle growth and decreases estradiol levels in vitro; however, the mechanism by which DEHP elicits these effects is unknown. The present study tested the hypothesis that DEHP directly alters regulators of the cell cycle, apoptosis, and steroidogenesis to inhibit antral follicle functionality. Antral follicles from adult CD-1 mice were cultured with vehiclemore » control or DEHP (1–100 μg/ml) for 24–96 h to establish the temporal effects of DEHP on the follicle. Following 24–96 h of culture, antral follicles were subjected to gene expression analysis, and media were subjected to measurements of hormone levels. DEHP increased the mRNA levels of cyclin D2, cyclin dependent kinase 4, cyclin E1, cyclin A2, and cyclin B1 and decreased the levels of cyclin-dependent kinase inhibitor 1A prior to growth inhibition. Additionally, DEHP increased the mRNA levels of BCL2-associated agonist of cell death, BCL2-associated X protein, BCL2-related ovarian killer protein, B-cell leukemia/lymphoma 2, and Bcl2-like 10, leading to an increase in atresia. Further, DEHP decreased the levels of progesterone, androstenedione, and testosterone prior to the decrease in estradiol levels, with decreased mRNA levels of side-chain cleavage, 17α-hydroxylase-17,20-desmolase, 17β-hydroxysteroid dehydrogenase, and aromatase. Collectively, DEHP directly alters antral follicle functionality by inhibiting growth, inducing atresia, and inhibiting steroidogenesis. - Highlights: • DEHP inhibits antral follicle growth by dysregulating cell cycle regulators. • DEHP induces antral follicle atresia by dysregulating apoptosis regulators. • DEHP inhibits the production of antral follicle produced sex steroid hormones.« less

Relationship of ovarian stimulation response with vascular endothelial growth factor and degree of granulosa cell apoptosis.

PubMed

Quintana, R; Kopcow, L; Marconi, G; Sueldo, C; Speranza, G; Barañao, R I

2001-09-01

The aim of this study was to evaluate the concentration of vascular endothelial growth factor (VEGF) in follicular fluid and in granulosa cell cultures in relation to the degree of apoptosis in granulosa cells from patients with different types of ovarian response to controlled ovarian hyperstimulation. We studied 30 women who underwent controlled ovarian hyperstimulation and oocyte retrieval. Group A comprised patients with 1-4 follicles (n = 10), group B patients with 5-14 follicles (n = 10) and group C patients with >15 follicles (n = 10). Mean (+/-SD) VEGF concentrations in follicular fluid were 1232 +/- 209, 813 +/- 198 and 396 +/- 103 pg/ml for groups A, B and C respectively (P > 0.01). Concentrations of VEGF in granulosa cell supernatant were 684 +/- 316, 1101 +/- 295 and 1596 +/- 227 pg/ml respectively (P < 0.05). Percentages of apoptotic cells in granulosa cells culture was 55.02 +/- 7.5, 23.98 +/- 4.4 and 14.2 +/- 2.3% respectively (A versus B, P < 0.01, A versus C, P < 0.006, B versus C, NS). Our findings showed that in patients with decreased ovarian response to controlled ovarian hyperstimulation, follicular fluid VEGF concentration is elevated, the concentration from granulosa cells culture supernatant is decreased and the percentage of apoptotic granulosa cells is increased, while opposite findings occurred in patients with normal or hyper-responses.

In vitro re-expression of the aryl hydrocarbon receptor (Ahr) in cultured Ahr-deficient mouse antral follicles partially restores the phenotype to that of cultured wild-type mouse follicles.

PubMed

Ziv-Gal, A; Gao, L; Karman, B N; Flaws, J A

2015-03-01

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of various endocrine disrupting chemicals. In female mice, global deletion of the Ahr (AhrKO) results in slow growth of ovarian antral follicles. No studies, however, have examined whether injection of the Ahr restores the phenotypes of cultured AhrKO ovarian antral follicles to wild-type levels. We developed a system to construct a recombinant adenovirus containing the Ahr to re-express the Ahr in AhrKO granulosa cells and whole antral follicles. We then compared follicle growth and levels of factors in the AHR signaling pathway (Ahr, Ahrr, Cyp1a1, and Cyp1b1) in wild-type, AhrKO, and Ahr re-expressed follicles. Further, we compared the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in wild-type, AhrKO, and Ahr re-expressed follicles. Ahr injection into AhrKO follicles partially restored their growth pattern to wild-type levels. Further, Ahr re-expressed follicles had significantly higher levels of Ahr, Ahrr, Cyp1a1, and Cyp1b1 compared to wild-type follicles. Upon TCDD treatment, only Cyp1a1 levels were significantly higher in Ahr re-expressed follicles compared to the levels in wild-type follicles. Our system of re-expression of the Ahr partially restores follicle growth and transcript levels of factors in the AHR signaling pathway to wild-type levels. Copyright © 2014 Elsevier Ltd. All rights reserved.

Ovarian cysts

MedlinePlus

... trying to get pregnant and you often get functional cysts, you can prevent them by taking hormone drugs (such as birth control pills). These medicines prevent follicles from growing. Alternative Names Physiologic ovarian ...

Ovarian parameters and ovarian blood flow of women living in the area of environmental crisis.

PubMed

Balmagambetova, Aru; Abdelazim, Ibrahim A; Bekmukhambetov, Erbol; Zhurabekova, Gulmira; Yehia, Amr H; AbuFaza, Mohannad

2016-05-01

Exposure to environmental hazards will destroy a number of ovarian primordial follicles, reduce ovarian reserve and subsequent reproductive ability. This study designed to evaluate ovarian parameters and ovarian blood flow of women living in the area of environmental crisis Shalkar city (Kazakhstan) compared to women living in Aktobe city (Kazakhstan). 220 women in their reproductive age studied and classified into two groups; study (Shalkar) group and control (Aktobe) group. Blood sample taken from studied women during follicular phase (day 3) for hormonal level evaluation including; follicle stimulating hormone (FSH) and anti-Mullerian hormone (AMH). Studied women evaluated using trans-vaginal ultrasound (TVS) to detect antral follicle count (AFC) during follicular scan and ovarian volume (OV), ovarian blood flow (OBF) using pulsatility index (PI) during follicular scan and luteal scan. Both ovaries AFC was significantly less in study (Shalkar) group compared to and control (Aktobe) group (p=0.0001). Mean ovarian volume was significantly less in Shalkar group in both follicular phase and luteal phase (5.86±0.23 and 6.19±0.22Cm(3); respectively) compared to Aktobe group (6.85±0.19 and 6.92±0.18Cm(3); respectively). In addition, mean ovarian pulsatility index was significantly high with subsequent decrease in ovarian blood flow in Shalkar group in both follicular phase and luteal phase (3.36±0.20 and 3.45±0.19Cm/s; respectively) compared to Aktobe group (2.96±0.16 and 2.92±0.15Cm/s; respectively). This study suggests definite environmental effect on ovarian parameters as indicated by decreased AFC, decreased both follicular and luteal OV and OBF in women living in environmental crisis Shalkar group compared to Aktobe group. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Should we isolate human preantral follicles before or after cryopreservation of ovarian tissue?

PubMed

Vanacker, Julie; Luyckx, Valérie; Amorim, Christiani; Dolmans, Marie-Madeleine; Van Langendonckt, Anne; Donnez, Jacques; Camboni, Alessandra

2013-04-01

To evaluate the survival and growth potential of human preantral follicles isolated before and after cryopreservation. Pilot study. Gynecology research unit in a university hospital. Six women aged 27 to 32 years. Six ovarian biopsy samples were cut into two equal parts, half subjected to slow-freezing followed by follicle isolation (cryo-iso group) and alginate-matrigel embedding, and half immediately processed for follicle isolation and alginate-matrigel embedding followed by slow-freezing (iso-cryo group) or used as fresh controls (fresh group). Follicle number, viability, diameter, and morphology. After 1,134 preantral follicles had been isolated from fresh biopsy samples and 1,132 from frozen specimens, the three groups were compared before and after 7 days of in vitro culture (IVC) in alginate-matrigel beads. No statistically significant differences in viability were found between the three groups before or after IVC, but follicle diameter increased in all three groups after IVC. Morphology analysis revealed well-preserved follicles in both the iso-cryo and cryo-iso groups after IVC. Human preantral follicles can be successfully cryopreserved before or after isolation without impairing their ability to survive and grow in vitro. This could lead to development of new protocols for follicle cryopreservation, IVC, and grafting in clinical and research settings for fertility preservation. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effect of turmeric on the viability, ovarian folliculogenesis, fecundity, ovarian hormones and response to luteinizing hormone of rabbits.

PubMed

Sirotkin, A V; Kadasi, A; Stochmalova, A; Balazi, A; Földesiová, M; Makovicky, P; Chrenek, P; Harrath, A H

2018-06-01

The present study investigated whether dietary turmeric (Curcuma longa L.) can improve rabbit reproduction, ovarian function, growth, or viability. Female New Zealand White rabbits were either fed a standard diet (n=15) or a diet enriched with 5 g (group E1) or 20 g (group E2) turmeric powder per 100 kg feed mixture (n=16 or 15, respectively). After 295 days, weight gain, conception and kindling rates, pup and mother viability, ovarian macro- and micro-morphometric indices, release of leptin in response to the addition LH, and the release of progesterone, testosterone and leptin by isolated ovarian fragments were analyzed. Dietary turmeric failed to affect ovarian length and weight but did increase the number of primary follicles (E2: 32.5% greater than control group), as well as the diameter of primary (E1: +19.4%, E2: +21.1%), secondary (E2: +41.4%), and tertiary (E1: +97.1%, E2: +205.1%) follicles. Turmeric also increased the number of liveborn (E1: +21.0%) and weaned (E1: +25.0%) pups and decreased the number of stillborn pups (E2: -87.5%) but did not affect weight gain, conception, or kindling rate. Furthermore, dietary turmeric decreased doe mortality during the first reproductive cycle (13.3% in control; 0% in E1; and 6.7% in E2) but not during the second cycle. In vitro, the ovaries of the turmeric-treated rabbits released more progesterone (E1: +85.7%, E2: +90.0%) and less testosterone (E2: -87.0%) and leptin (E2: -29.0%) than the ovaries of control rabbits. Moreover, LH decreased the leptin output of control rabbits but increased that of experimental rabbits. Therefore, it is likely that dietary turmeric improves pup viability and that it could promote rabbit fecundity by either (1) promoting the production of primary ovarian follicles or (2) stimulating the growth of follicles at all stages of folliculogenesis.

Human umbilical cord mesenchymal stem cells improve the reserve function of perimenopausal ovary via a paracrine mechanism.

PubMed

Li, Jia; Mao, QiuXian; He, JingJun; She, HaoQing; Zhang, Zhi; Yin, ChunYan

2017-03-09

Human umbilical cord mesenchymal stem cells (hUCMSCs) are a type of pluripotent stem cell which are isolated from the umbilical cord of newborns. hUCMSCs have great therapeutic potential. We designed this experimental study in order to investigate whether the transplantation of hUCMSCs can improve the ovarian reserve function of perimenopausal rats and delay ovarian senescence. We selected naturally aging rats confirmed by vaginal smears as models of perimenopausal rats, divided into the control group and the treatment group, and selected young fertile female rats as normal controls. hUCMSCs were transplanted into rats of the treatment group through tail veins. Enzyme-linked immunosorbent assay (ELISA) detected serum levels of sex hormones, H&E staining showed ovarian tissue structure and allowed follicle counting, immunohistochemistry and western blot analysis revealed ovarian expression of hepatocyte growth factor (HGF), vascular endothelial cell growth factor (VEGF), and insulin-like growth factor-1 (IGF-1), polymerase chain reaction (PCR) and western blot analysis revealed hUCMSCs expression of HGF, VEGF, and IGF-1. At time points of 14, 21, and 28 days after hUCMSCs transplantation, estradiol (E 2 ) and anti-Müllerian hormone (AMH) increased while follicle-stimulating hormone (FSH) decreased; ovarian structure improved and follicle number increased; ovarian expression of HGF, VEGF, and IGF-1 protein elevated significantly. Meanwhile, PCR and western blot analysis indicated hUCMSCs have the capacity of secreting HGF, VEGF, and IGF-1 cytokines. Our results suggest that hUCMSCs can promote ovarian expression of HGF, VEGF, and IGF-1 through secreting those cytokines, resulting in improving ovarian reserve function and withstanding ovarian senescence.

Cryopreservation of ovarian tissue for fertility preservation in young female oncological patients.

PubMed

Andersen, Claus Yding; Kristensen, Stine Gry; Greve, Tine; Schmidt, Kirsten Tryde

2012-05-01

Girls and women suffering from a cancer that requires treatment with gonadotoxic drugs may experience cessation of reproductive function as a side effect due to obliteration of the ovarian pool of follicles. Techniques are now available for fertility preservation, such as cryopreservation of mature oocytes, embryos or ovarian cortical tissue. Whereas collection of mature oocytes and embryos requires at least a 2-week period, ovarian tissue may on short notice be frozen prior to treatment and can be transplanted back into women with ovarian failure. Transplanted frozen/thawed tissue supports survival and growth of follicles, giving rise to menstrual cycles and hormone production for several years. Worldwide, the procedure has resulted in the birth of 15 healthy children. Many cancer patients including girls and young women want fertility preservation, and the techniques are now being further developed and implemented in several centers.

Coexpression of the Follicle Stimulating Hormone Receptor and Stem Cell Markers: A Novel Approach to Target Ovarian Cancer Stem Cells

DTIC Science & Technology

2016-03-01

1 AD_____________ Award Number: W81XWH-11-1-0623 TITLE: Coexpression of the Follicle Stimulating Hormone Receptor and Stem Cell Markers: A...Novel Approach to Target Ovarian Cancer Stem Cells PRINCIPAL INVESTIGATOR: David W. Schomberg, PhD CONTRACTING ORGANIZATION: Duke University...Durham, NC 27705 REPORT DATE: March 2016 TYPE OF REPORT: Final PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick

Comparisons of mRNA expression for insulin-like growth factor (IGF) type 2 receptor (IGF2R) and IGF-1 in small ovarian follicles between cattle selected and not selected for twin ovulations

USDA-ARS?s Scientific Manuscript database

Both IGF-1 and -2 stimulate ovarian follicular cell proliferation and antral follicle development. Actions of IGF-1 and -2 are mediated through the IGF type 1 receptor, whereas binding of IGF-2 to the IGF2R results in its degradation. Information on the role of IGF2R in regulating bovine follicula...

Plasma luteinizing hormone and the development of ovarian follicles after loss of clutch in female mallards (Anas platyrhynchos)

USGS Publications Warehouse

Donham, R.S.; Dane, C.W.; Farner, D.S.

1976-01-01

The plasma level of LH and the extent of development of ovarian follicles were analyzed in incubating female Mallards. In both wild and game-farm stock, incubation was associated with a significant decline in plasma levels of LH from those of laying females. Within 1 day after removal of eggs, LH levels had increased to levels indistinguishable from those of laying females. The mean diameter of the largest follicle in wild females on the tenth day of incubation was 5.3 mm; it was 5.2 mm in game-farm stock at the same stage. Three days following removal of eggs, the mean of the largest follicles of wild-stock hens had increased to 14.0 mm and those of game-farm stock to 12.7 mm.

Kisspeptin level in the aging ovary is regulated by the sympathetic nervous system.

PubMed

Fernandois, Daniela; Cruz, Gonzalo; Na, Eun Kyung; Lara, Hernán E; Paredes, Alfonso H

2017-01-01

Previous work has demonstrated that the increase in the activity of sympathetic nerves, which occurs during the subfertility period in female rats, causes an increase in follicular cyst development and impairs follicular development. In addition, the increase in ovarian sympathetic activity of aged rats correlates with an increased expression of kisspeptin (KISS1) in the ovary. This increase in KISS1 could participate in the decrease in follicular development that occurs during the subfertility period. We aimed to determine whether the blockade of ovarian sympathetic tone prevents the increase in KISS1 expression during reproductive aging and improves follicular development. We performed 2 experiments in rats: (1) an in vivo blockade of beta-adrenergic receptor with propranolol (5.0 mg/kg) and (2) an ovarian surgical denervation to modulate the sympathetic system at these ages. We measured Kisspeptin and follicle-stimulating hormone receptor (FSHR) mRNA and protein levels by qRT-PCR and western blot and counted primordial, primary and secondary follicles at 8, 10 and 12 months of age. The results showed that ovarian KISS1 decreased but FSHR increased after both propranolol administration and the surgical denervation in rats of 8, 10 and 12 months of age. An increase in FSHR was related to an increase in the number of smaller secondary follicles and a decreased number of primordial follicles at 8, 10 and 12 months of age. These results suggest that intraovarian KISS1 is regulated by sympathetic nerves via a beta-adrenergic receptor and participates locally in ovarian follicular development in reproductive aging. © 2017 Society for Endocrinology.

Effects of repeated transvaginal aspiration of immature follicles on mare health and ovarian status.

PubMed

Velez, I C; Arnold, C; Jacobson, C C; Norris, J D; Choi, Y H; Edwards, J F; Hayden, S S; Hinrichs, K

2012-12-01

Transvaginal ultrasound-guided follicle aspiration (TVA) is performed clinically but there is little information available on complications associated with this procedure. It is possible that TVA is associated with damage to the ovary and may induce peritonitis or peritoneal adhesions. This study was conducted to determine the effect of repeated TVA on mare health and ovarian status. Thirty-two mares were used for oocyte recovery via repeated TVA over a 3 year period; different mares were used each year. In Year 1, ovarian status was monitored in 11 mares by transrectal palpation and ultrasonography. In Year 2, 6 of 11 mares underwent abdominocentesis and were examined by laparoscopy after one TVA and again after multiple TVAs. In Year 3, 10 mares underwent multiple TVAs with either a 15 or a 12 gauge needle and the ovaries were removed for examination. Four hundred and twenty-seven aspiration sessions (390 via TVA and 37 via needle placement through the flank) and 3202 follicle punctures (3161 TVA and 41 flank) were performed. One mare developed an ovarian abscess. Transient rectal bleeding was evident after 16% of TVA sessions. No adhesions were found on laparoscopic or gross examination of ovaries and there were minimal changes on histological evaluation. Follicle aspiration carries a small possibility (< 0.5%) of ovarian abscess formation. There is a possibility of rectal abrasion or puncture but little gross or histological damage to the ovary. These results provide a basis for using prophylactic administration of antibiotics after TVA and for advising mare owners of the rare but potential complications associated with the procedure.

Cadmium exposure in newborn rats ovary induces developmental disorders of primordial follicles and the differential expression of SCF/c-kit gene.

PubMed

Zhang, Wenchang; Wu, Tingting; Zhang, Chenyun; Luo, Lingfeng; Xie, Meimei; Huang, Huiling

2017-10-05

Since the 1990s, the rising problem that gonad reproductive toxicity on adult female after exposing to cadmium (Cd), an environmental endocrine disruptor, has attracted high attention at home and abroad,and was systematically studied. Our research focuses on a further problem is that early cadmium exposure (during birth to before puberty) impact on development and function of ovarian cells and its possible mechanism. Our research focuses on the changes of ovarian cells growth and development after the newborn rat ovaries with cadmium exposure in vitro, and different expression of ovarian cells development-related factors, SCF/c-kit and changes of their DNA methylation status. We obtained ovaries from 4-day-old SD rats and cultured them in DMEM/F12 mixed with α-MEM media in vitro. Different doses of cadmium were designed as control, 0.5, 5, 10 and 50μM, and then the constituent ratio of ovarian follicle and follicular oocytes diameter were observed with microscope after 4-h exposure. We found that the increased constituent ratio of original follicle and decreased diameter of all levels of follicular oocytes(compared with control, with statistically significant differences, P<0.01).After the measurement of expression of SCF/c-kit by qRT-PCR and Western Blotting, the mRNA and protein expression of SCF/c-kit in ovarian were both decreased. We further found that the increased constituent ratio of growth follicle and increased diameter of oocytes under the treatment of adding SCF in cell culture media. Finally, MALDI-TOF-MS method showed DNA-low methylation status of SCF/c-kit promoter region after Cd exposure. Overall, we concluded that the exposure of cadimium (5-50μM) on newborn rats ovaries could inhibit follicle development.SCF/c-kit system might mediate follicle development damage caused by cadmium, which is associated with DNA hypomethylation of SCF/c-kit promoter region may be worthy of further study. Copyright © 2017. Published by Elsevier B.V.

Androgen receptor-mediated non-genomic effects of vinclozolin on porcine ovarian follicles and isolated granulosa cells: Vinclozolin and non-genomic effects in porcine ovarian follicles.

PubMed

Wartalski, Kamil; Knet-Seweryn, Malgorzata; Hoja-Lukowicz, Dorota; Tabarowski, Zbigniew; Duda, Malgorzata

2016-05-01

The present study investigated the influence of the androgen receptor (AR) agonists testosterone (T) and dihydrotestosterone (DHT), and vinclozolin (Vnz), a fungicide with antiandrogenic activity, on non-genomic signal transduction within ovarian follicles. Porcine granulosa cells (GCs) isolated from mature follicles were cultured for 48h. For the last 24h of culture, they were exposed to T (10(-7)M), DHT (10(-7)M), Vnz (1.4×10(-5)M), T and Vnz (T+Vnz), or DHT and Vnz (DHT+Vnz) at the same concentrations. To better imitate in vivo conditions, whole follicles (4-6mm in diameter) were incubated (24h) in an organ culture system with the same factors. Expression of AR mRNA and protein was determined by real-time PCR and western blot analyses. To demonstrate AR localization in cultured GCs and whole follicles, immunocytochemistry and immunohistochemistry were performed, respectively. To elucidate the possible non-genomic action of Vnz in GCs, protein expression and the activity of ERK1/2 and Akt kinases were determined by western blot and ELISA analyses. The immunocytochemistry and immunohistochemistry results showed that exposure of GCs and follicles to Vnz resulted in cytoplasmic and perinuclear AR localization. Real-time PCR and western blot analysis showed that AR mRNA and protein expression increased (P≤0.001) in GC cultures after combined treatment with an androgen and Vnz. In whole follicles, such treatment also increased AR mRNA with a decrease in the respective protein expression (P≤0.001). Moreover, addition of T or DHT with Vnz increased the activity of ERK1/2 and Akt kinases in cultured GCs (P≤0.001). The results suggest a novel mechanism for Vnz action in porcine ovarian follicles on both AR mRNA and protein levels. Thus, this environmental antiandrogen activates non-genomic signaling pathways, as indicated by the increased activity of both investigated kinases observed within minutes of Vnz addition. Given the widespread presence of Vnz in the environment, elucidation of its non-genomic action should be the subject of studies on female fertility. Copyright © 2016 Elsevier GmbH. All rights reserved.

The effects on ovarian activity of a monophasic oral contraceptive with 100 microg levonorgestrel and 20 microg ethinyl estradiol.

PubMed

Coney, P; DelConte, A

1999-11-01

An open-label, single-center, noncomparative study was conducted to determine the effects of a monophasic oral contraceptive containing 100 microg levonorgestrel and 20 microg ethinyl estradiol on ovarian activity. The subjects were 26 healthy women 20 to 35 years of age who had normal ovulatory cycles and were not at risk for becoming pregnant. For 3 treatment cycles, they took 1 tablet of active drug daily for 21 days followed by placebo tablets for 7 days. Follicle diameters and serum progesterone and 17beta-estradiol levels were measured before, during, and after treatment. In 2 (2.7%) of 73 cycles, luteinized unruptured follicles were present and in another 2 (2.7%) cycles, ovulation was confirmed by the disappearance of the enlarged follicle. Ovarian activity, as reflected by mean serum progesterone levels, was restored after treatment. The results of this study are in agreement with those of other studies that showed suppression of ovarian activity in women treated with a monophasic oral contraceptive containing 100 microg levonorgestrel and 20 microg ethinyl estradiol. These results indicate that low-dose 100 microg levonorgestrel and 20 microg ethinyl estradiol given for 21 days is effective in suppressing ovarian activity and they confirm the contraceptive efficacy observed in clinical trials (Pearl index of 0.8).

Equol inhibits growth, induces atresia, and inhibits steroidogenesis of mouse antral follicles in vitro

PubMed Central

Mahalingam, Sharada; Gao, Liying; Gonnering, Marni; Helferich, William; Flaws, Jodi A.

2016-01-01

Equol is a non-steroidal estrogen metabolite produced by microbial conversion of daidzein, a major soy isoflavone, in the gut of some humans and many animal species. Isoflavones and their metabolites can affect endogenous estradiol production, action, and metabolism, potentially influencing ovarian follicle function. However, no studies have examined the effects of equol on intact ovarian antral follicles, which are responsible for sex steroid synthesis and further development into ovulatory follicles. Thus, the present study tested the hypothesis that equol inhibits antral follicle growth, increases follicle atresia, and inhibits steroidogenesis in the adult mouse ovary. To test this hypothesis, antral follicles isolated from adult CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or equol (600 nM, 6 μM, 36 μM, 100 μM) for 48 and 96 h. Every 24 h, follicle diameters were measured to monitor growth. At 48 and 96 h, the culture medium was subjected to measurement of hormone levels, and the cultured follicles were subjected to gene expression analysis. Additionally, follicles were histologically evaluated for signs of atresia after 96 h of culture. The results indicate that equol (100 μM) inhibited follicle growth, altered the mRNA levels of bcl2-associated X protein and B cell leukemia/lymphoma 2, and induced follicle atresia. Further, equol decreased the levels of estradiol, testosterone, androstenedione, and progesterone, and it decreased mRNA levels of cholesterol side-chain cleavage, steroid 17-α-hydroxalase, and aromatase. Collectively, these data indicate that equol inhibits growth, increases atresia, and inhibits steroidogenesis of cultured mouse antral follicles. PMID:26876617

Niv versus dropping vitrification in cryopreservation of human ovarian tissue.

PubMed

Xiao, Z; Li, S W; Zhang, Y Y; Wang, Y; Li, L L; Fan, W

2014-01-01

The containers for vitrification of tissues include cryovials, copper grids, Pasteur pipettes, the solid-surface method and etc. Recently the acupuncture needle was used to achieve better result in vitrification of human ovarian tissue. To determine if the needle immersed vitrification method (NIV) is a promising approach to vitrify the human ovarian tissue. Human ovarian biopsies from five patients were vitrified using NIV and Dropping vitrification. After 14 days of in vitro culture, the incidence of apoptotic primordial follicles from fresh and vitrified groups was assessed by TUNEL assay. 17β-estradiol (E2) and progesterone (P4) were detected in the media after culturing of vitrified and fresh ovarian tissues. The incidence of apoptotic primordial follicles was significantly higher in the dropping vitrification group than in the NIV group (P < 0.05). E2 and P4 concentrations were significantly higher in NIV groups than in Dropping vitrification group (P < 0.05). NIV was an appropriate method to vitrify ovarian tissue by improving the growth potential of frozen-warmed ovarian tissue in vitro culture.

Histological and transcriptomic effects of 17α-methyltestosterone on zebrafish gonad development.

PubMed

Lee, Stephanie Ling Jie; Horsfield, Julia A; Black, Michael A; Rutherford, Kim; Fisher, Amanda; Gemmell, Neil J

2017-07-24

Sex hormones play important roles in teleost ovarian and testicular development. In zebrafish, ovarian differentiation appears to be dictated by an oocyte-derived signal via Cyp19a1a aromatase-mediated estrogen production. Androgens and aromatase inhibitors can induce female-to-male sex reversal, however, the mechanisms underlying gonadal masculinisation are poorly understood. We used histological analyses together with RNA sequencing to characterise zebrafish gonadal transcriptomes and investigate the effects of 17α-methyltestosterone on gonadal differentiation. At a morphological level, 17α-methyltestosterone (MT) masculinised gonads and accelerated spermatogenesis, and these changes were paralleled in masculinisation and de-feminisation of gonadal transcriptomes. MT treatment upregulated expression of genes involved in male sex determination and differentiation (amh, dmrt1, gsdf and wt1a) and those involved in 11-oxygenated androgen production (cyp11c1 and hsd11b2). It also repressed expression of ovarian development and folliculogenesis genes (bmp15, gdf9, figla, zp2.1 and zp3b). Furthermore, MT treatment altered epigenetic modification of histones in zebrafish gonads. Contrary to expectations, higher levels of cyp19a1a or foxl2 expression in control ovaries compared to MT-treated testes and control testes were not statistically significant during early gonad development (40 dpf). Our study suggests that both androgen production and aromatase inhibition are important for androgen-induced gonadal masculinisation and natural testicular differentiation in zebrafish.

Developmental programming: prenatal testosterone excess disrupts anti-Müllerian hormone expression in preantral and antral follicles.

PubMed

Veiga-Lopez, Almudena; Ye, Wen; Padmanabhan, Vasantha

2012-03-01

To investigate the impact of prenatal T excess on the expression of key ovarian regulators implicated in follicular recruitment and persistence using a large animal model of polycystic ovarian syndrome (PCOS). Interventional, animal model study. Academic research unit. A total of 25 female fetuses, 14 prepubertal female, and 24 adult female Suffolk sheep. Prenatal T treatment. Immunohistochemical determination of expression of anti-Müllerian hormone (AMH), kit ligand, and growth differentiation factor 9 (GDF9) in fetal, prepubertal, and adult ovarian tissues. Prenatal T treatment reduced the AMH protein expression in granulosa cells (GC) of preantral follicles and increased its expression in antral follicles compared with age-matched adult controls. These differences were not evident in prepubertal animals. Protein expression of GDF9 and kit ligand was not altered at any of the developmental time points studied. Prenatal T exposure is associated with changes in AMH expression in preantral and antral follicles in adult ovaries, similar to findings in women with PCOS. These findings indicate that abnormal folliculogenesis in PCOS may be at least in part mediated by changes in AMH expression. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

S100A8, An Oocyte-Specific Chemokine, Directs the Migration of Ovarian Somatic Cells During Mouse Primordial Follicle Assembly.

PubMed

Teng, Zhen; Wang, Chao; Wang, Yijing; Huang, Kun; Xiang, Xi; Niu, Wanbao; Feng, Lizhao; Zhao, Lihua; Yan, Hao; Zhang, Hua; Xia, Guoliang

2015-12-01

In the mammalian ovaries, the primordial follicle pool determines the reproductive capability over the lifetime of a female. The primordial follicle is composed of two cell members, namely the oocyte and the pre-granulosa cells that encircle the oocyte. However, it is unclear what factors are involved in the reorganization of the two distinct cells into one functional unit. This study was performed to address this issue. Firstly, in an in vitro reconstruction system, dispersed ovarian cells from murine fetal ovaries at 19.0 days post coitum (dpc) reassembled into follicle-like structures, independent of the physical distance between the cells, implying that either oocytes or ovarian somatic cells (OSCs) were motile. We then carried out a series of transwell assay experiments, and determined that it was in fact 19.0 dpc OSCs (as opposed to oocytes), which exhibited a significant chemotactic response to both fetal bovine serum and oocytes themselves. We observed that S100A8, a multi-functional chemokine, may participate in the process as it is mainly expressed in oocytes within the cysts/plasmodia. S100A8 significantly promoted the number of migrating OSCs by 2.5 times in vitro, of which 66.9% were FOXL2 protein-positive cells, implying that the majority of motile OSCs were pre-granulosa cells. In addition, an S100A8-specific antibody inhibited the formation of follicle-like reconstruction cell mass in vitro. And, the primordial follicle formation was reduced when S100a8-specific siRNA was applied onto in vitro cultured 17.5 dpc ovary. Therefore, S100A8 could be a chemokine of oocyte origin, which attracts OSCs to form the primordial follicles. © 2015 Wiley Periodicals, Inc.

Gap junctions are essential for murine primordial follicle assembly immediately before birth.

PubMed

Teng, Zhen; Wang, Chao; Wang, Yijing; Huang, Kun; Xiang, Xi; Niu, Wanbao; Feng, Lizhao; Zhao, Lihua; Yan, Hao; Zhang, Hua

2016-02-01

The reserve of primordial follicles determines the reproductive ability of the female mammal over its reproductive life. The primordial follicle is composed of two types of cells: oocytes and surrounding pre-granulosa cells. However, the underlying mechanism regulating primordial follicle assembly is largely undefined. In this study, we found that gap junction communication (GJC) established between the ovarian cells in the perinatal mouse ovary may be involved in the process. First, gap junction structures between the oocyte and surrounding pre-granulosa cells appear at about 19.0 dpc (days post coitum). As many as 12 gap junction-related genes are upregulated at birth, implying that a complex communication may exist between ovarian cells, because specifically silencing the genes of individual gap junction proteins, such as Gja1, Gja4 or both, has no influence on primordial follicle assembly. On the other hand, non-specific blockers of GJC, such as carbenoxolone (CBX) and 18α-glycyrrhetinic acid (AGA), significantly inhibit mouse primordial follicle assembly. We proved that the temporal window for establishment of GJC in the fetal ovary is from 19.5 dpc to 1 dpp (days postpartum). In addition, the expression of ovarian somatic cell (OSC)-specific genes, such as Notch2, Foxl2 and Irx3, was negatively affected by GJC blockers, whereas oocyte-related genes, such as Ybx2, Nobox and Sohlh1, were hardly affected, implying that the establishment of GJC during this period may be more important to OSCs than to oocytes. In summary, our results indicated that GJC involves in the mouse primordial follicle assembly process at a specific temporal window that needs Notch signaling cross-talking. © 2016 Society for Reproduction and Fertility.

Seasonal morphological changes in the ovary of the Jungle crow (Corvus macrorhynchos).

PubMed

Islam, Muhammad Nazrul; Zhu, Xiao Bo; Aoyama, Masato; Sugita, Shoei

2010-12-01

Morphometric and histological studies were conducted to examine the seasonal ovarian changes in the Jungle crow of the Kanto area, Japan, from December to June. The ovary weights, largest diameters and atresias of the ovarian follicles and steroid-producing cells were examined. Hematoxylin and eosin-stained ovary sections and ImageJ software were used. The most developed ovary weight increased 373-fold in April, compared to those in December, followed by a 29-fold decrease in June. The average largest follicle diameter of the December and the January ovaries were 1.03 ± 0.35 and 1.05 ± 0.3 mm, respectively. The average largest follicle diameter increased by 2-fold in February, 4-fold in March and 8-fold in April, compared to those of December and January. Thereafter, the average largest follicle diameter declined by 6-fold in June. The average ovary weight and the largest follicle diameter in April increased significantly (P < 0.05) compared to those of December and January, followed by a significant decrease (P < 0.05) in June. The ovary weight correlated well with the expansion of the largest follicular diameter. Non-bursting and bursting atresias of smaller follicles were more common in the December, January, February and June ovaries, and bursting atresias of larger follicles were more common in the March, April and May ovaries. Ovarian steroidogenic cells became heavily charged with lipids in December, January, February and June, and they depleted their lipids in March and April, which might be due to steroid synthesis. Our results indicate that there are significant seasonal histomorphologic variations in the Jungle crow ovary.

The role of the PI3K-Akt signaling pathway in the developmental competence of bovine oocytes.

PubMed

Andrade, Gabriella Mamede; da Silveira, Juliano Coelho; Perrini, Claudia; Del Collado, Maite; Gebremedhn, Samuel; Tesfaye, Dawit; Meirelles, Flávio Vieira; Perecin, Felipe

2017-01-01

The ovarian follicle encloses oocytes in a microenvironment throughout their growth and acquisition of competence. Evidence suggests a dynamic interplay among follicular cells and oocytes, since they are constantly exchanging "messages". We dissected bovine ovarian follicles and recovered follicular cells (FCs-granulosa and cumulus cells) and cumulus-oocyte complexes (COCs) to investigate whether the PI3K-Akt signaling pathway impacted oocyte quality. Following follicle rupture, COCs were individually selected for in vitro cultures to track the follicular cells based on oocyte competence to reach the blastocyst stage after parthenogenetic activation. Levels of PI3K-Akt signaling pathway components in FCs correlated with oocyte competence. This pathway is upregulated in FCs from follicles with high-quality oocytes that are able to reach the blastocyst stage, as indicated by decreased levels of PTEN and increased levels of the PTEN regulators bta-miR-494 and bta-miR-20a. Using PI3K-Akt responsive genes, we showed decreased FOXO3a levels and BAX levels in lower quality groups, indicating changes in cell cycle progression, oxidative response and apoptosis. Based on these results, the measurement of levels of PI3K-Akt pathway components in FCs from ovarian follicles carrying oocytes with distinct developmental competences is a useful tool to identify putative molecular pathways involved in the acquisition of oocyte competence.

Effect of tributyltin on the development of ovary in female cuvier (Sebastiscus marmoratus).

PubMed

Zhang, Jiliang; Zuo, Zhenghong; Chen, Yixin; Zhao, Yang; Hu, Shuai; Wang, Chonggang

2007-07-20

Organotin compounds, such as tributyltin (TBT) used as an antifouling biocide, can induce masculinization in female mollusks. However, few studies addressing the effect of TBT in fish have been reported. This study was conducted to investigate effects of TBT at environmental levels (1, 10, 100ng/L) on the development of ovary in female cuvier. TBT exposure elevated testosterone levels, increased the ratio of testosterone to 17beta-estradiol and decreased 17beta-estradiol levels in ovaries after 50 days compared to the control. Three stages of follicles (primary growth stage, yolk vesicle stage, vitellogenic stage) were observed in the ovaries of cuvier at the control and 1ng/L TBT group. The ovaries at the 10ng/L TBT group were characterized by the lack of vitellogenic stage follicles and instead had higher proportions of primary growth stage follicles. 100ng/L TBT resulted in follicles that were entirely at the earliest (primary growth stage) stages of development. There was a significant increase in apoptotic ovarian follicular cells judged by TUNEL-positive cell at the 10ng/L TBT group. The TUNEL-positive follicles were observed at the 100ng/L TBT group. The result in the present study showed that TBT at environmentally realistic concentrations can inhibit the ovarian development in fish. Besides the changes of sex hormone induced by TBT, apoptosis appears to be one mechanism affecting ovarian development.

Seasonal effects on fertility and ovarian follicular growth and maturation in camels (Camelus dromedarius).

PubMed

Sghiri, A; Driancourt, M A

1999-04-30

Camels are said to be seasonal breeders, but the extent to which season interferes with food supply to affect ovarian function is not fully documented. Hence, the three aims of this study were: (1) to define the breeding season of camels maintained in semi-arid conditions in southern Morocco; (2) to relate the proportion of females with active ovaries (i.e., with follicles > 5 mm), with ovulatory (11-17 mm) or cystic (> 18 mm) follicles to age and body conditions score; (3) to study the consequences of the interactions between age and body conditions score on the proportion of females ovulating and conceiving; and (4) to compare follicular maturation, using in vitro steroidogenesis by intact follicles as a marker during the transition into the breeding season (October) and peak breeding season (March). There was a clear breeding season in the two flocks studied, since over 80-90% of the matings occurred during the period from mid-November to mid-April. Collection of ovaries at slaughter (n = 238) demonstrated a significant seasonal effect on the proportion of females with active ovaries (increasing from 73.5% in October-December to 89% in January-May), but no changes in the proportion of females with ovulatory follicles. Lean females (BCS < 2.5) had a delayed initiation of ovarian function in October-December. In addition, the proportion of females with cystic follicles was also affected by season (peaking during April-May). Neither age nor body condition modulated the frequency of cysts. Finally, the proportion of females conceiving increased steadily as season progressed (peaking at 57% in April-May). Body condition score did not affect this proportion, but young females (< or = 5 years old) had a low ability to conceive. Morphological features of large follicles were unaffected by season. Ovulatory follicles contained around 10(7) granulosa and theca cells. In vitro testosterone output by intact follicles was unrelated to follicle size and season. In vitro oestradiol output increased with increasing follicle size and was larger in follicles obtained during peak breeding season than at its initiation. This may indicate that early breeding season follicles display a low aromatase activity in their granulosa cells. Whether the low oestradiol output of early breeding season follicles is resulting in the low fertility observed at this period remains to be determined.

Does obesity compromise ovarian reserve markers? A clinician's perspective.

PubMed

Malhotra, Neena; Bahadur, Anupama; Singh, Neeta; Kalaivani, Mani; Mittal, Suneeta

2013-01-01

The aim of the study was to ascertain if increasing body mass index (BMI) adversely affects ovarian reserve among infertile women of Asian origin undergoing in vitro fertilization (IVF). This prospective study on 183 women was carried out in the infertility clinic of All India Institute of Medical Sciences, New Delhi, India. Blood hormonal assay in all patients including follicle-stimulating hormone (FSH), luteinizing hormone (LH) and inhibin B was performed on day 2/3 of a spontaneous cycle. A transvaginal ultrasonographic examination on day 2-5 of the menstrual cycle was done for antral follicle count (AFC) and ovarian volume. A correlation between BMI and ovarian reserve parameters like FSH, LH, inhibin B, antral follicle count and ovarian volume was noted. Age was comparable in the three BMI groups. The mean duration of infertility was 8.38 years. Compared to the normal weight, the overweight and obese women had a statistically significantly low inhibin B (p < 0.0259). The AFC when taken together on both sides was not statistically significant between the groups; however, the overweight and obese women had a significantly low AFC (p < 0.0129) on the right side. Incorporating anti-mullerian hormone, a newer marker for ovarian reserve, may benefit these obese infertile women. Further work is required to elucidate the mechanisms underlying the effect of obesity on inhibin B as a marker of ovarian reserve in infertile women. The best marker to check the ovarian reserve is perhaps the woman's performance during an IVF cycle. However, considering the psychological and financial stress of the procedure, it may seem wise to consider counseling of obese women on their expected performance in the first cycle of IVF through such studies.

Females transplanted with ovaries subjected to hypoxic preconditioning show impair of ovarian function.

PubMed

Damous, Luciana Lamarão; Nakamuta, Juliana Sanajotti; Soares, José Maria; Maciel, Gustavo Arantes Rosa; Simões, Ricardo Dos Santos; Montero, Edna Frasson de Souza; Krieger, José Eduardo; Baracat, Edmund Chada

2014-03-20

Cryopreservation of the ovarian tissue has shown promising results. However, there remain controversial issues such as the short half-life of grafts. In this aspect, there are some evidences that preconditioning the ovarian tissue before transplantation is beneficial. To determine the effect of hypoxic preconditioning in vitro on ovarian tissue prior to transplantation. Eighteen female adult Wistar rats, were sorted into three experimental groups. Ovaries were maintained in DMEM low glucose serum free at 37°C with 5% CO2, at atmospheric oxigen concentration (normoxia) or 1% O2 (hypoxia) for 16 hours. Oxigen concentration was determined by injection of nitrogen in the incubator. Animals submitted to ovarian transplantation immediately after oophorectomy were the Control Group (C). After this, the ovaries were implanted in the retroperitoneum with nonabsorbable suture and animals evaluated for thirty days after transplantation. Beginning on postoperative (PO) day 11, a daily collection of vaginal smear was carried out. Analyses comprised morphological, morphometric (counting ovarian follicles and corpora lutea) and immunohistochemistry for cleaved caspase-3 (apoptosis). In normoxia and control groups all animals recovered their estrous cycles, while in the hypoxia group, two animals did not ovulate but, among those which did, resumption took longer than in the other groups (p < 0.05). The number of ovarian follicles and corpora lutea decreased significantly in the hypoxia group when compared to the other two groups (p < 0.001) and apoptosis was increased in the few ovarian follicles which remained viable (p < 0.001). The hypoxic preconditioning in vitro was not beneficial to the graft and worsened their viability, compromising its functionality or delaying the return of this.

Females transplanted with ovaries subjected to hypoxic preconditioning show impair of ovarian function

PubMed Central

2014-01-01

Background Cryopreservation of the ovarian tissue has shown promising results. However, there remain controversial issues such as the short half-life of grafts. In this aspect, there are some evidences that preconditioning the ovarian tissue before transplantation is beneficial. Objective To determine the effect of hypoxic preconditioning in vitro on ovarian tissue prior to transplantation. Methods Eighteen female adult Wistar rats, were sorted into three experimental groups. Ovaries were maintained in DMEM low glucose serum free at 37°C with 5% CO2, at atmospheric oxigen concentration (normoxia) or 1% O2 (hypoxia) for 16 hours. Oxigen concentration was determined by injection of nitrogen in the incubator. Animals submitted to ovarian transplantation immediately after oophorectomy were the Control Group (C). After this, the ovaries were implanted in the retroperitoneum with nonabsorbable suture and animals evaluated for thirty days after transplantation. Beginning on postoperative (PO) day 11, a daily collection of vaginal smear was carried out. Analyses comprised morphological, morphometric (counting ovarian follicles and corpora lutea) and immunohistochemistry for cleaved caspase-3 (apoptosis). Results In normoxia and control groups all animals recovered their estrous cycles, while in the hypoxia group, two animals did not ovulate but, among those which did, resumption took longer than in the other groups (p < 0.05). The number of ovarian follicles and corpora lutea decreased significantly in the hypoxia group when compared to the other two groups (p < 0.001) and apoptosis was increased in the few ovarian follicles which remained viable (p < 0.001). Conclusion The hypoxic preconditioning in vitro was not beneficial to the graft and worsened their viability, compromising its functionality or delaying the return of this. PMID:24655551

Sonographic evaluation of polycystic ovaries.

PubMed

Zhu, Ruo-Yan; Wong, Yee-Chee; Yong, Eu-Leong

2016-11-01

The morphological features of the ovaries in women with polycystic ovary syndrome (PCOS) have been well described by ultrasound imaging technology. These include enlarged ovary size, multiple small follicles of similar size, increased ovarian stromal volume and echogenicity, peripheral distribution of the follicles, and higher stromal blood flow. Ultrasound identification of the presence of polycystic ovarian morphology (PCOM) has been recognized as a component of PCOS diagnosis. With the advance of ultrasound technology, new definition has been proposed recently. There is, however, a paucity of data for the ovarian morphology in normal and PCOS adolescents. Magnetic resonance imaging has the potential to be an alternative imaging modality for diagnosing PCOM in adolescence. Copyright © 2016. Published by Elsevier Ltd.

Intraovarian transplantation of primordial follicles fails to rescue chemotherapy injured ovaries

PubMed Central

Park, Mi-Ryung; Choi, Yun-Jung; Kwon, Deug-Nam; Park, Chankyu; Bui, Hong-Thuy; Gurunathan, Sangiliyandi; Cho, Ssang-Goo; Song, Hyuk; Seo, Han Geuk; Min, Gyesik; Kim, Jin-Hoi

2013-01-01

Busulfan and cyclophosphamide (B/C)-treated mice exhibited a marked increase in apoptosis and a concomitant decrease in the ovarian weight. Histological and RT-PCR analysis indicate that the period of germ cell depletion in the B/C-treated ovaries coincides with decreased expression of genes Figla, Lhx8, Nobox, c-kit, and Sox3. However, depletion of the ovarian germ cells is mediated by autophagy-independent pathways that involve Fas/FasL-, TNF-, and/or p53-signalings. Treatment with B/C resulted in the cease of the reproductive function to produce their offspring during the 15th week post-treatment period in female mice. Furthermore, injection of the 3 × 106 GFP positive primordial follicles into the ovaries of the B/C treated mouse did not show apparent colonization of the transplanted follicles within the recipient ovaries. The present results suggest that B/C treatment is closely associated with an increased risk of premature ovarian failure. PMID:23463338

White spotting variant (Wv) mouse as an experimental model for ovarian aging and menopausal biology

PubMed Central

Smith, Elizabeth R.; Yeasky, Toni; Wei, Jain Qin; Miki, Roberto A.; Cai, Kathy Q.; Smedberg, Jennifer L.; Yang, Wan-Lin; Xu, Xiang-Xi

2011-01-01

Objective Menopause is a unique phenomenon in modern women, as most mammalian species possess a reproductive period comparable to their lifespan. Menopause is caused by the depletion of germ cell-containing ovarian follicles, and in laboratory studies is usually modeled in animals in which the ovarian function is removed by ovariectomy or chemical poisoning of the germ cells. Our objective was to explore and characterize the white spotting variant (Wv) mice that have reduced ovarian germ cell abundance, a result of a point mutation in the c-kit gene that decreases the kinase activity, as a genetic model for use in menopausal studies. Methods Physiological and morphological features associated with menopause were determined in female Wv/Wv mice compared to age-matched wildtype controls. Immunohistochemistry was used to evaluate the presence and number of follicles in paraffin-embedded ovaries. Bone density and body composition were evaluated using the PIXImus X-ray densitometer, and lipids, calcium, and hormone levels were determined in serum using antigen-specific EIAs. Heart and body weight were measured, and cardiac function was evaluated by transthoracic echocardiography. Results The ovaries of the Wv/Wv females have a greatly reduced number of normal germ cells at birth compared to wildtype mice. The remaining follicles are depleted by around 2 months, and the ovaries develop benign epithelial lesions that resemble morphological changes that occur during ovarian aging, whereas a normal mouse ovary has numerous follicles at all stages of development and retains some follicles even in advanced age. Wv mice have elevated plasma gonadotrophins and reduced estrogen and progesterone levels, a significant reduction in bone mass density, and elevated serum cholesterol and lipoprotein levels. Moreover, the Wv female mice have enlarged hearts and reduced cardiac function. Conclusions The reduction of c-kit activity in Wv mice leads to a substantially diminished follicular endowment in newborn mice and premature depletion of follicles in young mice, though the mutant females have a normal lifespan after cessation of ovarian function. The Wv female mice exhibit consistent physiological changes that resemble common features of postmenopausal women. These alterations include follicle depletion, morphological aging of the ovary, altered serum levels of cholesterol, gonadotropins, and steroid hormones, decreased bone density, and reduced cardiac function. These changes were not observed in male mice, either age-matched male Wv/Wv or WT mice, and are unlikely caused by global loss of c-kit function. The Wv mouse may be a genetic, intact-ovary model that mimics closely the phenotypes of human menopause to be used for further studies to understand mechanisms of menopausal biology. PMID:22228319

Effects of 4-Vinylcyclohexene Diepoxide on Peripubertal and Adult Sprague–Dawley Rats: Ovarian, Clinical, and Pathologic Outcomes

PubMed Central

Muhammad, F Salih; Goode, Amanda K; Kock, Nancy D; Arifin, Esther A; Cline, J Mark; Adams, Michael R; Hoyer, Patricia B; Christian, Patricia J; Isom, Scott; Kaplan, Jay R; Appt, Susan E

2009-01-01

Young rats treated daily with intraperitoneal 4-vinylcyclohexene diepoxide (VCD) undergo selective destruction of primordial follicles, resulting in gradual ovarian failure resembling the menopausal transition in women. To determine whether VCD has similar effects on ovaries of older rats, adult and peripubertal Sprague–Dawley rats were injected intraperitoneally daily for 30 d with vehicle or VCD at 40 or 80 mg/kg. Body weight, food intake, complete blood counts, and markers of liver injury and renal function were measured during VCD treatment. Complete gross necropsy and microscopic observations were performed on day 31, and ovarian follicles were counted. At 80 mg/kg, VCD destroyed primordial and primary follicles to a similar extent in both adult and peripubertal animals, although adult rats likely started with fewer follicles and therefore approached follicle depletion. Treatment with VCD did not affect body weight, but food intake was reduced in both adult and peripubertal rats treated with 80 mg/kg VCD. Adult rats treated with 80 mg/kg VCD had neutrophilia and increased BUN and creatinine; in addition, 4 of these rats were euthanized on days 25 or 26 due to peritonitis. VCD treatment did not increase alanine aminotransferase levels, a marker of liver injury, although the 80-mg/kg dose increased liver weights. In conclusion, VCD effectively destroys small preantral follicles in adult Sprague–Dawley rats, making them a suitable model of the menopausal transition of women. However, because adult rats were more sensitive to the irritant properties of VCD, the use of a lower dose should be considered. PMID:19295054

Histological and genotoxic evaluation of gold nanoparticles in ovarian cells of zebrafish ( Danio rerio)

NASA Astrophysics Data System (ADS)

Dayal, Navami; Thakur, Mansee; Patil, Poonam; Singh, Dipty; Vanage, Geeta; Joshi, D. S.

2016-10-01

Gold nanoparticles (AuNPs) have attracted a lot of attention due to their usage in consumer- and therapy-based biomedical applications. These particles are frequently the medium-sized particles within the range of 10-50 nm. A number of scientific reports have addressed the cytotoxic potential of these NPs. However, their genotoxic potential with respect to reproductive aspects remains unclear. For assessment of safety and risks associated with AuNPs to female reproductive system, adult female zebrafish (Danio rerio) were exposed in vivo to 20 μg/g/day of AuNPs of two different sizes. AuNPs of 15 nm (type I) and 47 nm (type II) in diameters were administered orally to female zebrafish for a period of 28 days (chronic). The ability of these AuNPs to gain access to female reproductive organs was confirmed by their accumulation pattern through inductive coupled plasma mass spectroscopy. Gonads were assessed for changes in ovarian morphology at histopathological level followed by the confirmation of bioaccumulation of AuNPs using transmission electron microscopy. Using comet assay, strand breaks in DNA of ovarian cells were investigated. Chronic exposure to type I and II AuNPs showed distinctive patterns of bioaccumulation in ovaries. Interestingly, accumulated NPs resulted in gross cellular alterations in different cell types of ovarian tissue. Comet assay analysis revealed extensive number of strand breaks in ovarian cells from the NP exposed fishes. In conclusion, AuNPs ranging between 10 and 50 nm are capable of gaining access to ovaries of zebrafish and potential enough to cause strand breaks in ovarian cells. The findings of the present study highlight the adverse effects of these NPs to female reproductive system. It opens up further avenues for research on effects of these NPs on F1 generation descending from the exposed fishes.

Stimulation of ovarian stem cells by follicle stimulating hormone and basic fibroblast growth factor during cortical tissue culture.

PubMed

Parte, Seema; Bhartiya, Deepa; Manjramkar, Dhananjay D; Chauhan, Anahita; Joshi, Amita

2013-04-01

Cryopreserved ovarian cortical tissue acts as a source of primordial follicles (PF) which can either be auto-transplanted or cultured in vitro to obtain mature oocytes. This offers a good opportunity to attain biological parenthood to individuals with gonadal insufficiency including cancer survivors. However, role of various intra- and extra-ovarian factors during PF growth initiation still remain poorly understood. Ovarian biology has assumed a different dimension due to emerging data on presence of pluripotent very small embryonic-like stem cells (VSELs) and ovarian germ stem cells (OGSCs) in ovary surface epithelium (OSE) and the concept of postnatal oogenesis. The present study was undertaken to decipher effect of follicle stimulating hormone (FSH) and basic fibroblast growth factor (bFGF) on the growth initiation of PF during organ culture with a focus on ovarian stem cells. Serum-free cultures of marmoset (n=3) and human (young and peri-menopausal) ovarian cortical tissue pieces were established. Cortical tissue pieces stimulated with FSH (0.5 IU/ml) or bFGF (100 ng/ml) were collected on Day 3 for histological and molecular studies. Gene transcripts specific for pluripotency (Oct-4A, Nanog), early germ cells (Oct-4, c-Kit, Vasa) and to reflect PF growth initiation (oocyte-specific Gdf-9 and Lhx8, and granulosa cells specific Amh) were studied by q-RTPCR. A prominent proliferation of OSE (which harbors stem cells) and transition of PF to primary follicles was observed after FSH and bFGF treatment. Ovarian stem cells were found to be released on the culture inserts and retained the potential to spontaneously differentiate into oocyte-like structures in extended cultures. q-RTPCR analysis revealed an increased expression of gene transcripts specific for VSELs, OGSCs and early germ cells suggestive of follicular transition. The present study shows that both FSH and bFGF stimulate stem cells present in OSE and also lead to PF growth initiation. Thus besides being a source of PF, cryopreserved ovarian cortical tissue could also be a source of stem cells which retain the ability to spontaneously differentiate into oocyte-like structures in vitro. Results provide a paradigm shift in the basic understanding of FSH action and also offer a new perspective to the field of oncofertility research.

Role of natriuretic peptide receptor 2-mediated signaling in meiotic arrest of zebrafish oocytes and its estrogen regulation through G protein-coupled estrogen receptor (Gper).

PubMed

Pang, Yefei; Thomas, Peter

2018-03-22

Natriuretic peptide type C (NPPC) and its receptor, natriuretic peptide receptor 2 (NPR2), have essential roles in maintaining meiotic arrest of oocytes in several mammalian species. However, it is not known if a similar mechanism exists in non-mammalian vertebrates. Using zebrafish as a model, we show that Nppc is expressed in ovarian follicle cells, whereas Npr2 is mainly detected in oocytes. Treatment of intact and defolliculated oocytes with 100 nM NPPC for 6 h caused a large increase in cGMP concentrations, and a significant decrease in oocyte maturation (OM), an effect that was mimicked by treatment with 8-Br-cGMP. Treatment with E2 and G-1, the specific GPER agonist, also increased cGMP levels. Cyclic AMP levels were also increased by treatments with 8-Br-cGMP, E2 and G1. The estrogen upregulation of cAMP levels was blocked by co-treatment with AG1478, an inhibitor of EGFR activation. Gene expression of npr2, but not nppc, was significantly upregulated in intact oocytes by 6 h treatments with 20 nM E2 and G-1. Both cilostamide, a phosphodiesterase 3 (PDE3) inhibitor, and rolipram, a PDE4 inhibitor, significantly decreased OM of intact and defolliculated oocytes, and enhanced the inhibitory effects of E2 and G-1 on OM. These findings indicate the presence of a Nppc/Npr2/cGMP pathway maintaining meiotic arrest in zebrafish oocytes that is upregulated by estrogen activation of Gper. Collectively, the results suggest that Nppc through Npr2 cooperates with E2 through Gper in upregulation of cGMP levels to inhibit phosphodiesterase activity resulting in maintenance of oocyte meiotic arrest in zebrafish. Copyright © 2018 Elsevier Inc. All rights reserved.

Impact of Obesity on Ovotoxicity Induced by 7,12-dimethylbenz[a]anthracene in Mice1

PubMed Central

Nteeba, Jackson; Ganesan, Shanthi; Keating, Aileen F.

2014-01-01

ABSTRACT Insulin, elevated during obesity, regulates xenobiotic biotransformation enzymes, potentially through phosphatidylinositol 3-kinase (PI3K) signaling, in extraovarian tissues. PI3K regulates oocyte viability, follicular activation, and ovarian chemical biotransformation. 7,12-Dimethylbenz[a]anthracene (DMBA), a carcinogen and ovotoxicant, destroys all stages of follicles, leading to premature ovarian failure. Obesity has been reported to promote DMBA-induced tumors, but it remains unknown whether obesity affects ovarian xenobiotic metabolism. Therefore, we investigated ovarian expression of xenobiotic metabolism genes—microsomal epoxide hydrolase (Ephx1), glutathione S-transferase (GST) class Pi (Gstp1) and class mu 1 (Gstm1), and PI3K-signaling members (protein kinase B [AKT] alpha [Akt1], beta [Akt2], and the forkhead transcription factor subfamily 3 [Foxo3])—in lean and obese female mice after DMBA exposure (1 mg/kg; intraperitoneal injection for 14 days). Relative to lean, obese mice had decreased (P < 0.05) healthy primordial and primary follicle numbers but increased (P < 0.05) secondary and preovulatory follicles numbers. Obesity increased (P < 0.05) Akt1, Akt2, Gstm1, and Ephx1 mRNA and pAKTSer473/Thr308, GSTM1, GSTP1, and EPHX1 protein levels. DMBA decreased (P < 0.05) ovarian weight in lean and obese mice, however, obese DMBA-treated females had a greater reduction (P < 0.05) in ovarian weight. In both lean and obese mice, DMBA decreased (P < 0.05) all stages of healthy follicle numbers, increased Gstp1 and Ephx1 mRNA as well as GSTM1, GSTP1, and EPHX1 protein levels, and decreased Akt1 and Akt2 mRNA as well as pAKTSer473 or pAKTThr308, FOXO3, and pFOXO3Ser253 protein expression. There was an additive effect between obesity and DMBA exposure for increased Gstm1 and Ephx1 mRNA as well as GSTM1 and EPHX1 protein expression. PMID:24501177

Two-step transplantation with adipose tissue-derived stem cells increases follicle survival by enhancing vascularization in xenografted frozen-thawed human ovarian tissue.

PubMed

Manavella, D D; Cacciottola, L; Pommé, S; Desmet, C M; Jordan, B F; Donnez, J; Amorim, C A; Dolmans, M M

2018-06-01

Do adipose tissue-derived stem cells (ASCs) enhance vascularization and follicle survival in xenografted ovarian tissue using a two-step transplantation approach? Higher rates of oxygenation and vascularization of ovarian tissue, as well as increased follicle survival rates, were detected in the early post-grafting period. ASCs have multilineage differentiation potential, proangiogenic properties and enhance vascularization in a peritoneal grafting site. Some studies suggest that using ASCs may improve ovarian tissue quality by enhancing graft angiogenesis. A total of 15 severe combined immunodeficient (SCID) mice were intraperitoneally grafted with frozen-thawed human ovarian tissue (OT) from five different patients. A peritoneal transplantation site had been previously prepared in a first step using either empty fibrin (Fi+OT group [n = 5]) or ASC-loaded fibrin (Fi/ASCs+OT group [n = 5]) for 14 days prior to grafting. Five mice underwent the standard one-step transplantation procedure and served as controls (OT group). Lithium phthalocyanine (LiPc) crystals were inserted into all grafted human ovarian tissue before transplantation. Levels of partial pressure of oxygen (pO2) in grafts were monitored in vivo by electron paramagnetic resonance (EPR) oximetry on Days 3 and 7. Samples for histology and immunohistochemistry (IHC) were collected after euthanizing the mice on Day 7 following EPR. One piece of ovarian tissue per patient was fixed for analysis to serve as non-grafted controls. Prospective experimental study conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to perform pO2 measurements (EPR oximetry), histological (haematoxylin and eosin staining), immunohistochemistry (anti-mouse and human double CD34 and anti-human Ki-67) and TUNEL analyses. A significant increase in pO2 was observed in all groups between Days 3 and 7 (P < 0.001). A significantly higher pO2 level was observed in the Fi/ASCs+OT group compared to the OT group on Day 7 (P = 0.028). Total CD34-positive vessel area on Day 7 was greater in the Fi/ASCs+OT group than in any other group (vs non-grafted group: P = 0.0014; vs OT group: P = 0.013; vs Fi+OT group: P = 0.018). Primordial follicle survival rates after grafting were higher in the Fi/ASCs+OT group than in the OT (P = 0.0059) or Fi+OT groups (P = 0.0307). TUNEL-positive follicle percentages after grafting were significantly lower in the Fi/ASCs+OT group than in any other grafted tissue (vs OT group: P = 0.045; vs Fi+OT group: P = 0.0268). Percentages of Ki-67-positive primordial follicles were significantly higher in all grafted groups compared to non-grafted tissue controls (P < 0.01). As demonstrated by our results, the proposed two-step ovarian tissue transplantation procedure using ASCs enhances vascularization in the early post-grafting period, leading to increased follicle survival rates and decreased apoptosis. However, mechanisms involved in the proangiogenic behavior of ASCs remain to be elucidated. Our results suggest that the proposed transplantation procedure with ASCs is a promising step towards potentially solving the problem of massive follicle loss after ovarian tissue grafting. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention T.0077.14, grant Télévie No. 7.6515.16 F to DDM and grant 5/4/150/5 awarded to MMD and CAA is research associate, FRS-FNRS), Fonds Spéciaux de Recherche, Fondation St Luc, and Foundation Against Cancer, and donations from the Ferrero family.

Definition and significance of polycystic ovarian morphology: a task force report from the Androgen Excess and Polycystic Ovary Syndrome Society.

PubMed

Dewailly, Didier; Lujan, Marla E; Carmina, Enrico; Cedars, Marcelle I; Laven, Joop; Norman, Robert J; Escobar-Morreale, Héctor F

2014-01-01

BACKGROUND The diagnosis of polycystic ovary syndrome (PCOS) relies on clinical, biological and morphological criteria. With the advent of ultrasonography, follicle excess has become the main aspect of polycystic ovarian morphology (PCOM). Since 2003, most investigators have used a threshold of 12 follicles (measuring 2-9 mm in diameter) per whole ovary, but that now seems obsolete. An increase in ovarian volume (OV) and/or area may also be considered accurate markers of PCOM, yet their utility compared with follicle excess remains unclear. METHODS Published peer-reviewed medical literature about PCOM was searched using PubMed.gov online facilities and was submitted to critical assessment by a panel of experts. Studies reporting antral follicle counts (AFC) or follicle number per ovary (FNPO) using transvaginal ultrasonography in healthy women of reproductive age were also included. Only studies that reported the mean or median AFC or FNPO of follicles measuring 2-9 mm, 2-10 mm or <10 mm in diameter, or visualized all follicles, were included. RESULTS Studies addressing women recruited from the general population and studies comparing control and PCOS populations with appropriate statistics were convergent towards setting the threshold for increased FNPO at ≥25 follicles, in women aged 18-35 years. These studies suggested maintaining the threshold for increased OV at ≥10 ml. Critical analysis of the literature showed that OV had less diagnostic potential for PCOM compared with FNPO. The review did not identify any additional diagnostic advantage for other ultrasound metrics such as specific measurements of ovarian stroma or blood flow. Even though serum concentrations of anti-Müllerian hormone (AMH) showed a diagnostic performance for PCOM that was equal to or better than that of FNPO in some series, the accuracy and reproducibility issues of currently available AMH assays preclude the establishment of a threshold value for its use as a surrogate marker of PCOM. PCOM does not associate with significant consequences for health in the absence of other symptoms of PCOS but, because of the use of inconsistent definitions of PCOM among studies, this question cannot be answered with absolute certainty. CONCLUSIONS The Task Force recommends using FNPO for the definition of PCOM setting the threshold at ≥25, but only when using newer technology that affords maximal resolution of ovarian follicles (i.e. transducer frequency ≥8 MHz). If such technology is not available, we recommend using OV rather than FNPO for the diagnosis of PCOM for routine daily practice but not for research studies that require the precise full characterization of patients. The Task Force recognizes the still unmet need for standardization of the follicle counting technique and the need for regularly updating the thresholds used to define follicle excess, particularly in diverse populations. Serum AMH concentration generated great expectations as a surrogate marker for the follicle excess of PCOM, but full standardization of AMH assays is needed before they can be routinely used for clinical practice and research. Finally, the finding of PCOM in ovulatory women not showing clinical or biochemical androgen excess may be inconsequential, even though some studies suggest that isolated PCOM may represent the milder end of the PCOS spectrum.

Peptidoglycan inhibits progesterone and androstenedione production in bovine ovarian theca cells.

PubMed

Magata, F; Horiuchi, M; Miyamoto, A; Shimizu, T

2014-08-01

Uterine bacterial infection perturbs uterine and ovarian functions in postpartum dairy cows. Peptidoglycan (PGN) produced by gram-positive bacteria has been shown to disrupt the ovarian function in ewes. The aim of this study was to determine the effect of PGN on steroid production in bovine theca cells at different stages of follicular development. Bovine theca cells isolated from pre- and post-selection ovarian follicles (<8.5mm and >8.5mm in diameter, respectively) were cultured in vitro and challenged with PGN. Steroid production was evaluated by measuring progesterone (P4) and androstenedione (A4) concentration in culture media after 48 h or 96 h of culture. Bovine theca cells expressed PGN receptors including Toll-like receptor 2 and nucleotide-binding oligomerization domain 1 and 2. Treatment with PGN (1, 10, or 50 μg/ml) led to a decrease in P4 and A4 production by theca cells in both pre- and post-selection follicles. The mRNA expression of steroidogenic enzymes were decreased by PGN treatment. Moreover, A4 production was further suppressed when theca cells of post-selection follicles were simultaneously treated by PGN and lipopolysaccharide (0.1, 1, or 10 μg/ml). These findings indicate that bacterial toxins may act locally on ovarian steroidogenic cells and compromise follicular development in postpartum dairy cows. Copyright © 2014 Elsevier Ltd. All rights reserved.

Fertility control of rodent pests: a review of the inhibitory effects of plant extracts on ovarian function.

PubMed

Tran, Tung Thanh; Hinds, Lyn A

2013-03-01

Plant extracts can inhibit fertility by adversely affecting, directly or indirectly, reproductive processes ranging from gonadal function and development to gestation. This review focuses on plant extracts that disrupt ovarian function in rodents. Extracts from at least 40 plant species exert some of their disruptive reproductive effects at the ovarian level. Of those, 13 plants induce a reduction in the number and type of ovarian follicles and also cause disruption to the oestrous cycle. Their effects are short term and reversible once treatment ceases. Protection of plant extracts to prevent their degradation before uptake in the gastrointestinal tract could enhance short-term efficacy but would not enhance the longevity of their effects. Identification and further testing of the specific chemicals responsible for reproductive effects would be beneficial. The adoption of a standard protocol for treatment and assessment of the inhibitory effects of potential control agents on reproductive function in rodents is essential. Treatment with higher concentrations of extracts in conjunction with other extracts or with other chemosterilants could have potential complementary effects and lead to more rapid and permanent changes in ovarian function. An orally delivered agent(s) that causes major depletion of all follicle types, and particularly of non-regenerating primordial follicles, could be an ideal fertility control product and serve as an additional tool for population control of pest rodents. Copyright © 2012 Society of Chemical Industry.

Mettl3 Mutation Disrupts Gamete Maturation and Reduces Fertility in Zebrafish.

PubMed

Xia, Hui; Zhong, Chengrong; Wu, Xingxing; Chen, Ji; Tao, Binbin; Xia, Xiaoqin; Shi, Mijuan; Zhu, Zuoyan; Trudeau, Vance L; Hu, Wei

2018-02-01

N 6 -methyladenosine (m 6 A), catalyzed by Mettl3 methyltransferase, is a highly conserved epigenetic modification in eukaryotic messenger RNA (mRNA). Previous studies have implicated m 6 A modification in multiple biological processes, but the in vivo function of m 6 A has been difficult to study, because mettl3 mutants are embryonic lethal in both mammals and plants. In this study, we have used transcription activator-like effector nucleases and generated viable zygotic mettl3 mutant, Z mettl3 m/m , in zebrafish. We find that the oocytes in Z mettl3 m/m adult females are stalled in early development and the ratio of full-grown stage (FG) follicles is significantly lower than that of wild type. Human chorionic gonadotropin-induced ovarian germinal vesicle breakdown in vitro and the numbers of eggs ovulated in vivo are both decreased as well, while the defects of oocyte maturation can be rescued by sex hormone in vitro and in vivo In Z mettl3 m/m adult males, we find defects in sperm maturation and sperm motility is significantly reduced. Further study shows that 11-ketotestosterone (11-KT) and 17β-estradiol (E2) levels are significantly decreased in Z mettl3 m/m , and defective gamete maturation is accompanied by decreased overall m 6 A modification levels and disrupted expression of genes critical for sex hormone synthesis and gonadotropin signaling in Z mettl3 m/m Thus, our study provides the first in vivo evidence that loss of Mettl3 leads to failed gamete maturation and significantly reduced fertility in zebrafish. Mettl3 and m 6 A modifications are essential for optimal reproduction in vertebrates. Copyright © 2018 by the Genetics Society of America.

Diminished ovarian reserve in women with transfusion-dependent beta-thalassemia major: Is iron gonadotoxic?

PubMed

Uysal, Aysel; Alkan, Gül; Kurtoğlu, Ayşegül; Erol, Onur; Kurtoğlu, Erdal

2017-09-01

Iron accumulation in the endocrine glands has been implicated in the aetiopathogenesis of decreased reproductive capacity in patients with beta-thalassemia major (β-TM). The aim of the current study was to investigate the serum concentration of anti-Müllerian hormone (AMH), a marker of ovarian reserve, in women with transfusion-dependent β-TM. In this case-control study, we recruited 43 women with transfusion-dependent TM and 44 age-matched healthy controls. Hormonal and haematological parameters, serum level of AMH, antral follicle count, and ovarian volume were assessed. Twenty-two of the 43 women were hypogonadotropic, 8 with primary amenorrhea and 14 with secondary amenorrhea. FSH, LH, estradiol, prolactin, and AMH levels; antral follicle count; and ovarian volume were significantly lower in women with TM compared with the control group (p<0.05 for all). AMH level and other ovarian reserve markers are significantly diminished in women with transfusion-dependent TM compared to age-matched controls. Our findings support a deleterious effect of iron overload on ovarian tissue. Published by Elsevier B.V.

Laparoscopic ovarian cystectomy of endometriomas does not affect the ovarian response to gonadotropin stimulation.

PubMed

Marconi, Guillermo; Vilela, Martín; Quintana, Ramiro; Sueldo, Carlos

2002-10-01

To evaluate the ovarian response cycles of IVF-ET in patients who previously underwent laparoscopic cystectomy for endometriomas. Retrospective study with prospective selection of participants and controls. Instituto de Ginecología y Fertilidad Buenos Aires, Argentina. Thirty-nine patients underwent an operation for ovarian endometriomas by atraumatic removal of the pseudocapsule with minimal bipolar cauterization of small bleeders and an IVF-ET cycle (group A) and 39 control patients of similar age underwent an IVF-ET cycle for tubal factor infertility (group B). Laparoscopic endometrioma cystectomy, IVF-ET cycle. E(2) levels, number of gonadotropin ampoules, follicles, oocytes retrieved, number and quality of embryos transferred, and clinical pregnancy rate. There were no differences in all the parameters studied (E(2) levels, number of follicles, oocytes retrieved, number and quality of embryos transferred, and clinical pregnancy rate) except for the number of gonadotropin ampoules needed for ovarian hyperstimulation, which was significantly higher in group A than in group B. Our results indicate that laparoscopic cystectomy for endometriomas is an appropriate treatment since it did not negatively affect the ovarian response for IVF-ET.

Improved selection of cortical ovarian strips for autotransplantation of ovarian tissue using full-field optical coherence tomography (FFOCT)

NASA Astrophysics Data System (ADS)

Stegehuis, Paulien L.; Peters, Inge T. A.; Eggermont, Jeroen; Kuppen, Peter J. K.; Trimbos, J. Baptist; Lelieveldt, Boudewijn P. F.; van de Velde, Cornelis J. H.; Bosse, Tjalling; Dijkstra, Jouke; Vahrmeijer, Alexander L.

2016-02-01

Premature ovarian failure is a major concern in women of reproductive age who undergo gonadotoxic cancer treatment. Autotransplantation of frozen-thawed cortical ovarian tissue allows the immediate start of cancer treatment, but risks reintroduction of cancer. Current tumor detection methods compromise the ovarian tissue's viability and can therefore only be used to exclude the presence of metastases in the cortical ovarian strips that are not transplanted. A non-invasive method is needed that can be used to exclude metastases in the actual ovarian autografts without affecting the tissue's viability. In this study we applied FFOCT - a non-fixative technique that uses white light interferometry to make highresolution images (1μm isotropic) of fresh tissue - to study healthy and malignant ovarian tissue. We created an image atlas of healthy ovarian tissues from premenopausal patients and ovarian tissues with breast cancer metastases. To get the best possible match between hematoxylin-and-eosin stained slides and FFOCT images formalinfixed paraffin-embedded tissue samples were deparaffinized and FFOCT images were acquired within a few minutes. FFOCT images were compared with histology images. All normal structures such as follicles in all phases, inclusion cysts, blood vessels, corpora lutea, and corpora albicantia were clearly recognizable. Ovarian metastases could be well distinguished from normal ovarian tissue. FFOCT is a promising technique in the field of fertility preservation: metastases can be detected and additionally cortical ovarian strips can be selected on the basis of high follicle density.

Cell Autonomous Phosphoinositide 3-Kinase Activation in Oocytes Disrupts Normal Ovarian Function Through Promoting Survival and Overgrowth of Ovarian Follicles

PubMed Central

Ebbert, Katherine; Cordeiro, Marilia H.; Romero, Megan; Zhu, Jie; Serna, Vanida Ann; Whelan, Kelly A.; Woodruff, Teresa K.

2015-01-01

In this study, we explored the effects of oocytic phosphoinositide 3-kinase (PI3K) activation on folliculogensis by generating transgenic mice, in which the oocyte-specific Cre-recombinase induces the expression of constitutively active mutant PI3K during the formation of primordial follicles. The ovaries of neonatal transgenic (Cre+) mice showed significantly reduced apoptosis in follicles, which resulted in an excess number of follicles per ovary. Thus, the elevation of phosphatidylinositol (3,4,5)-trisphosphate levels within oocytes promotes the survival of follicles during neonatal development. Despite the increase in AKT phosphorylation, primordial follicles in neonatal Cre+ mice remained dormant demonstrating a nuclear accumulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These primordial follicles containing a high level of nuclear PTEN persisted in postpubertal females, suggesting that PTEN is the dominant factor in the maintenance of female reproductive lifespan through the regulation of primordial follicle recruitment. Although the oocytic PI3K activity and PTEN levels were elevated, the activation of primordial follicles and the subsequent accumulation of antral follicles with developmentally competent oocytes progressed normally in prepubertal Cre+ mice. However, mature Cre+ female mice were anovulatory. Because postnatal day 50 Cre+ mice released cumulus-oocyte complexes with developmentally competent oocytes in response to super-ovulation treatment, the anovulatory phenotype was not due to follicular defects but rather endocrine abnormalities, which were likely caused by the excess number of overgrown follicles. Our current study has elucidated the critical role of oocytic PI3K activity in follicular function, as well as the presence of a PTEN-mediated mechanism in the prevention of immature follicle activation. PMID:25594701

Notch Signaling Regulates Ovarian Follicle Formation and Coordinates Follicular Growth

PubMed Central

Vanorny, Dallas A.; Prasasya, Rexxi D.; Chalpe, Abha J.; Kilen, Signe M.

2014-01-01

Ovarian follicles form through a process in which somatic pregranulosa cells encapsulate individual germ cells from germ cell syncytia. Complementary expression of the Notch ligand, Jagged1, in germ cells and the Notch receptor, Notch2, in pregranulosa cells suggests a role for Notch signaling in mediating cellular interactions during follicle assembly. Using a Notch reporter mouse, we demonstrate that Notch signaling is active within somatic cells of the embryonic ovary, and these cells undergo dramatic reorganization during follicle histogenesis. This coincides with a significant increase in the expression of the ligands, Jagged1 and Jagged2; the receptor, Notch2; and the target genes, Hes1 and Hey2. Histological examination of ovaries from mice with conditional deletion of Jagged1 within germ cells (J1 knockout [J1KO]) or Notch2 within granulosa cells (N2 knockout [N2KO]) reveals changes in follicle dynamics, including perturbations in the primordial follicle pool and antral follicle development. J1KO and N2KO ovaries also contain multi-oocytic follicles, which represent a failure to resolve germ cell syncytia, and follicles with enlarged oocytes but lacking somatic cell growth, signifying a potential role of Notch signaling in follicle activation and the coordination of follicle development. We also observed decreased cell proliferation and increased apoptosis in the somatic cells of both conditional knockout lines. As a consequence of these defects, J1KO female mice are subfertile; however, N2KO female mice remain fertile. This study demonstrates important functions for Jagged1 and Notch2 in the resolution of germ cell syncytia and the coordination of somatic and germ cell growth within follicles of the mouse ovary. PMID:24552588

Oocyte-somatic cell interactions in the human ovary-novel role of bone morphogenetic proteins and growth differentiation factors.

PubMed

Chang, Hsun-Ming; Qiao, Jie; Leung, Peter C K

2016-12-01

Initially identified for their capability to induce heterotopic bone formation, bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor β superfamily. Using cellular and molecular genetic approaches, recent studies have implicated intra-ovarian BMPs as potent regulators of ovarian follicular function. The bi-directional communication of oocytes and the surrounding somatic cells is mandatory for normal follicle development and oocyte maturation. This review summarizes the current knowledge on the physiological role and molecular determinants of these ovarian regulatory factors within the human germline-somatic regulatory loop. The regulation of ovarian function remains poorly characterized in humans because, while the fundamental process of follicular development and oocyte maturation is highly similar across species, most information on the regulation of ovarian function is obtained from studies using rodent models. Thus, this review focuses on the studies that used human biological materials to gain knowledge about human ovarian biology and disorders and to develop strategies for preventing, diagnosing and treating these abnormalities. Relevant English-language publications describing the roles of BMPs or growth differentiation factors (GDFs) in human ovarian biology and phenotypes were comprehensively searched using PubMed and the Google Scholar database. The publications included those published since the initial identification of BMPs in the mammalian ovary in 1999 through July 2016. Studies using human biological materials have revealed the expression of BMPs, GDFs and their putative receptors as well as their molecular signaling in the fundamental cells (oocyte, cumulus/granulosa cells (GCs) and theca/stroma cells) of the ovarian follicles throughout follicle development. With the availability of recombinant human BMPs/GDFs and the development of immortalized human cell lines, functional studies have demonstrated the physiological role of intra-ovarian BMPs/GDFs in all aspects of ovarian functions, from follicle development to steroidogenesis, cell-cell communication, oocyte maturation, ovulation and luteal function. Furthermore, there is crosstalk between these potent ovarian regulators and the endocrine signaling system. Dysregulation or naturally occurring mutations within the BMP system may lead to several female reproductive diseases. The latest development of recombinant BMPs, synthetic BMP inhibitors, gene therapy and tools for BMP-ligand sequestration has made the BMP pathway a potential therapeutic target in certain human fertility disorders; however, further clinical trials are needed. Recent studies have indicated that GDF8 is an intra-ovarian factor that may play a novel role in regulating ovarian functions in the human ovary. Intra-ovarian BMPs/GDFs are critical regulators of folliculogenesis and human ovarian functions. Any dysregulation or variations in these ligands or their receptors may affect the related intracellular signaling and influence ovarian functions, which accounts for several reproductive pathologies and infertility. Understanding the normal and pathological roles of intra-ovarian BMPs/GDFs, especially as related to GC functions and follicular fluid levels, will inform innovative approaches to fertility regulation and improve the diagnosis and treatment of ovarian disorders. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Minireview: roles of the forkhead transcription factor FOXL2 in granulosa cell biology and pathology.

PubMed

Pisarska, Margareta D; Barlow, Gillian; Kuo, Fang-Ting

2011-04-01

The forkhead transcription factor (FOXL2) is an essential transcription factor in the ovary. It is important in ovarian development and a key factor in female sex determination. In addition, FOXL2 plays a significant role in the postnatal ovary and follicle maintenance. The diverse transcriptional activities of FOXL2 are likely attributable to posttranslational modifications and binding to other key proteins involved in granulosa cell function. Mutations of FOXL2 lead to disorders of ovarian function ranging from premature follicle depletion and ovarian failure to unregulated granulosa cell proliferation leading to tumor formation. Thus, FOXL2 is a key regulator of granulosa cell function and a master transcription factor in these cells.

Minireview: Roles of the Forkhead Transcription Factor FOXL2 in Granulosa Cell Biology and Pathology

PubMed Central

Barlow, Gillian; Kuo, Fang-Ting

2011-01-01

The forkhead transcription factor (FOXL2) is an essential transcription factor in the ovary. It is important in ovarian development and a key factor in female sex determination. In addition, FOXL2 plays a significant role in the postnatal ovary and follicle maintenance. The diverse transcriptional activities of FOXL2 are likely attributable to posttranslational modifications and binding to other key proteins involved in granulosa cell function. Mutations of FOXL2 lead to disorders of ovarian function ranging from premature follicle depletion and ovarian failure to unregulated granulosa cell proliferation leading to tumor formation. Thus, FOXL2 is a key regulator of granulosa cell function and a master transcription factor in these cells. PMID:21248146

How Much Does AMH Really Vary in Normal Women?

PubMed Central

La Marca, Antonio; Grisendi, Valentina; Griesinger, Georg

2013-01-01

Anti-Mullerian Hormone (AMH) is an ovarian hormone expressed in growing follicles that have undergone recruitment from the primordial follicle pool but have not yet been selected for dominance. It is considered an accurate marker of ovarian reserve, able to reflect the size of the ovarian follicular pool of a woman of reproductive age. In comparison to other hormonal biomarkers such as serum FSH, low intra- and intermenstrual cycle variability have been proposed for AMH. This review summarizes the knowledge regarding within-subject variability, with particular attention on AMH intracycle variability. Moreover the impact of ethnicity, body mass index, and smoking behaviour on AMH interindividual variability will be reviewed. Finally changes in AMH serum levels in two conditions of ovarian quiescence, namely contraceptives use and pregnancy, will be discussed. The present review aims at guiding researchers and clinicians in interpreting AMH values and fluctuations in various research and clinical scenarios. PMID:24348558

Probiotics Can Induce Follicle Maturational Competence: The Danio rerio Case1

PubMed Central

Gioacchini, Giorgia; Giorgini, Elisabetta; Merrifield, Daniel L.; Hardiman, Gary; Borini, Andrea; Vaccari, Lisa; Carnevali, Oliana

2011-01-01

ABSTRACT In the present study, the effects of the probiotic Lactobacillus rhamnosus IMC 501 on the acquisition of oocyte maturational competence was examined in zebrafish (Danio rerio). L. rhamnosus administration induced the responsiveness of incompetent follicles (stage IIIa) to 17,20-dihydroxy-4-pregnen-3-one and their in vitro maturation. Acquisition of competence by the stage IIIa follicles was further validated by changes of lhr, mprb, inhbaa (activin betaA1), tgfb1, and gdf9 gene expression, which have recently emerged as key regulators of oocyte acquisition of maturational competence, and pou5f1 gene expression, which in other models has been shown to govern the establishment of developmental competence of oocytes. In addition, a DNA microarray experiment was conducted using the same follicles, and with relative gene ontology (GO) data analysis, the molecular effects of probiotic administration emerged. Molecular analysis using PCR-DGGE (denaturing gradient gel electrophoresis) approach, providing information about only the most abundant bacterial members of the microbial community, revealed that the probiotic was able to populate the gastrointestinal tract and modulate the microbial communities, causing a clear shift in them and specifically enhancing the presence of the lactic acid bacteria Streptococcus thermophilus. At the same time, PCR-DGGE analysis revealed that the probiotic was not directly associated with the ovaries. Finally, the effects of probiotic treatment on zebrafish follicle development were also analyzed by FPA (focal plane array) Fourier transform-infrared (FT-IR) imaging, a technique that provides the overall biochemical composition of samples. Changes were found above all in stage IIIa follicles from probiotic-exposed females; the modifications, observed in protein secondary structures as well as in hydration and in bands related to phosphate moieties, allowed us to hypothesize that probiotics act at this follicle stage, affecting the maturation phase. PMID:22088919

Normal female phenotype and ovarian development despite the ovarian expression of the sex-determining region of Y chromosome (SRY) in a 46,XX/69,XXY diploid/triploid mosaic child conceived after in vitro fertilization-intracytoplasmic sperm injection.

PubMed

Oktem, Ozgur; Paduch, Darius A; Xu, Kangpu; Mielnik, Anna; Oktay, Kutluk

2007-03-01

Diploid/triploid mosaicism (mixoploidy) is a rare chromosomal abnormality characterized by mental and growth retardation, hypotonia, and dysmorphic features such as facial asymmetry, low-set ears, and syndactyly. All 46,XX/69,XXY cases fall into three phenotypic groups: male with testicular development, ovotestis disorder of sex development (DSD), or undervirilized male DSD. All phenotypic females with diploid/triploid mosaic reported so far had 46,XX/69,XXX karyotype. We report an 8-year-old girl conceived after in vitro fertilization-intracytoplasmic sperm injection with normal internal/external genital and ovarian development despite 46,XX/69,XXY mosaicism and normal expression of sex-determining region of Y chromosome (SRY) in her gonads. Because of the increased risk of gonadoblastoma resulting from Y chromosome mosaicism, her ovaries were removed by laparoscopy. Ovarian tissue was analyzed histologically as well as by fluorescence in situ hybridization, PCR, and RT-PCR amplification to determine the localization of Y chromosome and expression of SRY and DAX1 mRNA. Methylation-specific PCR was used to assess the inactivation pattern of X chromosomes. By laparoscopy, internal female genital anatomy appeared to be normal. Cytogenetic and molecular methods confirmed the presence of intact and functionally active Y chromosome in the ovary. Strikingly, histological assessment of the gonads showed normal ovarian architecture with abundant primordial follicles despite the presence of the Y chromosome in ovarian follicles and the expression of SRY mRNA in gonadal tissue. This case illustrates that normal ovarian development is possible in the presence of Y chromosome in ovarian follicles and despite the expression of SRY in ovarian tissue. Furthermore, this is the first documented case of mixoploidy after in vitro fertilization-intracytoplasmic sperm injection and the only phenotypic female with 46,XX/69,XXY karyotype.

Ovarian morphology is associated with insulin resistance in women with polycystic ovary syndrome: a cross sectional study.

PubMed

Reid, Sara Pittenger; Kao, Chia-Ning; Pasch, Lauri; Shinkai, Kanade; Cedars, Marcelle I; Huddleston, Heather G

2017-01-01

Polycystic ovary syndrome (PCOS) is a very common disorder well known to be associated with insulin resistance and metabolic disease. Insulin resistance is likely involved in the promotion of the PCOS reproductive phenotype and may mediate some of the ovarian morphology seen in the disorder. The phenotype of each individual woman with PCOS can vary widely as can her metabolic risk. This is a cross-sectional study of patients seen in a multidisciplinary PCOS clinic at the University of California at San Francisco between 2006 and 2014. All participants underwent systematic evaluation with anthropometric measurements, comprehensive skin exam, transvaginal ultrasound and laboratory studies at the time of their initial visit to the clinic. Serum samples were stored and androgen studies were carried out on all stored samples at the University of Virginia. Logistic regression was employed to evaluate the association between ovarian volume or follicle number and metabolic parameters (fasting insulin, HOMA-IR, fasting glucose, 2 h glucose, waist circumference) and hyperandrogenism (free testosterone, total testosterone, DHEAS, acanthosis nigricans), controlling for age. Three-hundred thirteen patients seen during the study period met Rotterdam criteria for PCOS and had sufficient measurements for inclusion in our analysis. The odds ratio of elevated HOMA-IR for patients with a maximum ovarian volume >10 cc was 1.9 compared to those with a maximum ovarian volume of ≤10 cc (95% CI 1.0-3.4). The odds ratio of abnormal fasting insulin for patients with higher ovarian volume was 1.8 (95% CI 1.0-3.4) compared with those with lower ovarian volume. Follicle number was not significantly associated with any metabolic parameters. Increased ovarian volume is associated with markers of insulin resistance in PCOS. In concordance with prior studies, we did not find follicle number to be predictive of metabolic risk. Ovarian volume may serve as a useful tool to aid clinicians in their risk stratification and counseling of patients with PCOS.

Preliminary observations on whole-ovary xenotransplantation as an experimental model for fertility preservation.

PubMed

Nichols-Burns, Stephanie M; Lotz, Laura; Schneider, Heike; Adamek, Edyta; Daniel, Christoph; Stief, Andrea; Grigo, Christina; Klump, Dorothee; Hoffmann, Inge; Beckmann, Matthias W; Dittrich, Ralf

2014-11-01

Ovarian tissue preservation and retransplantation is a promising strategy to restore fertility in cancer survivors. Ischaemia accompanying ovarian tissue grafting, however, can lead to significant follicle loss. Transplantation of the whole ovary by vascular anastomosis has been considered as an alternative to prevent widespread ischaemic damage. In this study, the feasibility and function of transplanting whole ovary with intact vasculature were evaluated, with the goal of developing a xenograft model for studies using donated human ovaries. Whole-swine ovaries with vascular pedicles were perfused and transplanted as intact ovaries by anastomosis into irradiated ovariectomized nude rats (n = 10). The observation period was between 1 and 4 weeks. Fresh swine ovaries served as controls (n = 10). Ovarian stroma and follicle populations were assessed through histological examination in both transplanted and control ovaries. Most of the transplanted whole ovaries (n = 6) maintained stromal quality and all preantral follicle classes were represented, although follicle numbers decreased compared with fresh control. Four transplanted ovaries were fibrotic after 1-4 weeks within the nude rat. Our results demonstrate transplantation of whole-pig ovary into nude rats is possible and support development of this xenograft model system for human studies. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles.

PubMed

Lavranos, T C; Mathis, J M; Latham, S E; Kalionis, B; Shay, J W; Rodgers, R J

1999-08-01

We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells.

Effect of heat stress on the survival and development of in vitro cultured bovine preantral follicles and on in vitro maturation of cumulus-oocyte complex.

PubMed

Paes, V M; Vieira, L A; Correia, H H V; Sa, N A R; Moura, A A A; Sales, A D; Rodrigues, A P R; Magalhães-Padilha, D M; Santos, F W; Apgar, G A; Campello, C C; Camargo, L S A; Figueiredo, J R

2016-09-01

The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used in vitro preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after in vitro culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) from antral follicles (>2-6 mm) grown in vivo (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P < 0.05) the percentage of developing follicles (intermediate, primary, and secondary follicles) at 12 hours and increased levels of ROS at all evaluated time points (12, 24 hours, and D7), when compared to the control (experiment I). Heat stress did not affect (P > 0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the control (12 and 24 hours). In conclusion, HS caused: (1) early activation of primordial follicles; (2) an increase in ROS production by early preantral follicles enclosed in ovarian tissue and by COCs; (3) a short-term reduction of E2 production by COCs; and (4) an increase in P4 secretion from COCs. However, HS did not affect in vitro culture of advanced isolated secondary follicles. Experimental evidence indicates that preantral follicles are less sensitive to HS than COC. Copyright © 2016 Elsevier Inc. All rights reserved.

2,3,7,8-Tetrachlorodibenzo-p-dioxin activates the aryl hydrocarbon receptor and alters sex steroid hormone secretion without affecting growth of mouse antral follicles in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karman, Bethany N., E-mail: bklement@illinois.edu; Basavarajappa, Mallikarjuna S., E-mail: mbshivapur@gmail.com; Craig, Zelieann R., E-mail: zelieann@illinois.edu

The persistent environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an ovarian toxicant. These studies were designed to characterize the actions of TCDD on steroidogenesis and growth of intact mouse antral follicles in vitro. Specifically, these studies tested the hypothesis that TCDD exposure leads to decreased sex hormone production/secretion by antral follicles as well as decreased growth of antral follicles in vitro. Since TCDD acts through binding to the aryl hydrocarbon receptor (AHR), and the AHR has been identified as an important factor in ovarian function, we also conducted experiments to confirm the presence and activation of the AHR in our tissue culturemore » system. To do so, we exposed mouse antral follicles for 96 h to a series of TCDD doses previously shown to have effects on ovarian tissues and cells in culture, which also encompass environmentally relevant and pharmacological exposures (0.1–100 nM), to determine a dose response for TCDD in our culture system for growth, hormone production, and expression of the Ahr and Cyp1b1. The results indicate that TCDD decreases progesterone, androstenedione, testosterone, and estradiol levels in a non-monotonic dose response manner without altering growth of antral follicles. The addition of pregnenolone substrate (10 μM) restores hormone levels to control levels. Additionally, Cyp1b1 levels were increased by 3–4 fold regardless of the dose of TCDD exposure, evidence of AHR activation. Overall, these data indicate that TCDD may act prior to pregnenolone formation and through AHR transcriptional control of Cyp1b1, leading to decreased hormone levels without affecting growth of antral follicles. -- Highlights: ►TCDD disrupts sex steroid hormone levels, but not growth of antral follicles. ►Pregnenolone co-treatment by-passes TCDD-induced steroid hormone disruption. ►TCDD affects steroid hormone levels through an AHR pathway in antral follicles.« less

The natural insect peptide Neb-colloostatin induces ovarian atresia and apoptosis in the mealworm Tenebrio molitor.

PubMed

Czarniewska, Elżbieta; Rosiński, Grzegorz; Gabała, Elżbieta; Kuczer, Mariola

2014-01-30

The injection of Neb-colloostatin into T. molitor females causes gonadoinhibitory effects on ovarian development. This peptide inhibits intercellular space formation (patency) in follicular epithelium and results in slowed vitellogenesis, delayed ovulation, reduced number of eggs laid and presumably cell death in the terminal follicles. However, as does the form of cell death in the terminal follicle, the mode of action of Neb-colloostatin remains unknown. We tested Neb-colloostatin for a sterilizing effect on females of Tenebrio molitor. We report that injection of nanomolar doses of Neb-colloostatin induce ovarian follicle atresia in 4-day old females during their first gonadotropic cycle. Light microscope observations revealed morphological changes in the ovary: after Neb-colloostatin injection the terminal oocytes are significantly smaller and elicit massive follicle resorption, but the control terminal follicles possess translucent ooplasm in oocytes at different stages of vitellogenesis. A patency is visible in follicular epithelium of the control vitellogenic oocytes, whereas peptide injection inhibits intercellular space formation and, in consequence, inhibits vitellogenesis. Confocal and electron microscope examination showed that peptide injection causes changes in the morphology indicating death of follicular cells. We observed F-actin cytoskeleton disorganization, induction of caspase activity, changes in chromatin organization and autophagic vacuole formation. Moreover, the apical cytoplasm of follicular cells is filled with numerous free ribosomes, probably indicating a higher demand for protein biosynthesis, especially in preparation for autophagic vacuole formation. On the other hand, the process of polyribosomes formation is inhibited, indicating the contributing effect of this hormone. Neb-colloostatin induces atresia in the mealworm ovary. Degeneration of T. molitor follicles includes changes in morphology and viability of follicular cells, and oosorption as a consequence of these changes.

The natural insect peptide Neb-colloostatin induces ovarian atresia and apoptosis in the mealworm Tenebrio molitor

PubMed Central

2014-01-01

Background The injection of Neb-colloostatin into T. molitor females causes gonadoinhibitory effects on ovarian development. This peptide inhibits intercellular space formation (patency) in follicular epithelium and results in slowed vitellogenesis, delayed ovulation, reduced number of eggs laid and presumably cell death in the terminal follicles. However, as does the form of cell death in the terminal follicle, the mode of action of Neb-colloostatin remains unknown. Results We tested Neb-colloostatin for a sterilizing effect on females of Tenebrio molitor. We report that injection of nanomolar doses of Neb-colloostatin induce ovarian follicle atresia in 4-day old females during their first gonadotropic cycle. Light microscope observations revealed morphological changes in the ovary: after Neb-colloostatin injection the terminal oocytes are significantly smaller and elicit massive follicle resorption, but the control terminal follicles possess translucent ooplasm in oocytes at different stages of vitellogenesis. A patency is visible in follicular epithelium of the control vitellogenic oocytes, whereas peptide injection inhibits intercellular space formation and, in consequence, inhibits vitellogenesis. Confocal and electron microscope examination showed that peptide injection causes changes in the morphology indicating death of follicular cells. We observed F-actin cytoskeleton disorganization, induction of caspase activity, changes in chromatin organization and autophagic vacuole formation. Moreover, the apical cytoplasm of follicular cells is filled with numerous free ribosomes, probably indicating a higher demand for protein biosynthesis, especially in preparation for autophagic vacuole formation. On the other hand, the process of polyribosomes formation is inhibited, indicating the contributing effect of this hormone. Conclusion Neb-colloostatin induces atresia in the mealworm ovary. Degeneration of T. molitor follicles includes changes in morphology and viability of follicular cells, and oosorption as a consequence of these changes. PMID:24479487

DOE Office of Scientific and Technical Information (OSTI.GOV)

Madden, Jill A.; Hoyer, Patricia B.; Devine, Patrick J.

The finite ovarian follicle reserve can be negatively impacted by exposure to chemicals including the anti-neoplastic agent, cyclophosphamide (CPA). CPA requires bioactivation to phosphoramide mustard (PM) to elicit its therapeutic effects however; in addition to being the tumor-targeting metabolite, PM is also ovotoxic. In addition, PM can break down to a cytotoxic, volatile metabolite, chloroethylaziridine (CEZ). The aim of this study was initially to characterize PM-induced ovotoxicity in growing follicles. Using PND4 Fisher 344 rats, ovaries were cultured for 4 days before being exposed once to PM (10 or 30 μM). Following eight additional days in culture, relative to controlmore » (1% DMSO), PM had no impact on primordial, small primary or large primary follicle number, but both PM concentrations induced secondary follicle depletion (P < 0.05). Interestingly, a reduction in follicle number in the control-treated ovaries was observed. Thus, the involvement of a volatile, cytotoxic PM metabolite (VC) in PM-induced ovotoxicity was explored in cultured rat ovaries, with control ovaries physically separated from PM-treated ovaries during culture. Direct PM (60 μM) exposure destroyed all stage follicles after 4 days (P < 0.05). VC from nearby wells depleted primordial follicles after 4 days (P < 0.05), temporarily reduced secondary follicle number after 2 days, and did not impact other stage follicles at any other time point. VC was determined to spontaneously liberate from PM, which could contribute to degradation of PM during storage. Taken together, this study demonstrates that PM and VC are ovotoxicants, with different follicular targets, and that the VC may be a major player during PM-induced ovotoxicity observed in cancer survivors. - Highlights: • PM depletes all stage ovarian follicles in a temporal pattern. • A volatile ovotoxic compound is liberated from PM. • The volatile metabolite depletes primordial follicles.« less

Biochemical and hormonal analysis of follicular fluid and serum of female dromedary camels (Camelus dromedarius) with different sized ovarian follicles.

PubMed

El-Bahr, S M; Ghoneim, I M; Waheed, M M

2015-08-01

The current study aimed to compare some biochemical and hormonal constituents in follicular fluids and serum of female dromedary camels with different sized ovarian follicles. Therefore, follicular fluids from follicles sized 1.1-1.5cm (n=10), 1.6-2.1cm (n=10) and 2.2-2.5cm (n=10) and sera were harvested from 20 female camels. The concentrations of ascorbic acid, glucose, cholesterol and activities of acid phosphatase (ACP) and alkaline phosphatase (ALP) were not changed significantly neither in follicular fluids of all follicle sizes nor in sera of female camels with different sized follicles. The concentrations of estradiol-17β (E2) in the follicular fluid of follicles sized 2.2-2.5cm were significantly lower (P<0.01) than its corresponding value in follicular fluid of other follicle sizes. The concentrations of progesterone (P4), tri-iodothyronine (T3), thyroxin (T4), cortisol and insulin-like growth factor-1 (IGF-1) remained comparable in follicular fluids of all examined different sized follicles. The concentrations of E2, P4, T3, T4, cortisol and IGF-1 were similar in the serum of camels with different sized follicles. Interestingly, mean concentrations of P4 and IGF-1 in follicular fluids were higher than their corresponding values in sera of camels with different sized follicles and the mean concentrations of glucose, cholesterol, ALP and cortisol in sera were higher than their corresponding values in follicular fluids of the examined camels. With the exception of E2, there were no significant differences in biochemical and hormonal constituents between follicular fluids from different sized follicles. Copyright © 2015 Elsevier B.V. All rights reserved.

Co-expression of the Follicle Stimulating Hormone Receptor and Stem Cell Markers: A Novel Approach to Target Ovarian Cancer Stem Cells

DTIC Science & Technology

2012-09-01

ovarian cancer stem cell markers to consider it as a new experimental target for novel nanotechnology approaches capable of destroying ovarian cancer stem...FSHR mRNA after several generations in an amount consistent with stem cell characteristics. Nude mouse experiments to confirm co-expression in vivoare

Transitional gene expression profiling in ovarian follicle during ovulation in normal-cycle rats.

PubMed

Tsubota, Kenjiro; Kanki, Masayuki; Noto, Takahisa; Shiraki, Katsuhisa; Takeuchi, Ayano; Nakatsuji, Shunji; Seki, Jiro; Oishi, Yuji; Matsumoto, Masahiro; Nakayama, Hiroyuki

2011-06-01

Evaluation of ovarian toxicity requires an understanding of the physiological changes related to the estrous cycle in the ovary. The authors investigated the transitional gene expression profile of ovulatory follicles in rats that show normal estrous cyclicity. Ovaries were collected at 10:00 and 22:00 on the proestrus day and at 10:00 on the estrus day. Ovarian follicles or early corpora lutea were isolated using laser microdissection, and extracted total RNA was analyzed using microarray technology. Clustering analysis revealed four different expression patterns: transient up- or down-regulation only at 22:00 on the proestrus day (pattern 1), up- or down-regulation only at 10:00 on the estrus day (pattern 2), continuous increase at 22:00 on the proestrus day and at 10:00 on the estrus day (pattern 3), and up- or down-regulation at 22:00 on the proestrus day and level maintenance at 10:00 on the estrus day (pattern 4). In addition, these probe sets were functionally categorized in each pattern using the Ingenuity Pathways Analysis database. These data will aid in understanding the physiology of ovulation and may be useful in assessing ovarian toxicity and its mechanism, such as in investigations of chemical-induced ovulatory impairment.

Expression of regulator of G-protein signaling protein-2 gene in the rat ovary at the time of ovulation.

PubMed

Ujioka, T; Russell, D L; Okamura, H; Richards, J S; Espey, L L

2000-11-01

The ovulatory process in mammals begins when an endogenous surge in LH circulates to the ovary and couples with receptors in the plasma membranes of granulosa cells in mature ovarian follicles. This study provides evidence that the ovulatory stimulus includes induction of the gene for regulator of G-protein signaling protein-2 (RGS2). Immature Wistar rats were primed with 10 IU eCG s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG (a homolog of LH) s.c. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for reverse transcription-polymerase chain reaction differential display to detect gene expression in the stimulated ovarian tissue. Two of the amplified cDNAs that were upregulated within 2 h after the ovaries had been stimulated by hCG were homologous to segments of the mouse gene for RGS2. In situ hybridization indicated that the RGS2 mRNA was expressed in the granulosa layer of mature follicles. In conclusion, the gene for RGS2, which is known to regulate membrane signaling pathways, is transcribed in ovarian follicles in response to an ovulatory dose of gonadotropin.

Developmental programming: impact of prenatal testosterone excess on ovarian cell proliferation and apoptotic factors in sheep.

PubMed

Salvetti, Natalia R; Ortega, Hugo H; Veiga-Lopez, Almudena; Padmanabhan, Vasantha

2012-07-01

Prenatal testosterone (T) excess leads to reproductive dysfunctions in sheep, which include increased ovarian follicular recruitment and persistence. To test the hypothesis that follicular disruptions in T sheep stem from changes in the developmental ontogeny of ovarian proliferation and apoptotic factors, pregnant Suffolk sheep were injected twice weekly with T propionate or dihydrotestosterone propionate (DHT; a nonaromatizable androgen) from Days 30 to 90 of gestation. Changes in developmental expression of proliferating cell nuclear antigen (PCNA), BCL2, BAX, activated CASP3, and FAS/FASLG were determined at Fetal Days 90 and 140, 22 wk, 10 mo, and 21 mo of age by immunocytochemisty. Prenatal T treatment induced changes in expression of proliferative and apoptotic markers in a follicle-, age-, and steroid-specific manner. Changes in BAX were evident only during fetal life and PCNA, BCL2, and CASP3 only postnatally. Prenatal T and not DHT increased PCNA and decreased BCL2 in granulosa/theca cells of antral follicles at 10 and 21 mo but decreased CASP3 in granulosa/theca cells of antral follicles at 22 wk (prepubertal) and 10 and 21 mo. Both treatments decreased BAX immunostaining in granulosa cells of Fetal Day 90 primordial/primary follicles. Neither treatment affected FAS expression at any developmental time point in any follicular compartment. Effects on BAX appear to be programmed by androgenic actions and PCNA, BCL2, and CASP3 by estrogenic actions of T. Overall, the findings demonstrate that fetal exposure to excess T disrupts the ovarian proliferation/apoptosis balance, thus providing a basis for the follicular disruptions evidenced in these females.

Mechanisms influencing establishment of the ovarian reserve in heifers

USDA-ARS?s Scientific Manuscript database

Ultrasonography serves as a diagnostic tool for understanding what is occurring at the microscopic level in the bovine ovary. Situations exist, however, in which genetic or nutritional influences can alter the population of microscopic follicles without altering the number of antral follicles obser...

Equol inhibits growth, induces atresia, and inhibits steroidogenesis of mouse antral follicles in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mahalingam, Sharada, E-mail: mahalin2@illinois.edu; Gao, Liying, E-mail: lgao@uiuc.edu; Gonnering, Marni, E-mail: mgonne2@illinois.edu

Equol is a non-steroidal estrogen metabolite produced by microbial conversion of daidzein, a major soy isoflavone, in the gut of some humans and many animal species. Isoflavones and their metabolites can affect endogenous estradiol production, action, and metabolism, potentially influencing ovarian follicle function. However, no studies have examined the effects of equol on intact ovarian antral follicles, which are responsible for sex steroid synthesis and further development into ovulatory follicles. Thus, the present study tested the hypothesis that equol inhibits antral follicle growth, increases follicle atresia, and inhibits steroidogenesis in the adult mouse ovary. To test this hypothesis, antral folliclesmore » isolated from adult CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or equol (600 nM, 6 μM, 36 μM, and 100 μM) for 48 and 96 h. Every 24 h, follicle diameters were measured to monitor growth. At 48 and 96 h, the culture medium was subjected to measurement of hormone levels, and the cultured follicles were subjected to gene expression analysis. Additionally, follicles were histologically evaluated for signs of atresia after 96 h of culture. The results indicate that equol (100 μM) inhibited follicle growth, altered the mRNA levels of bcl2-associated X protein and B cell leukemia/lymphoma 2, and induced follicle atresia. Further, equol decreased the levels of estradiol, testosterone, androstenedione, and progesterone, and it decreased mRNA levels of cholesterol side-chain cleavage, steroid 17-α-hydroxalase, and aromatase. Collectively, these data indicate that equol inhibits growth, increases atresia, and inhibits steroidogenesis of cultured mouse antral follicles. - Highlights: • Equol exposure inhibits antral follicle growth. • Equol exposure increases follicle atresia. • Equol exposure inhibits sex steroid hormone levels. • Equol exposure inhibits mRNA levels of certain steroidogenic enzymes.« less

Robo1/2 regulate follicle atresia through manipulating granulosa cell apoptosis in mice

PubMed Central

Li, Jiangchao; Ye, Yuxiang; Zhang, Renli; Zhang, Lili; Hu, Xiwen; Han, Dong; Chen, Jiayuan; He, Xiaodong; Wang, Guang; Yang, Xuesong; Wang, Lijing

2015-01-01

Secreted Slit proteins and their Roundabout (Robo) receptors act as a repulsive cue to preventaxons from migrating to inappropriate locations during the development of the nervous system. Slit/Robo has also been implicated in reproductive system development, but the molecular mechanism of the Slit/Robo pathway in the reproductive system remains poorly understood. Using a transgenic mouse model, we investigated the function of the Slit/Robo pathway on ovarian follicle development and atresia. We first demonstrated that more offspring were born to mice with a partial knockout of the Robo1/2 genes in mice. We next showed that Robo1 and Robo2 are strongly expressed in ovarian granulosacells. Apoptosis in granulosa cells was reduced when Robo1/2 were partially knocked out, and this observation was further verified by in vitro Robo1/2 knockout experiments in mouse and human granulosa cells. We also found that ovarian angiogenesis wasenhanced by a partial lack of Robo1/2 genes. In summary, our data suggest that the Slit/Robo pathway can impact follicle development and atresia by influencinggranulosa cell apoptosis. PMID:25988316

Survival and growth of human preantral follicles after cryopreservation of ovarian tissue, follicle isolation and short-term xenografting.

PubMed

Paulini, Fernanda; Vilela, Janice M V; Chiti, Maria Costanza; Donnez, Jacques; Jadoul, Pascale; Dolmans, Marie-Madeleine; Amorim, Christiani A

2016-09-01

In women, chemotherapy and radiotherapy can be harmful to the ovaries, causing loss of endocrine and reproductive functions. When gonadotoxic treatment cannot be delayed, ovarian tissue cryobanking is the only way of preserving fertility. This technique, however, is not advisable for patients with certain types of cancer, because of the risk of reintroducing malignant cells present in the cryopreserved tissue. Our objective is therefore to develop a transplantable artificial ovary. To this end, cryopreserved human preantral follicles were isolated and embedded in fibrin formulations prepared with 50 mg/ml fibrinogen and 10 IU/ml thrombin supplemented or not with 3% hyaluronic acid, and respectively xenografted to specially created right and left peritoneal pockets in eight nude mice. On days 0 and 7, the animals were killed and the matrices retrieved. On day 7, no difference was observed in the recovery rate of follicles embedded in fibrin alone (23.4%) or fibrin-hyaluronic acid (20.5%). Ki67 staining confirmed growth of the grafted follicles and terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay revealed 100% of the follicles to be viable in both groups on day 7. In conclusion, fibrin seems to be a promising material for creation of an artificial ovary, supporting follicle survival and development. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Does methotrexate administration for ectopic pregnancy after in vitro fertilization impact ovarian reserve or ovarian responsiveness?

PubMed

Boots, Christina E; Gustofson, Robert L; Feinberg, Eve C

2013-12-01

To evaluate the effects of methotrexate (MTX) on the future fertility of women undergoing IVF by comparing ovarian reserve and ovarian responsiveness in the IVF cycle before and after an ectopic pregnancy (EP) treated with MTX. Retrospective cohort study. Private reproductive endocrinology and infertility practice. Sixty-six women undergoing IVF before and after receiving MTX for an EP. Methotrexate administration and ovarian stimulation. Markers of ovarian reserve (day 3 FSH, antral follicle count), measures of ovarian responsiveness (duration of stimulation, peak E2 level, total dose of gonadotropins, number of oocytes retrieved, fertilization rate), and time from MTX administration to subsequent IVF cycle. There were no differences after MTX administration in body mass index (BMI), FSH, or antral follicle count. A greater dose of gonadotropins was used in the cycle after MTX, but there were no differences in numbers of oocytes retrieved or high quality embryos transferred. As expected, there was a slight increase in age in the subsequent IVF cycle. The pregnancy rates (PR) were comparable to the average PRs within the practice when combining all age groups. Methotrexate remains the first line of therapy for medical management of asymptomatic EP and does not compromise ovarian reserve, ovarian responsiveness, or IVF success in subsequent cycles. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Comparison of the effects of laparoscopic bipolar electrocoagulation and intracorporeal suture application to ovarian reserve in benign ovarian cysts.

PubMed

Özgönen, Hakan; Erdemoglu, Evrim; Günyeli, Ilker; Güney, Mehmet; Mungan, Tamer

2013-04-01

Aim of the present study is to determine the effects of bipolar electrocoagulation and intracorporeal suture on the ovarian reserve after ovarian cystectomy. Sixty patients aged 18-42 years old and with a persistent adnexal mass were recruited to the study. Patients were randomized into suture hemostasis group or bipolar hemostasis group. Laparoscopic ovarian cystectomy was performed to all patients. Hemostasis was obtained by bipolar coagulation in 30 patients and by intracorporeal sutures in 30 patients. Serum levels of FSH, LH, estradiol, inhibin B and ultrasonographic measurements (antral follicle count and ovarian volume) were analyzed and recorded at day 3 of menstrual cycle, 1 and 3 months after the surgery. Basal FSH level measurement at the postoperative third month was significantly increased to 6.96 ± 1.86 mIU/ml (p < 0.05) in the bipolar electrocoagulation group. However, the decreased ovarian volume and antral follicle count was restored at the postoperative third month in the bipolar electrocoagulation group. Preoperative and postoperative FSH, LH, estradiol and inhibin B levels and ultrasonographic measurements were similar in the intracorporeal suture group. The unwanted effect of bipolar electrocoagulation on ovarian reserve is probably transient and causes minimal damage to ovary. FSH levels may be slightly elevated. Gentle use of bipolar electrocoagulation or intracorporeal are not found to effect ovarian reserve.

Ovarian structure and oogenesis of the extremophile viviparous teleost Poecilia mexicana (Poeciliidae) from an active sulfur spring cave in Southern Mexico.

PubMed

Torres-Martínez, Aarón; Hernández-Franyutti, Arlette; Uribe, Mari Carmen; Contreras-Sánchez, Wilfrido Miguel

2017-12-01

The structure of the ovary and oogenesis of Poecilia mexicana from an active sulfur spring cave is documented. Poecilia mexicana is the only poeciliid adapted to a subterranean environment with high hydrogen sulfide levels and extreme hypoxic conditions. Twenty females were captured throughout one year at Cueva del Azufre, located in the State of Tabasco in Southern Mexico. Ovaries were processed with histological techniques. P. mexicana has a single, ovoid ovary with ovigerous lamella that project to the ovarian lumen. The ovarian wall presents abundant loose connective tissue, numerous melanomacrophage centers and large blood vessels, possibly associated with hypoxic conditions. The germinal epithelium bordering the ovarian lumen contains somatic and germ cells forming cell nests projecting into the stroma. P. mexicana stores sperm in ovarian folds associated with follicles at different developmental phases. Oogenesis in P. mexicana consisted of the following stages: (i) oogonial proliferation, (ii) chromatin nucleolus, (iii) primary growth, subdivided into: (a) one nucleolus, (b) multiple nucleoli, (c) droplet oils-cortical alveoli steps; (iv) secondary growth, subdivided in: (a) early secondary growth, (b) late secondary growth, and (c) full grown. Follicular atresia was present in all stages of follicular development; it was characterized by oocyte degeneration, where follicle cells hypertrophy and differentiate in phagocytes. The ovary and oogenesis are similar to these seen in other poeciliids, but we found frequent atretic follicles, melanomacrophage centers, reduced fecundity and increased of offspring size. © 2017 Wiley Periodicals, Inc.

Developmental programming: prenatal steroid excess disrupts key members of intraovarian steroidogenic pathway in sheep.

PubMed

Padmanabhan, Vasantha; Salvetti, Natalia R; Matiller, Valentina; Ortega, Hugo H

2014-09-01

Prenatal testosterone (T) excess disrupts ovarian cyclicity and increases circulating estradiol levels as well as follicular recruitment and persistence culminating in multifollicular ovary similar to women with polycystic ovary syndrome. We tested whether prenatal T excess, by androgenic or estrogenic action, disrupts the steroid biosynthetic machinery in sheep in a cell-, follicle stage-, age-, and treatment-specific manner consistent with the ovarian disruptions and increased estradiol release. Impact of T/dihydrotestosterone (DHT) treatments from days 30-90 of gestation on steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, cytochrome P-450 17α-hydroxylase/C17, 20-lyase (CYP17A1), and cytochrome P-450 aromatase (CYP19A1) were examined on fetal day 90, 140 and 10 months (postpubertal), and 21 months (adult, no DHT group) of age by immunohistochemistry. All 4 markers changed in a cell-, follicle stage-, and age-specific manner. Both treatments increased steroidogenic acute regulatory protein expression in preantral follicles of postpubertal and adult females. Effects of prenatal T and DHT on 3β-hydroxysteroid dehydrogenase differed in a follicle- and age-specific manner. CYP17A1 was reduced in the theca interna of antral follicles by T, but not DHT, in 10- and 21-month-old females. CYP19A1 was reduced by both T and DHT at all ages barring an increase on fetal day 140. Reduced granulosa CYP19A1 and thecal CYP17A1 in adults likely disrupt the intrafollicular androgen/estrogen balance contributing to follicular persistence. The reduced thecal CYP17A1 expression suggests that the hyperandrogenic ovarian phenotype may originate from increased enzyme activity or alternatively via a different isoform of CYP17. The reduced CYP19A1 in antral follicles of adults indicates that the increased circulating estradiol release likely arises from the increased number of persisting follicles.

A new step toward the artificial ovary: survival and proliferation of isolated murine follicles after autologous transplantation in a fibrin scaffold.

PubMed

Luyckx, Valérie; Dolmans, Marie-Madeleine; Vanacker, Julie; Legat, Camille; Fortuño Moya, Cristina; Donnez, Jacques; Amorim, Christiani Andrade

2014-04-01

To create an artificial ovary to provide an alternative way of restoring fertility in patients who cannot benefit from transplantation of cryopreserved ovarian tissue due to the threat of reintroducing malignant cells. In vivo experimental study. Gynecology research unit in a university hospital. Six-week-old female NMRI mice. Autografting of isolated preantral follicles and ovarian cells (OCs) encapsulated in two fibrin matrices containing low concentrations of fibrinogen (F; mg/mL) and thrombin (T; IU/mL): F12.5/T1 and F25/T4. Follicular density and development, OC survival and proliferation, inflammatory response, and vascularization. After 1 week, the follicle recovery rate ranged from 30.8% (F25/T4) to 31.8% (F12.5/T1). With both fibrin formulations, all follicles were found to be alive or minimally damaged, as demonstrated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, and at the growing stage (primary, secondary, and antral follicles), confirmed by Ki67 immunostaining. Isolated OCs also survived and proliferated after grafting, as evidenced by <1% apoptotic cells and a high proportion of Ki67-positive cells. Vessels were found in both fibrin formulations, and the global vascular surface area varied from 1.35% (F25/T4) to 1.88% (F12.5/T1). Numerous CD45-positive cells were also observed in both F25/T4 and F12.5/T1 combinations. The present study is the first to show survival and growth of isolated murine ovarian follicles 1 week after autotransplantation of isolated OCs in a fibrin scaffold. The results indicate that fibrin is a promising candidate as a matrix for the construction of an artificial ovary. Xenotransplantation of isolated human follicles and OCs is the necessary next step to validate these findings. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Uterine and systemic inflammation influences ovarian follicular function in postpartum dairy cows

PubMed Central

Sá Filho, Ocilon G.; Absalon-Medina, Victor A.; Schneider, Augusto; Butler, W. R.; Gilbert, Robert O.

2017-01-01

The objective of this study was to determine the effects of uterine and systemic inflammatory responses to uterine bacterial contamination at calving in dairy cows on the growth and ovulatory outcomes of the first dominant follicle postpartum. Ovulatory capability of the first dominant follicle postpartum was predicted in 53 multiparous cows by using a combination of follicle growth characteristics and circulating estradiol concentrations. Endotoxin levels were assayed in follicular fluid samples that were aspirated the day after ovulatory outcome prediction. Plasma levels of haptoglobin, a proinflammatory acute phase protein, and paraoxonase, a negative acute phase protein were determined. Uterine bacteria and inflammation were evaluated in three uterine fluid samples from each cow collected on the day of calving, the day after follicle aspiration, and at 35 days postpartum. Cows that had a strong initial uterine inflammatory response (robust recruitment of polymorphonuclear leukocytes of ≥ 35% and cows with uterine pH < 8.5 on the day of calving) were more likely to have an ovulatory first dominant follicle. Follicular fluid endotoxin levels were higher in non-ovulatory cows compared with ovulatory cows. Endotoxin levels in circulation were not different between ovulatory groups but were higher prepartum than on day 7 and 14 postpartum. Systemic inflammation characterized by elevated haptoglobin concentrations was higher in non-ovulatory cows despite similar bacterial contamination and circulating endotoxin levels. Paraoxonase activity in follicular fluid was significantly associated with the paraoxonase activity in plasma, however, plasma paraoxonase concentrations were not different between non-ovulatory and ovulatory cows. Cows with a higher uterine bacterial load on the day of calving had slower ovarian follicle growth. In summary, a robust uterine inflammatory response on the day of calving was positively associated with ovarian function while elevated systemic inflammation during the early postpartum period was negatively associated with the ovulatory status of the first dominant follicle postpartum. PMID:28542500

Systemic compensatory response to neonatal estradiol exposure does not prevent depletion of the oocyte pool in the rat.

PubMed

Chalmey, Clémentine; Giton, Frank; Giton, Franck; Chalmel, Frédéric; Fiet, Jean; Jégou, Bernard; Mazaud-Guittot, Séverine

2013-01-01

The formation of ovarian follicles is a finely tuned process that takes place within a narrow time-window in rodents. Multiple factors and pathways have been proposed to contribute to the mechanisms triggering this process but the role of endocrine factors, especially estrogens, remains elusive. It is currently hypothesized that removal from the maternal hormonal environment permits follicle formation at birth. However, experimentally-induced maintenance of high 17β-estradiol (E2) levels leads to subtle, distinct, immediate effects on follicle formation and oocyte survival depending on the species and dose. In this study, we examined the immediate effects of neonatal E2 exposure from post-natal day (PND) 0 to PND2 on the whole organism and on ovarian follicle formation in rats. Measurements of plasma E2, estrone and their sulfate conjugates after E2 exposure showed that neonatal female rats rapidly acquire the capability to metabolize and clear excessive E2 levels. Concomitant modifications to the mRNA content of genes encoding selected E2 metabolism enzymes in the liver and the ovary in response to E2 exposure indicate that E2 may modify the neonatal maturation of these organs. In the liver, E2 treatment was associated with lower acquisition of the capability to metabolize E2. In the ovary, E2 depleted the oocyte pool in a dose dependent manner by PND3. In 10 µg/day E2-treated ovaries, apoptotic oocytes were observed in newly formed follicles in addition to areas of ovarian cord remodeling. At PND6, follicles without any visible oocyte were present and multi-oocyte follicles were not observed. Our study reveals a major species-difference. Indeed, neonatal exposure to E2 depletes the oocyte pool in the rat ovary, whereas in the mouse it is well known to increase oocyte survival.

Evidence of a local negative role for cocaine and amphetamine regulated transcript (CART), inhibins and low molecular weight insulin like growth factor binding proteins in regulation of granulosa cell estradiol production during follicular waves in cattle

PubMed Central

Kobayashi, Yasuhiro; Jimenez-Krassel, Fermin; Ireland, James J; Smith, George W

2006-01-01

The ability of ovarian follicles to produce large amounts of estradiol is a hallmark of follicle health status. Estradiol producing capacity is lost in ovarian follicles before morphological signs of atresia. A prominent wave like pattern of growth of antral follicles is characteristic of monotocous species such as cattle, horses and humans. While our knowledge of the role of pituitary gonadotropins in support of antral follicle growth and development is well established, the intrinsic factors that suppress estradiol production and may help promote atresia during follicular waves are not well understood. Numerous growth factors and cytokines have been reported to suppress granulosa cell estradiol production in vitro, but the association of expression of many such factors in vivo with follicle health status and their physiological significance are not clear. The purpose of this review is to discuss the in vivo and in vitro evidence supporting a local physiological role for cocaine and amphetamine regulated transcript, inhibins and low molecular weight insulin like growth factor binding proteins in negative regulation of granulosa cell estradiol production, with emphasis on evidence from the bovine model system. PMID:16611367

Successful vitrification and autografting of baboon (Papio anubis) ovarian tissue.

PubMed

Amorim, Christiani A; Jacobs, Sophie; Devireddy, Ram V; Van Langendonckt, Anne; Vanacker, Julie; Jaeger, Jonathan; Luyckx, Valérie; Donnez, Jacques; Dolmans, Marie-Madeleine

2013-08-01

Can a vitrification protocol using an ethylene glycol/dimethyl sulphoxide-based solution and a cryopin successfully cryopreserve baboon ovarian tissue? Our results show that baboon ovarian tissue can be successfully cryopreserved with our vitrification protocol. Non-human primates have already been used as an animal model to test vitrification protocols for human ovarian tissue cryopreservation. Ovarian biopsies from five adult baboons were vitrified, warmed and autografted for 5 months. After grafting, follicle survival, growth and function and also the quality of stromal tissue were assessed histologically and by immunohistochemistry. The influence of the vitrification procedure on the cooling rate was evaluated by a computer model. After vitrification, warming and long-term grafting, follicles were able to grow and maintain their function, as illustrated by Ki67, anti-Müllerian hormone (AMH) and growth differentiation factor-9 (GDF-9) immunostaining. Corpora lutea were also observed, evidencing successful ovulation in all the animals. Stromal tissue quality did not appear to be negatively affected by our cryopreservation procedure, as demonstrated by vascularization and proportions of fibrotic areas, which were similar to those found in fresh ungrafted ovarian tissue. Despite our promising findings, before applying this technique in a clinical setting, we need to validate it by achieving pregnancies. In addition to encouraging results obtained with our vitrification procedure for non-human ovarian tissue, this study also showed, for the first time, expression of AMH and GDF-9 in ovarian follicles. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie No. 7.4507.10, grant 3.4.590.08 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, Fondation St Luc, Foundation Against Cancer, and Department of Mechanical Engineering at Louisiana State University (support to Ram Devireddy), and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors has any competing interests to declare.

The local effects of ovarian diathermy in an ovine model of polycystic ovary syndrome.

PubMed

Connolly, Fiona; Rae, Michael T; Butler, Mairead; Klibanov, Alexander L; Sboros, Vassilis; McNeilly, Alan S; Duncan, W Colin

2014-01-01

In order to develop a medical alternative to surgical ovarian diathermy (OD) in polycystic ovary syndrome (PCOS) more mechanistic information is required about OD. We therefore studied the cellular, molecular and vascular effects of diathermy on the ovary using an established ovine model of PCOS. Pregnant sheep were treated twice weekly with testosterone propionate (100 mg) from day 30-100 gestation. Their female offspring (n = 12) were studied during their second breeding season when the PCOS-like phenotype, with anovulation, is fully manifest. In one group (n = 4) one ovary underwent diathermy and it was collected and compared to the contralateral ovary after 24 hours. In another group a treatment PCOS cohort underwent diathermy (n = 4) and the ovaries were collected and compared to the control PCOS cohort (n = 4) after 5 weeks. Ovarian vascular indices were measured using contrast-enhanced ultrasound and colour Doppler before, immediately after, 24 hours and five weeks after diathermy. Antral follicles were assessed by immunohistochemistry and ovarian stromal gene expression by quantitative RT-PCR 24 hours and 5 weeks after diathermy. Diathermy increased follicular atresia (P<0.05) and reduced antral follicle numbers after 5 weeks (P<0.05). There was an increase in stromal CCL2 expression 24 hours after diathermy (P<0.01) but no alteration in inflammatory indices at 5 weeks. Immediately after diathermy there was increased microbubble transit time in the ovarian microvasculature (P = 0.05) but this was not seen at 24 hours. However 24 hours after diathermy there was a reduction in the stromal Doppler blood flow signal (P<0.05) and an increased ovarian resistance index (P<0.05) both of which persisted at 5 weeks (P<0.01; P<0.05). In the ovine model of PCOS, OD causes a sustained reduction in ovarian stromal blood flow with an increased ovarian artery resistance index associated with atresia of antral follicles.

The Local Effects of Ovarian Diathermy in an Ovine Model of Polycystic Ovary Syndrome

PubMed Central

Connolly, Fiona; Rae, Michael T.; Butler, Mairead; Klibanov, Alexander L.; Sboros, Vassilis; McNeilly, Alan S.; Duncan, W. Colin

2014-01-01

In order to develop a medical alternative to surgical ovarian diathermy (OD) in polycystic ovary syndrome (PCOS) more mechanistic information is required about OD. We therefore studied the cellular, molecular and vascular effects of diathermy on the ovary using an established ovine model of PCOS. Pregnant sheep were treated twice weekly with testosterone propionate (100 mg) from day 30–100 gestation. Their female offspring (n = 12) were studied during their second breeding season when the PCOS-like phenotype, with anovulation, is fully manifest. In one group (n = 4) one ovary underwent diathermy and it was collected and compared to the contralateral ovary after 24 hours. In another group a treatment PCOS cohort underwent diathermy (n = 4) and the ovaries were collected and compared to the control PCOS cohort (n = 4) after 5 weeks. Ovarian vascular indices were measured using contrast-enhanced ultrasound and colour Doppler before, immediately after, 24 hours and five weeks after diathermy. Antral follicles were assessed by immunohistochemistry and ovarian stromal gene expression by quantitative RT-PCR 24 hours and 5 weeks after diathermy. Diathermy increased follicular atresia (P<0.05) and reduced antral follicle numbers after 5 weeks (P<0.05). There was an increase in stromal CCL2 expression 24 hours after diathermy (P<0.01) but no alteration in inflammatory indices at 5 weeks. Immediately after diathermy there was increased microbubble transit time in the ovarian microvasculature (P = 0.05) but this was not seen at 24 hours. However 24 hours after diathermy there was a reduction in the stromal Doppler blood flow signal (P<0.05) and an increased ovarian resistance index (P<0.05) both of which persisted at 5 weeks (P<0.01; P<0.05). In the ovine model of PCOS, OD causes a sustained reduction in ovarian stromal blood flow with an increased ovarian artery resistance index associated with atresia of antral follicles. PMID:25343339

Oxygen consumption rate of early pre-antral follicles from vitrified human ovarian cortical tissue

PubMed Central

ISHIKAWA, Takayuki; KYOYA, Toshihiko; NAKAMURA, Yusuke; SATO, Eimei; TOMIYAMA, Tatsuhiro; KYONO, Koichi

2014-01-01

The study of human ovarian tissue transplantation and cryopreservation has advanced significantly. Autotransplantation of human pre-antral follicles isolated from cryopreserved cortical tissue is a promising option for the preservation of fertility in young cancer patients. The purpose of the present study was to reveal the effect of vitrification after low-temperature transportation of human pre-antral follicles by using the oxygen consumption rate (OCR). Cortical tissues from 9 ovaries of female-to-male transsexuals were vitrified after transportation (6 or 18 h). The follicles were enzymatically isolated from nonvitrified tissue (group I, 18 h of transportation), vitrified-warmed tissue (group II, 6 and 18 h of transportation) and vitrified-warmed tissue that had been incubated for 24 h (group III, 6 and 18 h of transportation). OCR measurement and the LIVE/DEAD viability assay were performed. Despite the ischemic condition, the isolated pre-antral follicles in group I consumed oxygen, and the mean OCRs increased with developmental stage. Neither the transportation time nor patient age seemed to affect the OCR in this group. Meanwhile, the mean OCR was significantly lower (P < 0.05) in group II but was comparable to that of group I after 24 h of incubation. The integrity of vitrified-warmed primordial and primary follicles was clearly corroborated by the LIVE/DEAD viability assay. These results demonstrate that the OCR can be used to directly estimate the effect of vitrification on the viability of primordial and primary follicles and to select the viable primordial and primary follicles from vitrified-warmed follicles. PMID:25262776

Effects of eCG and FSH on ovarian response, recovery rate and number and quality of oocytes obtained by ovum pick-up in Holstein cows.

PubMed

Sendag, Sait; Cetin, Yunus; Alan, Muhammet; Hadeler, Klaus-Gerd; Niemann, Heiner

2008-06-01

The goal of the present study was to compare the ovarian response, oocyte yields per animal, and the morphological quality of oocytes collected by ultrasound guided follicular aspiration from Holstein cows treated either with FSH or eCG. Twenty four normal cyclic, German Holstein cows were randomly divided into two groups. Fourteen cows received 3000 IU eCG on day-4 prior to ovum pick-up (OPU) (day 0), 2 days later (day-2), 625 microg cloprostenol was administered. On day-1 GnRH was administered i.m. and 24h later OPU (day 0) was performed. In ten cows a total dose of 500 IU follicle stimulating hormone (Pluset) was administered intramuscularly in a constant dosage for 4 days with intervals of 12h, starting on day-5. Luteolysis was induced by application of 625 microg cloprostenol on day-2. On day-1 (24h after the last FSH treatment) GnRH was administered i.m. and 24h later OPU (day 0) was performed. Ovarian follicles were visualized on the ultrasound monitor, counted and recorded. All visible antral follicles were punctured. Recovered oocytes were graded morphologically based on the cumulus investment. Average follicle number in ovaries was higher in FSH group than eCG group (p<0.05). Oocyte yields per animal did not differ between FSH and eCG groups. The proportion of grade A oocytes was higher in the FSH group in the than eCG group (p<0.05). Likewise, rate of grade C oocytes in FSH group were lower than eCG group (p<0.05). In conclusion, these results suggest that ovarian response, follicle number in ovaries and oocyte quality are affected by the type of gonadotropin and FSH is better alternative than eCG for OPU treatment.

Glutathione S-transferase class mu regulation of apoptosis signal-regulating kinase 1 protein during VCD-induced ovotoxicity in neonatal rat ovaries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharya, Poulomi; Madden, Jill A.; Sen, Nivedita

2013-02-15

4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied.more » Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30 μM) for 2–8 days; 2) VCD (30 μM) for 2 days, followed by incubation in control media for 4 days (acute VCD exposure); or 3) LY294002 (20 μM) for 6 days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P < 0.05) after 6 days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P < 0.05) relative to control after 6 days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P < 0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P < 0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1. - Highlights: ► GSTM protein increases in response to ovarian VCD exposure. ► VCD increases Ask1 mRNA at the onset of follicle loss. ► Ovarian GSTM binds more ASK1 protein during VCD-induced ovotoxicity. ► PI3K regulates ovarian GSTM protein.« less

Long-term alteration of follicular steroid concentrations in relation to subclinical endometritis in postpartum dairy cows.

PubMed

Green, M P; Ledgard, A M; Beaumont, S E; Berg, M C; McNatty, K P; Peterson, A J; Back, P J

2011-11-01

The focus of this study was to investigate the effect of subclinical endometritis (scEndo) on ovarian follicular steroid concentrations in early postpartum pasture-fed dairy cows. Mixed-age lactating dairy cows (n = 169) were examined to ascertain uterine health status on d 21 postpartum (±3 d). From this herd, a cohort of scEndo and uninfected cows (n = 47) were selected using uterine cytology to determine scEndo. To ensure cows with scEndo were selected for the study, a conservative threshold [>18% polymorphonuclear (PMN) cells among uterine nucleated cells] was chosen as a selection threshold. Ovarian follicular dynamics were assessed by ultrasonography on d 21, 42, and 63 postpartum. On the latter 2 d, all follicles >4 mm in diameter were ablated, and 4 d later, the largest (F1) and second largest (F2) follicles were measured and their follicular fluid aspirated. Hematological variables and plasma metabolites were measured also on these days to further characterize scEndo cows. On d 21, the prevalence of scEndo was approximately 9% in this herd; by d 42 infections had self-resolved in the majority (81%) of those cows classified as having scEndo on d 21. The scEndo cows had a delayed return to cyclicity; however, no effect was evident on ovarian follicle size or growth rate. Weeks after scEndo had self-resolved and cyclicity was restored, decreased (P = 0.07) testosterone and increased (P = 0.07) cortisol concentrations were evident in F1 follicles of scEndo compared with uninfected cows. Progesterone concentrations of F1 increased (P < 0.05) in 11- to 16-mm diameter follicles of scEndo cows, whereas estradiol, androstendione, and dehydroepiandrosterone concentrations were decreased (P < 0.05) in F1 8- to 10-mm diameter follicles of scEndo cows. These 3 steroids also differed (P < 0.05) between F1 follicle size categories of scEndo but not uninfected cows. On d 21, mean plasma albumin concentration was decreased (P = 0.02) in scEndo cows. In summary, early postpartum scEndo had surprisingly long-term influences on the steroid concentrations of ovarian follicles long after infections had self-resolved. This is likely to affect oocyte quality and may partially explain the reduced conception rates and longer interval between calving and conception that are often associated with scEndo, although more detailed investigations are required to substantiate this theory.

Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system.

PubMed

McLaughlin, M; Albertini, D F; Wallace, W H B; Anderson, R A; Telfer, E E

2018-03-01

Can complete oocyte development be achieved from human ovarian tissue containing primordial/unilaminar follicles and grown in vitro in a multi-step culture to meiotic maturation demonstrated by the formation of polar bodies and a Metaphase II spindle? Development of human oocytes from primordial/unilaminar stages to resumption of meiosis (Metaphase II) and emission of a polar body was achieved within a serum free multi-step culture system. Complete development of oocytes in vitro has been achieved in mouse, where in vitro grown (IVG) oocytes from primordial follicles have resulted in the production of live offspring. Human oocytes have been grown in vitro from the secondary/multi-laminar stage to obtain fully grown oocytes capable of meiotic maturation. However, there are no reports of a culture system supporting complete growth from the earliest stages of human follicle development through to Metaphase II. Ovarian cortical biopsies were obtained with informed consent from women undergoing elective caesarean section (mean age: 30.7 ± 1.7; range: 25-39 years, n = 10). Laboratory setting. Ovarian biopsies were dissected into thin strips, and after removal of growing follicles were cultured in serum free medium for 8 days (Step 1). At the end of this period secondary/multi-laminar follicles were dissected from the strips and intact follicles 100-150 μm in diameter were selected for further culture. Isolated follicles were cultured individually in serum free medium in the presence of 100 ng/ml of human recombinant Activin A (Step 2). Individual follicles were monitored and after 8 days, cumulus oocyte complexes (COCs) were retrieved by gentle pressure on the cultured follicles. Complexes with complete cumulus and adherent mural granulosa cells were selected and cultured in the presence of Activin A and FSH on membranes for a further 4 days (Step 3). At the end of Step 3, complexes containing oocytes >100 μm diameter were selected for IVM in SAGE medium (Step 4) then fixed for analysis. Pieces of human ovarian cortex cultured in serum free medium for 8 days (Step 1) supported early follicle growth and 87 secondary follicles of diameter 120 ± 6 μm (mean ± SEM) could be dissected for further culture. After a further 8 days, 54 of the 87 follicles had reached the antral stage of development. COCs were retrieved by gentle pressure from the cultured follicles and those with adherent mural granulosa cells (n = 48) were selected and cultured for a further 4 days (Step 3). At the end of Step 3, 32 complexes contained oocytes >100 μm diameter were selected for IVM (Step 4). Nine of these complexes contained polar bodies within 24 h and all polar bodies were abnormally large. Confocal immuno-histochemical analysis showed the presence of a Metaphase II spindle confirming that these IVG oocytes had resumed meiosis but their developmental potential is unknown. This is a small number of samples but provides proof of concept that complete development of human oocytes can occur in vitro. Further optimization with morphological evaluation and fertilization potential of IVG oocytes is required to determine whether they are normal. The ability to develop human oocytes from the earliest follicular stages in vitro through to maturation and fertilization would benefit fertility preservation practice. Funded by MRC Grants (G0901839 and MR/L00299X/1). No competing interests.

Neurotrophin NT3 promotes ovarian primordial to primary follicle transition

PubMed Central

Nilsson, Eric; Dole, Gretchen; Skinner, Michael K

2017-01-01

Neurotrophins are growth factors that are known to have a role in promoting cell survival and differentiation. The focus of the current study is to examine the role of neurotrophins in regulating ovarian primordial follicle development. Ovaries from 4-day old rats were placed into organ culture and cultured for 10 days in the absence or presence of neurotrophin-3 (NT3), brain-derived neurotrophic factor (BDNF), or nerve growth factor (NGF). Treatment of ovaries with NT3 resulted in a significant (P<0.01) increase in primordial follicle development (i.e. primordial to primary follicle transition). Treatment with BDNF at high doses of 100–250 ng/ml also significantly (P<0.01) increased primordial follicle development, but NGF had no effect. Immunohistochemical studies determined that NT3 was present in granulosa cells, interstitial tissue, and in the oocytes of primordial and primary follicles. The NT3 receptor NTRK3 was present in oocytes at all stages of development. Analysis of ovaries that contain predominantly primordial follicles demonstrated the transcripts for NT3, NTRK3, NGF, and the BDNF/neurotrophin-4 (NT4) receptor NTRK2 are expressed, while BDNF, NT4, and the NGF receptor NTRK1 are not detectable. Inhibition of the NTRK3 receptor with the tyrphostin AG 879 resulted in oocyte death and a significant (P<0.01) reduction in follicle pool size. Inhibition of the NTRK receptors with K252a slowed primordial to primary follicle transition. A microarray analysis demonstrated that a small number of genes were differentially expressed after NT3 treatment. Observations indicate that the neurotrophin NT3, acting through the NTRK3 receptor in oocytes, promotes the primordial to primary follicle transition. PMID:19584175

The hedgehog system in ovarian follicles of cattle selected for twin ovulations and births: evidence of a link between the IGF and hedgehog systems

USDA-ARS?s Scientific Manuscript database

Hedgehog signaling is involved in regulation of ovarian function in Drosophila but its role in regulating mammalian ovarian folliculogenesis is less clear. Therefore, gene expression of Indian hedgehog (IHH) and its type 1 receptor, patched 1 (PTCH1), were quantified in bovine granulosa (GC) or the...

Graft-versus-host disease targets ovary and causes female infertility in mice.

PubMed

Shimoji, Sonoko; Hashimoto, Daigo; Tsujigiwa, Hidetsugu; Miyawaki, Kohta; Kato, Koji; Takahashi, Shuichiro; Ogasawara, Reiki; Jiromaru, Takashi; Iwasaki, Hiromi; Miyamoto, Toshihiro; Akashi, Koichi; Teshima, Takanori

2017-03-02

Infertility associated with ovarian failure is a serious late complication for female survivors of allogeneic hematopoietic stem cell transplantation (SCT). Although pretransplant conditioning regimen has been appreciated as a cause of ovarian failure, increased application of reduced-intensity conditioning allowed us to revisit other factors possibly affecting ovarian function after allogeneic SCT. We have addressed whether donor T-cell-mediated graft-versus-host disease (GVHD) could be causally related to female infertility in mice. Histological evaluation of the ovaries after SCT demonstrated donor T-cell infiltration in close proximity to apoptotic granulosa cells in the ovarian follicles, resulting in impaired follicular hormone production and maturation of ovarian follicles. Mating experiments showed that female recipients of allogeneic SCT deliver significantly fewer newborns than recipients of syngeneic SCT. GVHD-mediated ovary insufficiency and infertility were independent of conditioning. Pharmacologic GVHD prophylaxis protected the ovary from GVHD and preserved fertility. These results demonstrate for the first time that GVHD targets the ovary and impairs ovarian function and fertility and has important clinical implications in young female transplant recipients with nonmalignant diseases, in whom minimally toxic regimens are used. © 2017 by The American Society of Hematology.

Immunoregulation of follicular renewal, selection, POF, and menopause in vivo, vs. neo-oogenesis in vitro, POF and ovarian infertility treatment, and a clinical trial

PubMed Central

2012-01-01

The immune system plays an important role in the regulation of tissue homeostasis ("tissue immune physiology"). Function of distinct tissues during adulthood, including the ovary, requires (1) Renewal from stem cells, (2) Preservation of tissue-specific cells in a proper differentiated state, which differs among distinct tissues, and (3) Regulation of tissue quantity. Such morphostasis can be executed by the tissue control system, consisting of immune system-related components, vascular pericytes, and autonomic innervation. Morphostasis is established epigenetically, during morphogenetic (developmental) immune adaptation, i.e., during the critical developmental period. Subsequently, the tissues are maintained in a state of differentiation reached during the adaptation by a “stop effect” of resident and self renewing monocyte-derived cells. The later normal tissue is programmed to emerge (e.g., late emergence of ovarian granulosa cells), the earlier its function ceases. Alteration of certain tissue differentiation during the critical developmental period causes persistent alteration of that tissue function, including premature ovarian failure (POF) and primary amenorrhea. In fetal and adult human ovaries the ovarian surface epithelium cells called ovarian stem cells (OSC) are bipotent stem cells for the formation of ovarian germ and granulosa cells. Recently termed oogonial stem cells are, in reality, not stem but already germ cells which have the ability to divide. Immune system-related cells and molecules accompany asymmetric division of OSC resulting in the emergence of secondary germ cells, symmetric division, and migration of secondary germ cells, formation of new granulosa cells and fetal and adult primordial follicles (follicular renewal), and selection and growth of primary/preantral, and dominant follicles. The number of selected follicles during each ovarian cycle is determined by autonomic innervation. Morphostasis is altered with advancing age, due to degenerative changes of the immune system. This causes cessation of oocyte and follicular renewal at 38 +/-2 years of age due to the lack of formation of new granulosa cells. Oocytes in primordial follicles persisting after the end of the prime reproductive period accumulate genetic alterations resulting in an exponentially growing incidence of fetal trisomies and other genetic abnormalities with advanced maternal age. The secondary germ cells also develop in the OSC cultures derived from POF and aging ovaries. In vitro conditions are free of immune mechanisms, which prevent neo-oogenesis in vivo. Such germ cells are capable of differentiating in vitro into functional oocytes. This may provide fresh oocytes and genetically related children to women lacking the ability to produce their own follicular oocytes. Further study of "immune physiology" may help us to better understand ovarian physiology and pathology, including ovarian infertility caused by POF or by a lack of ovarian follicles with functional oocytes in aging ovaries. The observations indicating involvement of immunoregulation in physiological neo-oogenesis and follicular renewal from OSC during the fetal and prime reproductive periods are reviewed as well as immune system and age-independent neo-oogenesis and oocyte maturation in OSC cultures, perimenopausal alteration of homeostasis causing disorders of many tissues, and the first OSC culture clinical trial. PMID:23176151

Reproduction impairment and endocrine disruption in female zebrafish after long-term exposure to MC-LR: A life cycle assessment.

PubMed

Hou, Jie; Li, Li; Wu, Ning; Su, Yujing; Lin, Wang; Li, Guangyu; Gu, Zemao

2016-01-01

Microcystin-LR (MC-LR) has been found to cause reproductive and developmental impairments as well as to disrupt sex hormone homeostasis of fish during acute and sub-chronic toxic experiments. However, fish in natural environments are continuously exposed to MC-LR throughout their entire life cycle as opposed to short-term exposure. Here, we tested the hypothesis that the mechanism by which MC-LR harms female fish reproduction and development within natural water bodies is through interference of the reproductive endocrine system. In the present study, zebrafish hatchlings (5 d post-fertilization) were exposed to 0, 0.3, 3 and 30 μg/L MC-LR for 90 d until reaching sexual maturity. Female zebrafish were selected, and the changes in growth and developmental indicators, ovarian ultrastructure as well as the levels of gonadal steroid hormones and vitellogenin (VTG) were examined along with the transcription of related genes in the hypothalamic-pituitary-gonadal-liver axis (HPGL-axis). The results showed for the first time, a life cycle exposure to MC-LR caused growth inhibition, decreased ovary weight and ovarian ultra-pathological lesions. Decreased ovarian testosterone levels indicated that MC-LR disrupted sex steroid hormone balance. Significantly up-regulated transcription of brain FSHβ and LHβ along with ovarian ERα, FSHR and LHR suggested positive feedback regulation in the HPGL-axis was induced as a compensatory mechanism for MC-LR damage. It was also noted that ovarian VTG content and hepatic ERα and VTG1 expression were all down-regulated, which might be responsible for reduced vitellus storage noted in our histological observations. Our findings indicate that a life cycle exposure to MC-LR impairs the development and reproduction of female zebrafish by disrupting the transcription of related HPGL-axis genes, suggesting that MC-LR has potential adverse effects on fish reproduction and thus population dynamics in MCs-contaminated aquatic environment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Insect water-specific aquaporins in developing ovarian follicles of the silk moth Bombyx mori: role in hydration during egg maturation.

PubMed

Maruyama, Mariya; Kambara, Kohei; Naka, Hideshi; Azuma, Masaaki

2015-08-01

Egg formation in terrestrial insects is an absorptive process, accommodated not only by packing proteins and lipids into yolk but also by filling chorions with water. An osmotic swelling of ovarian follicles takes place during oocyte maturation. This study investigated the role of the aquaporin (AQP) water channel in the osmotic uptake of water during oogenesis in the silk moth Bombyx mori Linnaeus, 1758. Using the antibodies that specifically recognize previously characterized AQPs, two water-specific subtypes-AQP-Bom1 and AQP-Bom3-belonging to the Drosophila integral protein (DRIP) and Pyrocoelia rufa integral protein (PRIP) subfamilies of the insect AQP clade, respectively, were identified in the developing ovaries of B. mori. During oocyte growth, Bombyx PRIP was distributed at the oocyte plasma membrane, where it likely plays a role in water uptake and oocyte swelling, and may be responsible for oocyte hydration during fluid absorption by ovarian follicles. During the transition from vitellogenesis to choriogenesis during oocyte maturation, Bombyx DRIP expression became abundant in peripheral yolk granules underlying the oocyte plasma membrane. The restricted DRIP localization was not observed in non-diapause-destined follicles, where DRIP was evenly distributed in medullary yolk granules. There was no difference in PRIP distribution between diapause- and non-diapause-destined follicles. The diapause-destined oocytes encase DRIP protein in the peripheral yolk granules, where DRIP might be inert. This would be reflected in the metabolic arrest associated with diapause after fertilization and egg oviposition. © 2015 Marine Biological Laboratory.

Involvement of androgens in ovarian health and disease

PubMed Central

Lebbe, M.; Woodruff, T.K.

2013-01-01

In women, ovary and adrenal gland produce androgens. Androgens are essential drivers of the primordial to antral follicle development, prior to serving as substrate for estrogen production in the later stages of folliculogenesis. Androgens play a crucial role in the follicular–stromal intertalk by fine tuning the extracellular matrix and vessel content of the ovarian stroma. Local auto-and paracrine factors regulate androgen synthesis in the pre-antral follicle. Androgen excess is a hallmark of polycystic ovary syndrome and is a key contributor in the exaggerated antral follicle formation, stromal hyperplasia and hypervascularity. Hyperandrogenaemia overrides the follicular–stromal dialog, resulting in follicular arrest and disturbed ovulation. On the other hand, androgen deficiency is likely to have a negative impact on fertility as well, and further research is needed to examine the benefits of androgen-replacement therapy in subfertility. PMID:24026057

Repeated superovulation increases the risk of osteoporosis and cardiovascular diseases by accelerating ovarian aging in mice.

PubMed

Zhang, Jinjin; Lai, Zhiwen; Shi, Liangyan; Tian, Yong; Luo, Aiyue; Xu, Zheyuan; Ma, Xiangyi; Wang, Shixuan

2018-05-22

Superovulation procedures and assisted reproductive technologies have been widely used to treat couples who have infertility problems. Although generally safe, the superovulation procedures are associated with a series of complications, such as ovarian hyper-stimulation syndrome, thromboembolism, and adnexal torsion. The role of long-term repeated superovulation in ovarian aging and especially in associated disorders such as osteoporosis and cardiovascular diseases is still unclear. In this study, we sought to determine if repeated superovulation by ten cycles of treatment with pregnant mare serum gonadotropin/human chorionic gonadotropin could affect ovarian reserve, ovarian function, bone density and heart function. Ovarian reserve and function were reflected by the size of the primordial follicle pool, anti-Mullerian hormone expressions, hormone levels and fertility status. Furthermore, we examined bone density and heart function by microCT and cardiovascular ultrasonography, respectively. After repeated superovulation, the size of the primordial follicle pool and the expression of anti-mullerian hormone decreased, along with the concentrations of estrogen and progesterone. Mice exposed to repeated superovulation showed an obvious decrease in fertility and fecundity. Furthermore, both bone density and heart ejection fraction significantly decreased. These results suggest that repeated superovulation may increase the risk of osteoporosis and cardiovascular diseases by accelerating ovarian aging.

Sphingosine-1-phosphate prevents chemotherapy-induced human primordial follicle death.

PubMed

Li, Fang; Turan, Volkan; Lierman, Sylvie; Cuvelier, Claude; De Sutter, Petra; Oktay, Kutluk

2014-01-01

Can Sphingosine-1-phosphate (S1P), a ceramide-induced death pathway inhibitor, prevent cyclophosphamide (Cy) or doxorubicin (Doxo) induced apoptotic follicle death in human ovarian xenografts? S1P can block human apoptotic follicle death induced by both drugs, which have differing mechanisms of cytotoxicity. S1P has been shown to decrease the impact of chemotherapy and radiation on germinal vesicle oocytes in animal studies but no human translational data exist. Experimental human ovarian xenografting to test the in vivo protective effect of S1P on primordial follicle survival in the chemotherapy setting. The data were validated by assessing the same protective effect in the ovaries of xenografted mice in parallel. Xenografted mice were treated with Cy (75 mg/kg), Cy+S1P (200 μM), Doxo (10 mg/kg), Doxo+S1P or vehicle only (Control). S1P was administered via continuous infusion using a mini-osmotic pump beginning 24 h prior to and ending 72 h post-chemotherapy. Grafts were then recovered and stained with anti-caspase 3 antibody for the detection of apoptosis in primordial follicles. The percentage of apoptotic to total primordial follicles was calculated in each group. Both Cy and Doxo resulted in a significant increase in apoptotic follicle death in human ovarian xenografts compared with controls (62.0 ± 3.9% versus 25.7 ± 7.4%, P < 0.01 and 76.7 ± 7.4% versus 25.7 ± 7.4%, P < 0.01, respectively). This chemotherapy-induced apoptotic death was reduced both in the Cy+S1P (32.7 ± 4.4%, P < 0.01) and the Doxo+S1P group (27.1 ± 7.6%, P < 0.01) compared with Cy and Doxo groups, respectively. In the Doxo+S1P and Cy+S1P groups, the percentages of apoptotic follicles were similar to those of vehicle-treated controls (P > 0.05). The findings from the ovaries of the severe combined immunodeficient mice mirrored the findings with human tissue. The functionality of the rescued human ovarian follicles needs to be evaluated in future studies though the studies in rodents showed that rescued oocytes can result in healthy offspring. In addition, the impact of S1P on cancer cells should be further studied. S1P and its future analogs hold promise for preserving fertility by pharmacological means for patients undergoing chemotherapy. This research is supported by NIH's NICHD and NCI (5R01HD053112-06 and 5R21HD061259-02) and the Flemish Foundation for Scientific Research (FWO-Vlaanderen, grant number FWO G0.065.11N10). The authors have no conflicts of interest to disclose.

Polycystic ovary syndrome: possible involvement of androgen-induced, chemerin-mediated ovarian recruitment of monocytes/macrophages.

PubMed

Lima, Patricia D A; Nivet, Anne-Laure; Wang, Qi; Chen, Yi-An; Leader, Arthur; Cheung, Annie; Tzeng, Chii-Ruey; Tsang, Benjamin K

2018-04-24

Polycystic ovary syndrome (PCOS) is a continuum of endocrine and reproductive disorders characterized by hyperandrogenism, antral follicle growth arrest and chronic inflammation. Macrophages play key role in inflammation and the balance between M1 (inflammatory) and M2 (anti-inflammatory) macrophages determines physiological/pathological outcomes. Here, we investigated if hyperandrogenism increases ovarian chemerin altering the balance of M1 and M2 macrophages and the granulosa cell death. Ovarian chemerin was up-regulated by 5α-dihydrotestosterone (DHT) in lean and overweight rats; while increased serum chemerin levels were only evident in overweight rats, suggesting that the serum chemerin may be reflective of a systemic response and associated with obesity, whereas increased ovarian chemerin expression is a localized response independent of the metabolic status. DHT altered follicle dynamics while increased the M1: M2 macrophages ratio in antral and pre-ovulatory follicles. While ovarian M1 macrophages expressing chemokine-like receptor 1 (CMKLR1) were increased, CMKLR1 + monocytes, which migrated towards chemerin-rich environment, were markedly decreased after 15 days of DHT. Androgen-induced granulosa cell apoptosis was dependent on the presence of macrophages. In humans, chemerin levels in follicular fluid, but not in serum, was higher in lean PCOS patients compared to BMI-matched controls and was associated with increased M1: M2 ratio. Our results support the concept that in PCOS, hyperandrogenemia increases chemerin expression while promotes CMKLR1 + monocytes recruitment and deregulates the immunological niche of ovaries. This study established a new immunological perspective in PCOS at the ovarian level. Hyperandrogenism is associated with up-regulation of chemerin and macrophage unbalance in the ovaries.

Advanced glycation end products and their receptor contribute to ovarian ageing.

PubMed

Stensen, Mette Haug; Tanbo, Tom; Storeng, Ritsa; Fedorcsak, Peter

2014-01-01

Do advanced glycation end products (AGE) and the receptor for advanced glycation end products (RAGE) affect the cells of the human ovarian follicle? AGE accumulate on the surface of ovarian granulosa-lutein (GL) cells and monocytes by binding to RAGE and other receptors with possible functional effects on these cells. AGE and RAGE are expressed in granulosa and theca cells, as well as in luteinized cells derived from the ovary. In this prospective cohort study, human follicle fluid-derived cells were isolated from aspirates of ovarian follicles of women who underwent assisted reproduction treatment. Immunofluorescence microscopy and multi-colour flow cytometry were used to determine the presence of AGE and RAGE on the surface of follicular fluid-derived cells and to characterize downstream effects of RAGE activation. GL cells and ovarian monocytes were found to contain AGE and RAGE and to bind AGE-bovine serum albumin (BSA) in correlation with the patients' chronological age. AGE-BSA and BSA failed to induce significantly the cleavage of caspase-3, phosphorylation of nuclear factor-κB or the binding of annexin V (the latter was marginally increased). AGE-fibronectin was found to induce detachment of cultured GL cells in vitro. The impact of AGE and RAGE in the ovary, shown here in cells in culture, remains to be affirmed in clinical settings. The ligands of RAGE and their effects in the ovary remain uncertain but this study implies that AGEs in the form of structural long-lived extracellular matrix proteins, rather than soluble AGEs, may play a role in the decline of ovarian function during ageing. The project was funded by the Norwegian Resource Centre for Women's Health, Oslo University Hospital. The authors have no conflicts of interests.

The influence of a slow-release multi-trace element ruminal bolus on trace element status, number of ovarian follicles and pregnancy outcomes in synchronized Afshari ewes

PubMed Central

Abdollahi, E.; Kohram, H.; Shahir, M. H.; Nemati, M. H.

2015-01-01

Published data on the effects of ruminal bolus on the number of ovulatory follicles in ewes does not exist. The present study determined the effects of a ruminal bolus on trace element status, follicular dynamics and reproductive performance in ewes. Eighty Afshari cycling ewes were synchronized during breeding season using CIDR for 14 days and assigned to 4 groups (n=20); group 1 received a single Ferrobloc bolus four weeks prior to CIDR insertion following 400 IU eCG on CIDR removal, group 2 received two boluses four weeks prior to CIDR insertion following 400 IU eCG on CIDR removal, group 3 received only 400 IU eCG on CIDR removal and group 4 (control) received no bolus and no eCG. Transrectal ultrasonography was done to monitor the ovarian follicles on the day of CIDR removal and a day later. Results showed that boluses increased the status of copper, selenium and iodine on mating day and days 90 to 100 of gestation. Ruminal bolus did not significantly increase the number of different classes of ovarian follicles in ewes fed a diet meeting all trace mineral requirements. All ewes eventually became pregnant with 1 or 2 boluses but the multiple births rate (80%) was higher (P<0.05) after 2 boluses compared to the other groups. PMID:27175153

Scavenger receptor-B1 and luteal function in mice.

PubMed

Jiménez, Leonor Miranda; Binelli, Mario; Bertolin, Kalyne; Pelletier, R Marc; Murphy, Bruce D

2010-08-01

During luteinization, circulating high-density lipoproteins supply cholesterol to ovarian cells via the scavenger receptor-B1 (SCARB1). In the mouse, SCARB1 is expressed in cytoplasm and periphery of theca, granulosa, and cumulus cells of developing follicles and increases dramatically during formation of corpora lutea. Blockade of ovulation in mice with meloxicam, a prostaglandin synthase-2 inhibitor, resulted in follicles with oocytes entrapped in unexpanded cumulus complexes and with granulosa cells with luteinized morphology and expressing SCARB1 characteristic of luteinization. Mice bearing null mutation of the Scarb1 gene (SCARB1(-/-)) had ovaries with small corpora lutea, large follicles with hypertrophied theca cells, and follicular cysts with blood-filled cavities. Plasma progesterone concentrations were decreased 50% in mice with Scarb1 gene disruption. When SCARB1(-/-) mice were treated with a combination of mevinolin [an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR)] and chloroquine (an inhibitor of lysosomal processing of low-density lipoproteins), serum progesterone was further reduced. HMGR protein expression increased in SCARB1(-/-) mice, independent of treatment. It was concluded that theca, granulosa, and cumulus cells express SCARB1 during follicle development, but maximum expression depends on luteinization. Knockout of SCARB1(-/-) leads to ovarian pathology and suboptimal luteal steroidogenesis. Therefore, SCARB1 expression is essential for maintaining normal ovarian cholesterol homeostasis and luteal steroid synthesis.

Ovarian expressed microsomal epoxide hydrolase: Role in detoxification of 4-vinylcyclohexene diepoxide and regulation by phosphatidylinositol-3 kinase signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharya, Poulomi, E-mail: poulomib@iastate.edu; Sen, Nivedita, E-mail: nsen@email.arizona.edu; Hoyer, Patricia B., E-mail: Hoyer@u.arizona.edu

2012-01-01

4-vinylcyclohexene diepoxide (VCD) is a metabolite of 4-vinylcyclohexene (VCH) which has the potential to be formed in the ovary through CYP2E1 activity. VCD specifically destroys primordial and small primary follicles in the rodent ovary. Mouse ovaries exposed to VCD demonstrate increased mRNA and protein expression of microsomal epoxide hydrolase (mEH), and an inactive tetrol metabolite (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane) can be formed in mouse ovarian follicles, potentially through detoxification action of mEH. In contrast, mEH can bioactivate another ovotoxic chemical, 7,12-dimethylbenz[a]anthracene (DMBA) to a more toxic compound, DMBA-3,4-diol-1,2-epoxide. Thus, the present study evaluated a functional role for mEH during detoxification of VCD. Additionally,more » because inhibition of the phosphatidyinositol-3 kinase (PI3K) signaling pathway in a previous study protected primordial follicles from VCD-induced destruction, but accelerated DMBA-induced ovotoxicity, a role for PI3K in ovarian mEH regulation was evaluated. Using a post-natal day (PND) 4 Fischer 344 rat whole ovary culture system inhibition of mEH using cyclohexene oxide during VCD exposure resulted in a greater (P < 0.05) loss of primordial and small primary follicles relative to VCD-treated ovaries. Also, relative to controls, meh mRNA was increased (P < 0.05) on day 4 of VCD (30 μM) exposure, followed by increased (P < 0.05) mEH protein after 6 days. Furthermore, inhibition of PI3K signaling increased mEH mRNA and protein expression. Thus, these results support a functional role for mEH in the rat ovary, and demonstrate the involvement of PI3K signaling in regulation of ovarian xenobiotic metabolism by mEH. -- Highlights: ► Ovarian mEH functions to metabolize VCD to a less toxic compound. ► mEH expression is increased in a temporal pattern in response to VCD exposure. ► PI3K signaling is involved in regulation of ovarian mEH expression.« less

Pregnenolone co-treatment partially restores steroidogenesis, but does not prevent growth inhibition and increased atresia in mouse ovarian antral follicles treated with mono-hydroxy methoxychlor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Craig, Zelieann R., E-mail: zelieann@illinois.edu; Hannon, Patrick R., E-mail: phannon2@illinois.edu; Flaws, Jodi A., E-mail: jflaws@illinois.edu

Mono-hydroxy methoxychlor (mono-OH MXC) is a metabolite of the pesticide, methoxychlor (MXC). Although MXC is known to decrease antral follicle numbers, and increase follicle death in rodents, not much is known about the ovarian effects of mono-OH MXC. Previous studies indicate that mono-OH MXC inhibits mouse antral follicle growth, increases follicle death, and inhibits steroidogenesis in vitro. Further, previous studies indicate that CYP11A1 expression and production of progesterone (P{sub 4}) may be the early targets of mono-OH MXC in the steroidogenic pathway. Thus, this study tested whether supplementing pregnenolone, the precursor of progesterone and the substrate for HSD3B, would preventmore » decreased steroidogenesis, inhibited follicle growth, and increased follicle atresia in mono-OH MXC-treated follicles. Mouse antral follicles were exposed to vehicle (dimethylsulfoxide), mono-OH MXC (10 μg/mL), pregnenolone (1 μg/mL), or mono-OH MXC and pregnenolone together for 96 h. Levels of P{sub 4}, androstenedione (A), testosterone (T), estrone (E{sub 1}), and 17β-estradiol (E{sub 2}) in media were determined, and follicles were processed for histological evaluation of atresia. Pregnenolone treatment alone stimulated production of all steroid hormones except E{sub 2}. Mono-OH MXC-treated follicles had decreased sex steroids, but when given pregnenolone, produced levels of P{sub 4}, A, T, and E{sub 1} that were comparable to those in vehicle-treated follicles. Pregnenolone treatment did not prevent growth inhibition and increased atresia in mono-OH MXC-treated follicles. Collectively, these data support the idea that the most upstream effect of mono-OH MXC on steroidogenesis is by reducing the availability of pregnenolone, and that adding pregnenolone may not be sufficient to prevent inhibited follicle growth and survival. - Highlights: • Mono-OH MXC inhibited antral follicle steroidogenesis, growth, and survival. • Pregnenolone partially restored steroidogenesis in mono-OH MXC-treated follicles. • Pregnenolone did not prevent mono-OH MXC-induced inhibition of growth and survival.« less

[Effect of Transcutaneuos Acupoint Electrostimulation on Serum Sex Hormone Levels and Expression of Ovarian Steroid Hormone Metabolic Enzymes in Polycystic Ovary Syndrome Rats].

PubMed

Zhou, Jian-yong; Zhang, Xiao-yue; Yu, Mei-ling; Lu, Sheng-feng; Chen, Xia

2016-02-01

To observe the effect of transcutaneuos acupoint electrostimulation(TAES) on ovarian serum sex hormone levels and ovarian follicle granular cell aromatase cytochrome P 450 (P 450 arom) protein and follicle theca cell cytochrome P 450 17 α-hydroxylase/c 17-20 lyase cytochrome P 450 (P 450 c 17 α) protein expression in polycystic ovary syndrome (PCOS)rats, so as to explore its mechanisms underlying improvement of PCOS. METHODS Forty SD rats were randomly divided into four groups: normal control, model, medication and TAES (10 rats/group). The PCOS model was established by giving (gavage) the animals with letrozole solution (1.0 mg/kg, once daily for 21 consecutive days). Rats of the medication group were treated with Clomiphene (1 mg/kg) once daily for 7 days, and those of the TAES group were treated with electrical stimulation (2 Hz, 3 mA) of "Guanyuan" (CV 4) and "Sanyinjiao" (SP 6) areas for 30 min, once daily for 7 consecutive days. The rats body weight and bilateral ovarian weight were detected, and the ovarian structure and follicular development degree were observed under light microscope after H. E. stain, and the serum testosterone (T), estradiol (E2), luteotrophic hormone (LH) and follicle-stimulating hormone (FSH) contents were detected using radioimmunoassay. The expression of ovarian P 450 arom (for production of estrogen)protein and P 450 c 17 α (for production of androgen) protein was detected by using immunohistochemical stain and Western blot, respectively. The body weight, bilateral ovary weight, serum T and LH contents, and ratio of LH/FSH, and ovarian P 450 c 17 α immunoactivity and protein expression levels in the model group were all significantly increased compared with the normal control group (P < 0.01), and the levels of serum E2 and ovarian P 450 arom immunoactivity and protein expression were significantly decreased after modeling (P < 0.01). Following the treatment, the increased body weight, ovary weight, serum T and LH contents, ratio of LH/FSH, and ovarian P 450 c 17 α immunoactivity and protein expression levels, and the decreased ovarian P 450 arom immunoactivity and protein expression levels were reversed in the TAES group (P < 0.01, P < 0.05) rather than in the medication group, except serum T and ratio of LH/FSH in the medication group. No significant differences were found between the medication and TAES groups in the serum T and ratio of LH/FSH (P > 0.05). In addition, the increased vesicular follicle number, the decreased corepus luteum number and the thickness of granular cell layer were markedly improved in the TAES group. TAES intervention can reduce both body weight and ovarian weight and regulate the levels of serum sex hormones and ovarian P 450 c 17 α and P 450 arom protein expression levels in PCOS rats, which may contribute to its effect in improving PCOS.

BMP signalling in human fetal ovary somatic cells is modulated in a gene-specific fashion by GREM1 and GREM2

PubMed Central

Bayne, Rosemary A.; Donnachie, Douglas J.; Kinnell, Hazel L.; Childs, Andrew J.; Anderson, Richard A.

2016-01-01

STUDY QUESTION Do changes in the expression of bone morphogenetic proteins (BMPs) 2 and 4, and their antagonists Gremlin1 (GREM1) and Gremlin2 (GREM2) during human fetal ovarian development impact on BMP pathway activity and lead to changes in gene expression that may influence the fate and/or function of ovarian somatic cells? STUDY FINDING BMPs 2 and 4 differentially regulate gene expression in cultured human fetal ovarian somatic cells. Expression of some, but not all BMP target genes is antagonised by GREM1 and GREM2, indicating the existence of a mechanism to fine-tune BMP signal intensity in the ovary. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker of immature ovarian somatic cells, is identified as a novel transcriptional target of BMP4. WHAT IS KNOWN ALREADY Extensive re-organisation of the germ and somatic cell populations in the feto-neonatal ovary culminates in the formation of primordial follicles, which provide the basis for a female's future fertility. BMP growth factors play important roles at many stages of ovarian development and function. GREM1, an extracellular antagonist of BMP signalling, regulates the timing of primordial follicle formation in the mouse ovary, and mRNA levels of BMP4 decrease while those of BMP2 increase prior to follicle formation in the human fetal ovary. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Expression of genes encoding BMP pathway components, BMP antagonists and markers of ovarian somatic cells were determined by quantitative (q)RT-PCR in human fetal ovaries (from 8 to 21 weeks gestation) and fetal ovary-derived somatic cell cultures. Ovarian expression of GREM1 protein was confirmed by immunoblotting. Primary human fetal ovarian somatic cell cultures were derived from disaggregated ovaries by differential adhesion and cultured in the presence of recombinant human BMP2 or BMP4, with or without the addition of GREM1 or GREM2. MAIN RESULTS AND THE ROLE OF CHANCE We demonstrate that the expression of BMP antagonists GREM1, GREM2 and CHRD  increases in the lead-up to primordial follicle formation in the human fetal ovary, and that the BMP pathway is active in cultured ovarian somatic cells. This leads to differential changes in the expression of a number of genes, some of which are further modulated by GREM1 and/or GREM2. The positive transcriptional regulation of LGR5 (a marker of less differentiated somatic cells) by BMP4 in vitro suggests that increasing levels of GREM1 and reduced levels of BMP4 as the ovary develops in vivo may act to reduce LGR5 levels and allow pre-granulosa cell differentiation. LIMITATIONS, REASONS FOR CAUTION While we have demonstrated that markers of different somatic cell types are expressed in the cultured ovarian somatic cells, their proportions may not represent the same cells in the intact ovary which also contains germ cells. WIDER IMPLICATIONS OF THE FINDINGS This study extends previous work identifying germ cells as targets of ovarian BMP signalling, and suggests BMPs may regulate the development of both germ and somatic cells in the developing ovary around the time of follicle formation. LARGE SCALE DATA Not applicable. STUDY FUNDING/COMPETING INTERESTS This work was supported by The UK Medical Research Council (Grant No.: G1100357 to RAA), and Medical Research Scotland (Grant No. 345FRG to AJC). The authors have no competing interests to declare. PMID:27385727

BMP signalling in human fetal ovary somatic cells is modulated in a gene-specific fashion by GREM1 and GREM2.

PubMed

Bayne, Rosemary A; Donnachie, Douglas J; Kinnell, Hazel L; Childs, Andrew J; Anderson, Richard A

2016-09-01

Do changes in the expression of bone morphogenetic proteins (BMPs) 2 and 4, and their antagonists Gremlin1 (GREM1) and Gremlin2 (GREM2) during human fetal ovarian development impact on BMP pathway activity and lead to changes in gene expression that may influence the fate and/or function of ovarian somatic cells? BMPs 2 and 4 differentially regulate gene expression in cultured human fetal ovarian somatic cells. Expression of some, but not all BMP target genes is antagonised by GREM1 and GREM2, indicating the existence of a mechanism to fine-tune BMP signal intensity in the ovary. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker of immature ovarian somatic cells, is identified as a novel transcriptional target of BMP4. Extensive re-organisation of the germ and somatic cell populations in the feto-neonatal ovary culminates in the formation of primordial follicles, which provide the basis for a female's future fertility. BMP growth factors play important roles at many stages of ovarian development and function. GREM1, an extracellular antagonist of BMP signalling, regulates the timing of primordial follicle formation in the mouse ovary, and mRNA levels of BMP4 decrease while those of BMP2 increase prior to follicle formation in the human fetal ovary. Expression of genes encoding BMP pathway components, BMP antagonists and markers of ovarian somatic cells were determined by quantitative (q)RT-PCR in human fetal ovaries (from 8 to 21 weeks gestation) and fetal ovary-derived somatic cell cultures. Ovarian expression of GREM1 protein was confirmed by immunoblotting. Primary human fetal ovarian somatic cell cultures were derived from disaggregated ovaries by differential adhesion and cultured in the presence of recombinant human BMP2 or BMP4, with or without the addition of GREM1 or GREM2. We demonstrate that the expression of BMP antagonists GREM1, GREM2 and CHRD  increases in the lead-up to primordial follicle formation in the human fetal ovary, and that the BMP pathway is active in cultured ovarian somatic cells. This leads to differential changes in the expression of a number of genes, some of which are further modulated by GREM1 and/or GREM2. The positive transcriptional regulation of LGR5 (a marker of less differentiated somatic cells) by BMP4 in vitro suggests that increasing levels of GREM1 and reduced levels of BMP4 as the ovary develops in vivo may act to reduce LGR5 levels and allow pre-granulosa cell differentiation. While we have demonstrated that markers of different somatic cell types are expressed in the cultured ovarian somatic cells, their proportions may not represent the same cells in the intact ovary which also contains germ cells. This study extends previous work identifying germ cells as targets of ovarian BMP signalling, and suggests BMPs may regulate the development of both germ and somatic cells in the developing ovary around the time of follicle formation. Not applicable. This work was supported by The UK Medical Research Council (Grant No.: G1100357 to RAA), and Medical Research Scotland (Grant No. 345FRG to AJC). The authors have no competing interests to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Protein concentration in pre-ovulatory follicular fluid related to ovarian stimulation.

PubMed

Suchanek, E; Mujkic-Klaric, A; Grizelj, V; Simunic, V; Kopjar, B

1990-05-01

Sixty follicular fluids obtained from 26 women with either clomiphene citrate and human menopausal gonadotropins (hMG) or hMG-induced ovulation were analyzed for the contents of total proteins, fibrinogen, plasminogen, antithrombin III, ceruloplasmin, alpha-2 macroglobulin, alpha-1 antitrypsin and immunoglobulins (IgG, IgA, IgM). Concentrations of these proteins was correlated to the type of ovarian follicle growth induction. Follicular fluids from patients stimulated with clomiphene citrate-hMG contained significantly higher concentrations of ceruloplasmin than those treated with hMG alone. No significant differences in the concentrations of other proteins were noted between the two types of ovarian induction. A multivariate data analysis resulted in three Varimax factors (VRX I) suggesting that proteins with antiprotease activity in the follicular fluid may play a role in human follicle maturation. Follicular fluid Ig may reflect the degree of follicular wall permeability under hMG treatment. Accordingly, it may be assumed that a combination of different proteins described by VRX factors could be used for evaluation of ovarian stimulation.

Expression and regulation of the tumor suppressor, SEF, during folliculogenesis in humans and mice.

PubMed

Lutwak, Ela; Price, Christopher A; Abramovich, Sagit-Sela; Rabinovitz, Shiri; Granot, Irit; Dekel, Nava; Ron, Dina

2014-11-01

Similar expression to FGF (Sef or IL17-RD), is a tumor suppressor and an inhibitor of growth factors as well as of pro-inflammatory cytokine signaling. In this study, we examined the regulation of Sef expression by gonadotropins during ovarian folliculogenesis. In sexually immature mice, in situ hybridization (ISH) localized Sef gene expression to early developing oocytes and granulosa cells (GC) but not to theca cells. Sef was also expressed in mouse ovarian endothelial cells, in the fallopian tube epithelium as well as in adipose tissue venules. SEF protein expression, determined by immunohistochemistry (IHC), correlated well with Sef mRNA expression in GC, while differential expression was noticed in oocytes. High Sef mRNA but undetectable SEF protein levels were observed in the oocytes of primary/secondary follicles, while an inverse correlation was found in the oocytes of preantral and small antral follicles. Sef mRNA expression dropped after pregnant mare's serum gonadotropin (PMSG) administration, peaked at 6-8 h after human chorionic gonadotropin (hCG) treatment, and declined by 12 h after this treatment. ISH and IHC localized the changes to oocytes and mural GC following PMSG treatment, whereas Sef expression increased in mural GC and declined in granulosa-lutein cells upon hCG treatment. The ovarian expression of SEF was confirmed using human samples. ISH localized SEF transcripts to human GC of antral follicles but not to corpora lutea. Furthermore, SEF mRNA was detected in human GC recovered from preovulatory follicles. These results are the first to demonstrate SEF expression in a healthy ovary during folliculogenesis. Hormonal regulation of its expression suggests that SEF may be an important factor involved in intra-ovarian control mechanisms. © 2014 Society for Reproduction and Fertility.

Depressive Symptomatology, Psychological Stress, and Ovarian Reserve: A Role for Psychological Factors in Ovarian Aging?

PubMed Central

Bleil, Maria E.; Adler, Nancy E.; Pasch, Lauri A.; Sternfeld, Barbara; Gregorich, Steven E.; Rosen, Mitchell P.; Cedars, Marcelle I.

2012-01-01

Objective To examine psychological factors in relation to antral follicle count (AFC), a marker of ovarian reserve, in a multiethnic sample of 683 pre-menopausal women in the Ovarian Aging (OVA) Study. Methods In cross-sectional analyses, linear regression was performed to determine whether AFC decline across women varied over levels of depression as well as depression in combination with psychological stress. The total and subscale scores of the Center for Epidemiological Studies Depression Scale (CESD) were used to measure depression and the Perceived Stress Scale was used to measure psychological stress. Results Following covariate-adjustment, the 2-way interaction of age-x-positive affect and the 3-way interaction of age-x-positive affect-x-stress were related to AFC (b = 0.047, p = .036; b = 0.012, p = .099, respectively). In stratified analyses, stress was related to AFC in women with low positive affect (b = −.070, p = .021) but not in women with high positive affect (b = .018, p = .54). AFC decline across women was progressively higher in women with low positive affect who reported low (−0.747 follicles/year), mid (−0.920 follicles/year), and high (−1.112 follicles/year) levels of stress. Results examining the CESD total and remaining subscale scores were all non-significant (p’s > .05). Conclusions Cross-sectional evidence suggests that 1) women with low positive affect may experience accelerated AFC decline and 2) low positive affect may be a vulnerability factor, or, alternatively, high positive affect may be a protective factor, in moderating the negative effects of psychological stress on AFC decline. PMID:22760086

Regulation and mechanism of leptin on lipid metabolism in ovarian follicle cells from yellow catfish Pelteobagrus fulvidraco.

PubMed

Zhang, Li-Han; Tan, Xiao-Ying; Wu, Kun; Zhuo, Mei-Qin; Song, Yu-Feng; Chen, Qing-Ling

2015-10-01

The present study was conducted to determine the effect of leptin on lipid metabolism in ovarian follicle cells of yellow catfish Pelteobagrus fulvidraco. For that purpose, primary ovarian follicle cells were isolated from yellow catfish, cultured and subjected to different treatments (control, 0.1% DMSO, 500ng/ml leptin, 500ng/ml leptin plus 100μM wortmannin, 500ng/ml leptin plus 50nM AG490, respectively) for 48h. Intracellular triglyceride (TG) content, the activities (CPT I, FAS, G6PD, and 6PGD) and/or expression level of several enzymes (CPT I, FAS, G6PD, 6PGD, ACCa and ACCb), as well as the mRNA expression of transcription factors (PPARα, PPARγ and SREBP-1) involved in lipid metabolism were determined. Recombinant human leptin (rt-hLEP) incubation significantly reduced intracellular TG content, activities and mRNA levels of FAS, G6PD and 6PGD, SREBP-1 and PPARγ, but enhanced activity and mRNA level of CPT I, PPARα and ACCa. Specific inhibitors AG490 and wortmannin of JAK-STAT and IRS-PI3K signaling pathways prevented leptin-induced changes, indicating that JAK-STAT and IRS-PI3K signaling pathways were involved in the process of leptin-induced changes of lipid metabolism. Based on these observations above, for the first time, our study indicated that leptin reduced lipid deposition by activating lipolysis and suppressing lipogenesis in ovarian follicles of yellow catfish, and both JAK-STAT and IRS-PI3K signaling pathways were involved in the changes of leptin-induced lipid metabolism. Copyright © 2015 Elsevier Inc. All rights reserved.

Successful slush nitrogen vitrification of human ovarian tissue.

PubMed

Talevi, Riccardo; Barbato, Vincenza; Fiorentino, Ilaria; Braun, Sabrina; De Stefano, Cristofaro; Ferraro, Raffaele; Sudhakaran, Sam; Gualtieri, Roberto

2016-06-01

To study whether slush nitrogen vitrification improves the preservation of human ovarian tissue. Control vs. treatment study. University research laboratory. Ovarian biopsies collected from nine women (aged 14-35 years) during laparoscopic surgery for benign gynecologic conditions. None. Ovarian cortical strips of 2 × 5 × 1 mm were vitrified with liquid or slush nitrogen. Fresh and vitrified cortical strips were analyzed for cryodamage and viability under light, confocal, and transmission electron microscopy. Compared with liquid nitrogen, vitrification with slush nitrogen preserves [1] follicle quality (grade 1 follicles: fresh control, 50%; liquid nitrogen, 27%; slush nitrogen, 48%); [2] granulosa cell ultrastructure (intact cells: fresh control, 92%; liquid nitrogen, 45%; slush nitrogen, 73%), stromal cell ultrastructure (intact cells: fresh control, 59.8%; liquid nitrogen, 24%; slush nitrogen, 48.7%), and DNA integrity (TUNEL-positive cells: fresh control, 0.5%; liquid nitrogen, 2.3%; slush nitrogen, 0.4%); and [3] oocyte, granulosa, and stromal cell viability (oocyte: fresh control, 90%; liquid nitrogen, 63%; slush nitrogen, 87%; granulosa cells: fresh control, 93%; liquid nitrogen, 53%; slush nitrogen, 81%; stromal cells: fresh control, 63%; liquid nitrogen, 30%; slush nitrogen, 52%). The histology, ultrastructure, and viability of follicles and stromal cells are better preserved after vitrification with slush nitrogen compared with liquid nitrogen. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Protective effect of alpha-lipoic acid in methotrexate-induced ovarian oxidative injury and decreased ovarian reserve in rats.

PubMed

Soylu Karapinar, Oya; Pinar, Neslihan; Özcan, Oğuzhan; Özgür, Tümay; Dolapçıoğlu, Kenan

2017-08-01

To determine whether the possible oxidative effect of methotrexate (Mtx) on ovary and to evaluate the effectiveness of alpha lipoic acid (ALA), which may be useful in many oxidative stress models. Thirty-two female Wistar-albino rats were randomly divided into four groups; control group, alpha lipoic acid group (ALA 100 mg/kg, 10 days), multiple dose Mtx group (Mtx 1 mg/kg 1, 3, 5, 7 days) and Mtx and ALA group (Mtx 1 mg/kg 1, 3, 5, 7 days and ALA 100 mg/kg, 10 days). Serum total antioxidant status (TAS), total oxidant status (TOS) and oxidative stress index (OSI), tumor necrosis factor-alpha (TNF-α), tissue malondialdehyde (MDA) and activities of glutathione peroxidase (GSH-Px) and catalase (CAT) and anti-Mullerian hormone (AMH) and total ovarian follicle count were evaluated. Mtx administration caused a significant decrease in TAS, a significant increase in TOS and OSI, a significant increase in MDA levels and a decrease in GSH-Px and CAT activity. Moreover the proinflammatory cytokine (TNF-α) was increased in the Mtx group. And AMH values and total follicle count were significantly decreased in Mtx group. However, ALA treatment reversed biochemical results and AMH levels and total follicle count. Alpha lipoic acid ameliorates methotrexate induced oxidative damage of ovarian in rats.

Comparison of In Vitro- and Chorioallantoic Membrane (CAM)-Culture Systems for Cryopreserved Medulla-Contained Human Ovarian Tissue

PubMed Central

Isachenko, Vladimir; Mallmann, Peter; Petrunkina, Anna M.; Rahimi, Gohar; Nawroth, Frank; Hancke, Katharina; Felberbaum, Ricardo; Genze, Felicitas; Damjanoski, Ilija; Isachenko, Evgenia

2012-01-01

At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hen's eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5–2.0×1.0–1.2×0.8–1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips. PMID:22479331

Localization of vascular endothelial growth factor in the zona pellucida of developing ovarian follicles in the rat: a possible role in destiny of follicles.

PubMed

Celik-Ozenci, Ciler; Akkoyunlu, Gokhan; Kayisli, Umit Ali; Arici, Aydin; Demir, Ramazan

2003-11-01

There is increasing evidence that in many species angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), may have important roles in folliculogenesis. The aim of this study is to determine the localization of VEGF and its receptors, Flt-1 and KDR, and bFGF expression in the rat ovary and to evaluate their distributions throughout the different follicular stages. Out of 20 virginal female rats, 10 were studied during the natural ovarian cycle without any ovulation induction. The other 10 were superovulated and their ovaries were studied by western analysis and immunohistochemistry. Granulosa cells (GC) and oocytes of primordial follicles were negative for VEGF. In early primary follicles, VEGF was present in the oocyte but its immunoreactivity was weak, while newly developing zona pellucida (ZP) of primary follicles was negative for VEGF. Subsequently, with the commencement of antral spaces between GC of the secondary follicle, ZP of some secondary follicles became strongly positive for VEGF, forming a continuous ring around the oocyte. In preovulatory mature follicles granulosa and theca interna (TI) cells showed a weak immunoreactivity for VEGF. Western blot analyses have also demonstrated that VEGF, a 26-kDa protein, was present in follicles. Moreover, in ovulated cumulus-oocyte complex we observed a halo-like immunoreactivity of VEGF around the fully mature oocyte. The immunoreactivity for Flt-1 and KDR receptors in growing follicles was mostly limited to GC and TI cells. Anti-bFGF did not exhibit any immunoreactivity in ZP of follicles at any stage. Its expression was weak in GC of the follicles at different stages, whereas, it could be localized to some extent in the blood capillaries of TI of antral follicles and in blood vessels localized in the stroma. Interestingly, VEGF immunoreactivity in the ZP of some secondary follicles is very striking. Accordingly, the possibility that VEGF may be an important regulatory molecule for the dominant follicle selection or atresia should be considered.

Ultrastructure and composition of Call-Exner bodies in bovine follicles.

PubMed

van Wezel, I L; Irving-Rodgers, H F; Sado, Y; Ninomiya, Y; Rodgers, R J

1999-05-01

Call-Exner bodies are present in ovarian follicles of a range of species including human and rabbit, and in a range of human ovarian tumors. We have also found structures resembling Call-Exner bodies in bovine preantral and small antral follicles. Hematoxylin and eosin staining of single sections of bovine ovaries has shown that 30% of preantral follicles with more than one layer of granulosa cells and 45% of small (less than 650 microns) antral follicles have at least one Call-Exner body composed of a spherical eosinophilic region surrounded by a rosette of granulosa cells. Alcian blue stains the spherical eosinophilic region of the Call-Exner bodies. Electron microscopy has demonstrated that some Call-Exner bodies contain large aggregates of convoluted basal lamina, whereas others also contain regions of unassembled basal-lamina-like material. Individual chains of the basal lamina components type IV collagen (alpha 1 to alpha 5) and laminin (alpha 1, beta 2 and delta 1) have been immunolocalized to Call-Exner bodies in sections of fresh-frozen ovaries. Bovine Call-Exner bodies are presumably analogous to Call-Exner bodies in other species but are predominantly found in preantral and small antral follicles, rather than large antral follicles. With follicular development, the basal laminae of Call-Exner bodies change in their apparent ratio of type IV collagen to laminin, similar to changes observed in the follicular basal lamina, suggesting that these structures have a common cellular origin.

Rapamycin preserves the follicle pool reserve and prolongs the ovarian lifespan of female rats via modulating mTOR activation and sirtuin expression.

PubMed

Zhang, Xing-mei; Li, Li; Xu, Jin-jie; Wang, Na; Liu, Wei-juan; Lin, Xuan-hao; Fu, Yu-cai; Luo, Li-li

2013-07-01

To maintain the normal length of female reproductive life, the majority of primordial follicles must be maintained in a quiescent state for later use. In this study, we aimed to study the effects of rapamycin on primordial follicle development and investigate the role of mTOR and sirtuin signaling. Rats were treated every other day with an intraperitoneal injection of rapamycin (5mg/kg) or vehicle. After 10weeks of treatment, ovaries were harvested for hematoxylin and eosin (HE) staining, and analysis by immunohistochemistry and Western blotting. HE staining showed that the number and percentage of primordial follicles in the rapamycin-treated group were twice the control group (P<0.001). Immunohistochemical analysis showed that mTOR and phosphorylated-p70S6K were extensively expressed in surviving follicles with strong staining observed in the cytoplasm of the oocyte. Western blotting showed decreased expression of phosphorylated mTOR and phosphorylated p70S6K in the rapamycin-treated group, and increased the expression of both SIRT1 and SIRT6 compared to the control group (P<0.05). Taken together, these results suggest that rapamycin may inhibit the transition from primordial to developing follicles and preserve the follicle pool reserve, thus extending the ovarian lifespan of female rats via the modulation of mTOR and sirtuin signalings. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterization of Chicken MMP13 Expression and Genetic Effect on Egg Production Traits of Its Promoter Polymorphisms.

PubMed

Yuan, Zhenjie; Chen, Yuxia; Chen, Qiuyue; Guo, Miao; Kang, Li; Zhu, Guiyu; Jiang, Yunliang

2016-05-03

Extracelluar matrix undergoes constant remodeling, cell-cell, and cell-matrix interactions during chicken ovarian follicle growth, which is coordinated by matrix metalloproteinases (MMPs), and their associated endogenous inhibitors (TIMPs). Transcriptome analysis revealed upregulation of MMP13 in sexually mature chicken ovaries. In this study, we found that the expression of MMP13 in chicken ovary was stably elevated from 60 d to 159 d, and was significantly higher at 159 d than at the other three developmental stages (P < 0.05). The expression of MMP13 mRNA increased from SW (small white follicles) to F5 (fifth largest follicles), then decreased to F1 (first largest follicles), and dramatically increased again in POF1 (newly postovulatory follicles) follicles (P < 0.05). The MMP13 protein was localized in stroma cells and primordial follicles of sexually immature chicken ovaries, in the theca cell layers of all sized follicles of sexually mature chicken ovaries. Furthermore, we identified a positive element (positions -1863 to -1036) controlling chicken MMP13 transcription, and, in this region, six single nucleotide polymorphisms were found and genotyped in chicken populations. In the White Recessive Rock population, hens with A(-1356)-C(-1079)/A(-1356)-C(-1079) genotype had earlier "age at first laying" than those with G(-1356)-T(-1079)/G(-1356)-T(-1079) genotype (P < 0.05), and exhibited significantly lower transcriptional activity (P < 0.01). Collectively, chicken MMP13 plays an important role in ovarian follicle growth and regression, and polymorphisms in its promoter region could be used as molecular markers for improving the trait "age at first laying" in chicken breeding. Copyright © 2016 Yuan et al.

Evaluation of the ultrasound image attributes of developing ovarian follicles in the four follicular waves of the interovulatory interval in ewes

PubMed Central

Toosi, B.M.; Seekallu, S.V.; Pierson, R.A.; Rawlings, N.C.

2010-01-01

Computer-assisted quantitative echotextural analysis was applied to ultrasound images of antral follicles in the follicular waves of an interovulatory interval in sheep. The ewe has three or four waves per cycle. Seven healthy, cyclic Western White Face ewes (Ovis aris) underwent daily, transrectal, ovarian ultrasonography for an interovulatory interval. Follicles in the third wave of the ovulatory interval had a longer static phase than that of those in Waves 1 and 2 (P < 0.05). The numeric pixel value for the wall of anovulatory follicles emerging in the third wave of the cycle was significantly higher than that for Waves 1 and 2 at the time of emergence (156.7 ± 8.09, 101.6 ± 3.72, and 116.5 ± 13.93, respectively), and it decreased as follicles in Wave 3 reached maximum follicular diameter (P < 0.05). The numeric pixel value of the antrum in the ovulatory follicles decreased as follicular diameter increased to ≥5 mm in diameter (P < 0.05). The pixel heterogeneity of the follicular antrum in Wave 1 increased from the end of the growth phase to the end of the regression phase for follicles in that wave (P < 0.05). The total area for the wall and antrum of the follicles studied were correlated with follicular diameter in all follicular waves (r = 0.938, P < 0.01 and r = 0.941, P < 0.01 for the wall and antrum, respectively). Changes in image attributes of the follicular wall and antrum indicate potential morphologic and functional differences among antral follicles emerging at different stages of the interovulatory interval in cyclic ewes. PMID:19665782

Anti-Müllerian hormone and polycystic ovary syndrome.

PubMed

Bhide, Priya; Homburg, Roy

2016-11-01

Anti-Müllerian hormone (AMH) is expressed by the granulosa cells of the pre-antral and small antral follicles in the ovary. It is significantly higher in women with polycystic ovary syndrome (PCOS) due to an increased number of antral follicles and also a higher production per antral follicle. It is postulated to have an inhibitory role in folliculogenesis and may play an important role in the pathophysiology of anovulation associated with PCOS. Measurement of the serum AMH levels is very useful for the identification of PCOS and has been suggested as a diagnostic criterion. An international standardisation of the AMH assay, large population-based studies and a global consensus are needed before its incorporation into routine diagnosis. Serum AMH levels add significant value to the clinical markers for the prediction of hyperresponse to controlled ovarian stimulation for in vitro fertilisation treatment and development of ovarian hyperstimulation syndrome. Copyright © 2016. Published by Elsevier Ltd.

[Current progress and future direction in the biology of ovarian germ stem cells in mammals].

PubMed

Li, Chao-Hui; Guo, Kun; Zheng, Ping

2012-12-01

Whether or not oogenesis continues after birth in mammalian ovaries remains controversial. Since the 1950's, it has been generally accepted that oogenesis takes place during embryogenesis in mammals and ceases at birth. At birth, germ cells in mammalian ovaries have progressed to the diplotene stage of meiotic prophase and have formed primordial follicles with surrounding somatic cells. These primordial follicles represent follicle reserves of the reproductive life. However, this view has been recently challenged by a growing body of evidence showing the isolation and propagation of germ stem cells from mouse and human ovaries. These ovarian germ stem cells are capable of regenerating functional oocytes when transplanted back into recipient ovaries. Despite the discovery of the potential germ stem cells in mammalian ovaries, it remains uncertain whether these cells exist and function in ovaries under physiological conditions. Herein we review the current progress and future direction in this infant area.

Dopamine inhibits reproduction in female zebrafish (Danio rerio) via three pituitary D2 receptor subtypes.

PubMed

Fontaine, Romain; Affaticati, Pierre; Yamamoto, Kei; Jolly, Cécile; Bureau, Charlotte; Baloche, Sylvie; Gonnet, Françoise; Vernier, Philippe; Dufour, Sylvie; Pasqualini, Catherine

2013-02-01

In many teleosts, the stimulatory control of gonadotrope axis by GnRH is opposed by an inhibitory control by dopamine (DA). The functional importance of this inhibitory pathway differs widely from one teleostean species to another. The zebrafish (Danio rerio) is a teleost fish that has become increasingly popular as an experimental vertebrate model. However, the role of DA in the neuroendocrine control of its reproduction has never been studied. Here the authors evaluated in sexually regressed female zebrafish the effects of in vivo treatments with a DA D2 receptor (D2-R) antagonist domperidone, or a GnRH agonist, alone and in combination, on the pituitary level of FSHβ and LHβ transcripts, the gonadosomatic index, and the ovarian histology. Only the double treatment with GnRH agonist and domperidone could induce an increase in the expression of LHβ, in the gonadosomatic index, and a stimulation of ovarian vitellogenesis, indicating that removal of dopaminergic inhibition is required for the stimulatory action of GnRH and reactivation of ovarian function to occur. Using double immunofluorescent staining on pituitary, the authors showed in this species the innervation of LH cells by tyrosine-hydroxylase immunoreactive fibers. Finally, using in situ hybridization and immunofluorescence, the authors showed that the three subtypes of zebrafish DA D2-R (D2a, D2b, and D2c) were expressed in LH-producing cells, suggesting that they all may be involved in mediating this inhibition. These results show for the first time that, in zebrafish, DA has a direct and potent inhibitory action capable of opposing the stimulatory effect of GnRH in the neuroendocrine control of reproduction.

Life and death of female gametes during oogenesis and folliculogenesis.

PubMed

Krysko, Dmitri V; Diez-Fraile, Araceli; Criel, Godelieve; Svistunov, Andrei A; Vandenabeele, Peter; D'Herde, Katharina

2008-09-01

The vertebrate ovary is an extremely dynamic organ in which excessive or defective follicles are rapidly and effectively eliminated early in ontogeny and thereafter continuously throughout reproductive life. More than 99% of follicles disappear, primarily due to apoptosis of granulosa cells, and only a minute fraction of the surviving follicles successfully complete the path to ovulation. The balance between signals for cell death and survival determines the destiny of the follicles. An abnormally high rate of cell death followed by atresia can negatively affect fertility and eventually lead irreversibly to premature ovarian failure. In this review we provide a short overview of the role of programmed cell death in prenatal differentiation of the primordial germ cells and in postnatal folliculogenesis. We also discuss the issue of neo-oogenesis. Next, we highlight molecules involved in regulation of granulosa cell apoptosis. We further discuss the potential use of scores for apoptosis in granulosa cells and characteristics of follicular fluid as prognostic markers for predicting the outcome of assisted reproduction. Potential therapeutic strategies for combating premature ovarian failure are also addressed.

Preliminary assessment of the relative toxicity of 1,5-diazido-3-nitrazapentane 90-day dermal application male and female rabbits study number 75-51-y809-90, May 1990 - June 1992. Phase 2. Final report, May 1990-June 1992

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haight, E.A.; Harvey, J.G.; Beall, P.

1992-06-01

A 90-day application of DANPE has the potential to cause testicular hypospermatogenesis in male rabbits and an apparent inhibition of mature ovarian follicles formation in female rabbits. A no-observed-adverse-effect-level was not achieved in the male rabbits but was achieved in female rabbits at 27.3 mg/kg dose level.... 1,5-Diazido-3-Nitrazapentane, Dermal, Approximate Lethal Dose (ALD), Testicular hypospermatogenesis, Ovarian follicle No-observed-adverse-effect-level (NOAEL).

Ovary structure in a presocial insect, Elasmucha grisea (Heteroptera, Acanthosomatidae).

PubMed

Ogorzałek, Antoni; Trochimczuk, Artur

2009-11-01

First generation egg clusters of Elasmucha grisea are more closely guarded than second generation clusters. The ovaries of this species are structured to enhance this behavior. The population of E. grisea from S-W Poland breeds in the spring (May-June) and late summer (July-August). The second generation clutches contain fewer eggs and are destroyed 3-4 days after oviposition by predators and parasitoids. The ovary structure in the studied species differs from that found in other Heteroptera. The average number of ovarioles per ovary is 24 while in the other investigated species the number of ovarioles per ovary is 6-7. Lateral oviducts are elongated and the ovarioles are arranged in a pennate pattern. Each ovariole contains only one growing ovarian follicle. Differentiation of the ovarioles and ovarian follicles is synchronised thus enabling simultaneous oviposition. A comparative analysis of the ovary structure during the life cycle, particularly the presence of atresive ovarian follicles in the ovarioles of egg- and nymph guarding females, as well as the shape and structure of the apical part of the tropharium all support the hypothesis of cooperation between females in E. grisea. A similar ovary structure has been observed in the Coccoidea (Hemiptera, Homoptera) which indicates presocial behavior.

A Taenia crassiceps metacestode factor enhances ovarian follicle atresia and oocyte degeneration in female mice.

PubMed

Solano, S; Zepeda, N; Copitin, N; Fernandez, A M; Tato, P; Molinari, J L

2015-01-01

The histopathological effects of Taenia crassiceps infection or T. crassiceps metacestode factor inoculation on the mouse ovary were determined using six female mice in three groups: infected mice, mice inoculated with the metacestode factor and control mice. The control group was subcutaneously inoculated with healthy peritoneal fluid. The infected group was intraperitoneally inoculated with 40 T. crassiceps metacestodes, and the metacestode factor group was subcutaneously inoculated with T. crassiceps metacestode factor (MF). Light and electron microscopy and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling) assays revealed a significant increase in ovarian follicular atresia (predominantly in antral/preovulatory stages of development), oocyte degeneration (P< 0.05), and a decrease in the amount of corpus luteum in follicles of mice infected and inoculated with MF compared with the control group. Significant abnormalities of the granulosa cells and oocytes of the primordial, primary and secondary ovarian follicles occurred in both treated mouse groups (P< 0.05) compared with no degeneration in the control group. These pathological changes in female mice either infected with T. crassiceps metacestodes or inoculated with T. crassiceps MF may have consequences for ovulation and fertility.

Zearalenone exposure impairs ovarian primordial follicle formation via down-regulation of Lhx8 expression in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Guo-Liang

Zearalenone (ZEA) is an estrogenic mycotoxin mainly produced as a secondary metabolite by numerous species of Fusarium. Previous work showed that ZEA had a negative impact on domestic animals with regard to reproduction. The adverse effects and the mechanisms of ZEA on mammalian ovarian folliculogenesis remain largely unknown, particularly its effect on primordial follicle formation. Thus, we investigated the biological effects of ZEA exposure on murine ovarian germ cell cyst breakdown and primordial follicle assembly. Our results demonstrated that newborn mouse ovaries exposed to 10 or 30 μM ZEA in vitro had significantly less germ cell numbers compared to themore » control group. Moreover, the presence of ZEA in vitro increased the numbers of TUNEL and γH2AX positive cells within mouse ovaries and the ratio of mRNA levels of the apoptotic genes Bax/Bcl-2. Furthermore, ZEA exposure reduced the mRNA of oocyte specific genes such as LIM homeobox 8 (Lhx8), newborn ovary homeobox (Nobox), spermatogenesis and oogenesis helix-loop-helix (Sohlh2), and factor in the germline alpha (Figlα) in a dose dependent manner. Exposure to ZEA led to remarkable changes in the Lhx8 3′-UTR DNA methylation dynamics in oocytes and severely impaired folliculogenesis in ovaries after transplantation under the kidney capsules of immunodeficient mice. In conclusion, ZEA exposure impairs mouse primordial follicle formation in vitro. - Highlights: • First time to evaluate the impact of ZEA on primordial follicle formation • ZEA exposure increases oocyte apoptosis and delays germ cell cyst breakdown. • ZEA exposure impairs the expression of LHX8 by affecting its DNA methylation.« less

Does reduced peri-pubertal nutrient intake influence the ovarian reserve in beef heifers?

USDA-ARS?s Scientific Manuscript database

Reducing nutrient intake in beef heifers after weaning decreases production costs; however, the impact of reduced nutrient intake on the ovarian reserve has not been investigated in this species. In rodent models, caloric restriction increased the number of primordial follicles and decreased the num...

Progress in understanding human ovarian folliculogenesis and its implications in assisted reproduction.

PubMed

Yang, Dong Zi; Yang, Wan; Li, Yu; He, Zuanyu

2013-02-01

To highlight recent progress in understanding the pattern of follicular wave emergence of human menstrual cycle, providing a brief overview of the new options for human ovarian stimulation and oocyte retrieval by making full use of follicular physiological waves of the patients either with normal or abnormal ovarian reserve. Literature review and editorial commentary. There has been increasing evidence to suggest that multiple (two or three) antral follicular waves are recruited during human menstrual cycle. The treatment regimens designed based on the theory of follicular waves, to promote increased success with assisted reproduction technology (ART) and fertility preservation have been reported. These new options for human ovarian stimulation and oocyte retrieval by making full use of follicular waves of the patients either with normal or abnormal ovarian reserve lead to new thinking about the standard protocols in ART and challenge the traditional theory that a single wave of antral follicles grows only during the follicular phase of the menstrual cycle. The understanding of human ovarian folliculogenesis may have profound implications in ART and fertility preservation. Further studies are needed to evaluate the optimal regimens in ART based on the theory of follicular waves and to identify non-invasive markers for predicting the outcome and the potential utilities of follicles obtained from anovulatory follicular waves in ART.

Evaluation of the effects of fulvestrant and micronized progesterone on the post-operative adhesion formation and ovarian reserve in rat model with immunohistochemical and biochemical analysis.

PubMed

Oner, Gokalp; Ulug, Pasa; Demirci, Elif; Kumtepe, Yakup; Gündogdu, Cemal

2015-01-01

To evaluate the effects of fulvestrant and micronized progesterone on post-operative adhesion formation and ovarian reserve in a rat uterine horn adhesion. In this prospective randomized controlled trial, 32 female Wistar albino rats were randomly divided into four groups including control group (Group 1), the control adhesion group (Group 2), 1 mg/kg daily intramuscular fulvestrant received group (Group 3) and 1 mg/kg daily oral micronized progesterone received group (Group 4). The extent and severity of adhesions were scored and samples were taken from adhesion areas to investigate the grades of adhesions according to the immunohistochemical scoring system. Ovarian reserves were measured with anti-Müllerian hormone (AMH) and histological ovarian follicles count. The extent, severity and total adhesion scores were reduced in all treatment groups compared to control adhesion group (Group 2). Similarly, immunohistochemical adhesion scores were lower in the treatment groups. AMH and follicle count were significantly found lower in adhesion groups compared with control group. However, treatment groups were found to have higher ovarian reserve compared to control adhesion group (Group 2). Fulvestrant and micronized progesterone were found to reduce post-operative adhesion formations and have decreased detrimental effects of adhesion formation on ovarian reserve.

3 to 5 Years Later: Long-term Effects of Prophylactic Bilateral Salpingectomy on Ovarian Function.

PubMed

Venturella, Roberta; Lico, Daniela; Borelli, Massimo; Imbrogno, Maria G; Cevenini, Gabriele; Zupi, Errico; Zullo, Fulvio; Morelli, Michele

2017-01-01

Preliminary data on the effects of prophylactic bilateral salpingectomy (PBS) show that postoperative ovarian function is preserved up to 3 months after surgery. The confirmation of PBS safety on ovarian function even many years after surgery is essential to reassure the medical community that this new strategy, recently proposed for the prevention of ovarian cancer, is at least able to avoid the risk of premature surgical menopause. We investigated whether the addition of PBS during total laparoscopic hysterectomy (TLH) causes long-term effects on ovarian function. An observational study (Canadian Task Force classification II-3). Department of Obstetrics and Gynecology, "Magna Graecia" University, Catanzaro, Italy. Seventy-nine patients who underwent TLH plus salpingectomy between September 2010 and September 2012 at our institution have been recalled to be submitted to ovarian reserve evaluation in February 2015. Eight of 79 women refused to participate in this follow-up study. The ovarian age of PBS patients has been determined through OvAge (OvAge sr., Catanzaro, Italy), a statistical model that combines antimüllerian hormone, follicle-stimulating hormone, 3-dimensional antral follicle count, vascular index, flow index, and vascular flow index values. The control group consisted of a large population of 652 healthy women (with intact uterus and adnexa) previously enrolled to build the OvAge model. Comparisons between ovarian ages of PBS patients and the control group have been assessed by analysis of covariance linear statistical modeling. The main outcome measurement was the differences in the behavior within OvAge/age relation between PBS and control women. Descriptive statistics of those 71 enrolled PBS patients are the following: age, 49.61 ± 2.15 years; OvAge, 49.22 ± 2.57 years; follicle-stimulating hormone, 43.02 ± 19.92 mU/mL; antimüllerian hormone, 0.12 ± 0.20 ng/mL; 3-dimensional antral follicle count, 1.91 ± 1.28; vascular index, 2.80% ± 5.32%; flow index, 19.37 ± 5.88; and vascular flow index, 0.56 ± 1.12. Analysis of covariance disclosed that PBS and control women do not exhibit different behaviors (p = .900) within OvAge/age relation. According to our model, the addition of PBS to TLH in the late reproductive years does not modify the ovarian age of treated women up to 3 to 5 years after surgery. Copyright © 2016 AAGL. Published by Elsevier Inc. All rights reserved.

Initiation of follicular atresia: gene networks during early atresia in pig ovaries.

PubMed

Zhang, Jinbi; Liu, Yang; Yao, Wang; Li, Qifa; Liu, Hong-Lin; Pan, Zengxiang

2018-05-09

In mammals, more than 99% of ovarian follicles undergo a degenerative process known as atresia. The molecular events involve in atresia initiation remain incompletely understood. The objective of this study was to analyze differential gene expression profiles of medium antral ovarian follicles during early atresia in pig. The transcriptome evaluation was performed on cDNA microarrays using healthy and early atretic follicle samples and was validated by quantitative PCR. Annotation analysis applying current database (sus scrofa 11.1) revealed 450 significantly differential expressed genes between healthy and early atretic follicles. Among them, 142 were significantly up-regulated in early atretic with respect to healthy group and 308 were down-regulated. Similar expression trends were observed between microarray data and qRT-PCR confirmation, which indicated the reliability of the microarray analysis. Further analysis of the differential expressed genes revealed the most significantly affected biological functions during early atresia including blood vessel development, regulation of DNA-templated transcription in response to stress and negative regulation of cell adhesion. The pathway and interaction analysis suggested that atresia initiation associates with 1) a crosstalk of cell apoptosis, autophagy, and ferroptosis rather than change of typical apoptosis markers, 2) dramatic shift of steroidogenic enzymes, 3) deficient glutathione metabolism, and 4) vascular degeneration. The novel gene candidates and pathways identified in the current study will lead to a comprehensive view of the molecular regulation of ovarian follicular atresia and a new understanding of atresia initiation.

Malathion-induced granulosa cell apoptosis in caprine antral follicles: an ultrastructural and flow cytometric analysis.

PubMed

Bhardwaj, Jitender K; Saraf, Priyanka

2014-12-01

Organophosphate pesticides (OPs) like malathion interfere with normal ovarian function resulting in an increased incidence of atresia and granulosa cell apoptosis that plays a consequential role in the loss of ovarian follicles or follicular atresia. The aim of present study was to assess malathion-induced (100 nM) reproductive stress, ultrastructural damage and changes in apoptosis frequency in ovarian granulosa cells of antral follicles. Transmission electron microscopy (TEM) was employed for ultrastructural characterization, oxidative stress was evaluated using thiobarbituric acid reactive substances (TBARS) assay to measure lipid peroxidation, and apoptosis was quantified via flow cytometry. By TEM, apoptosis was identified by the presence of an indented nuclear membrane with blebbing, pyknotic crescent-shaped fragmented nuclei, increased vacuolization, degenerating mitochondria, and lipid droplets. The results indicate a significant increase in malondialdehyde (MDA) level (nmols/g wet tissue) at a 100 nM dose of malathion i.e. 7.57±0.033*, 8.53±0.12*, and 12.87±0.78** at 4, 6, or 8 h, respectively, as compared with controls (6.07±0.033, p<0.01*, p<0.05**) showing a positive correlation between malathion-induced lipid peroxidation and percentage of granulosa cell apoptosis (r=1; p<0.01). The parallel use of these three methods enabled us to determine the role of malathion in inducing apoptosis as a consequence of cytogenetic damage and oxidative stress generated in granulosa cells of antral follicles.

Cryopreservation of human ovarian tissue.

PubMed

Fabbri, Raffaella; Pasquinelli, Gianandrea; Bracone, Graziella; Orrico, Catia; Di Tommaso, Barbara; Venturoli, Stefano

2006-01-01

New and often aggressive treatment schemes allow the successful healing of many young patients with cancer, but the price the young women have to pay is high: many of them lose ovarian function and fertility. Due to the improved long-term survival of adolescents and young women with malignancies undergoing gonadotoxic chemotherapy, preservation of future fertility has been the focus of recent ubiquitarian interest. A feasible solution is the cryopreservation of ovarian tissue. Ovarian tissue, after thawing, can be used in three different ways: 1. grafted into its normal site (orthotopic); 2. grafted into a site other than its normal position (heterotopic), necessitating recourse to in vitro fertilization (IVF); 3. grown and in vitro matured in order to obtain metaphase II oocytes for an IVF program. It is believed that protein supplementation, in cryopreservation solution, is essential for improving ovarian tissue cryopreservation. The aim of this study was to evaluate the ultrastructural appearance of human ovarian tissue cryopreserved in 1.5 M 1,2 propanediol (PROH), 0.2 M sucrose using different protein sources: fetal calf serum (FCS), plasmanate or syntetic serum substitute (SSS). Fresh and frozen/thawed ovarian tissues were compared by transmission electron microscope (TEM), to evaluate the appearance of stromal and follicle cells as affected by different protein sources. Our data indicate that FCS is a better protein support for ovarian tissue cryopreservation when compared to SSS or Plasmanate. In addition the follicles are more resistant to the cryopreservation with respect to stroma.

Sphingosine-1-phosphate prevents chemotherapy-induced human primordial follicle death

PubMed Central

Li, Fang; Turan, Volkan; Lierman, Sylvie; Cuvelier, Claude; De Sutter, Petra; Oktay, Kutluk

2014-01-01

STUDY QUESTION Can Sphingosine-1-phosphate (S1P), a ceramide-induced death pathway inhibitor, prevent cyclophosphamide (Cy) or doxorubicin (Doxo) induced apoptotic follicle death in human ovarian xenografts? SUMMARY ANSWER S1P can block human apoptotic follicle death induced by both drugs, which have differing mechanisms of cytotoxicity. WHAT IS KNOWN ALREADY S1P has been shown to decrease the impact of chemotherapy and radiation on germinal vesicle oocytes in animal studies but no human translational data exist. STUDY DESIGN, SIZE, DURATION Experimental human ovarian xenografting to test the in vivo protective effect of S1P on primordial follicle survival in the chemotherapy setting. The data were validated by assessing the same protective effect in the ovaries of xenografted mice in parallel. PARTICIPANTS/MATERIALS, SETTING, METHODS Xenografted mice were treated with Cy (75 mg/kg), Cy+S1P (200 μM), Doxo (10 mg/kg), Doxo+S1P or vehicle only (Control). S1P was administered via continuous infusion using a mini-osmotic pump beginning 24 h prior to and ending 72 h post-chemotherapy. Grafts were then recovered and stained with anti-caspase 3 antibody for the detection of apoptosis in primordial follicles. The percentage of apoptotic to total primordial follicles was calculated in each group. MAIN RESULTS AND THE ROLE OF CHANCE Both Cy and Doxo resulted in a significant increase in apoptotic follicle death in human ovarian xenografts compared with controls (62.0 ± 3.9% versus 25.7 ± 7.4%, P < 0.01 and 76.7 ± 7.4% versus 25.7 ± 7.4%, P < 0.01, respectively). This chemotherapy-induced apoptotic death was reduced both in the Cy+S1P (32.7 ± 4.4%, P < 0.01) and the Doxo+S1P group (27.1 ± 7.6%, P < 0.01) compared with Cy and Doxo groups, respectively. In the Doxo+S1P and Cy+S1P groups, the percentages of apoptotic follicles were similar to those of vehicle-treated controls (P > 0.05). The findings from the ovaries of the severe combined immunodeficient mice mirrored the findings with human tissue. LIMITATIONS, REASONS FOR CAUTION The functionality of the rescued human ovarian follicles needs to be evaluated in future studies though the studies in rodents showed that rescued oocytes can result in healthy offspring. In addition, the impact of S1P on cancer cells should be further studied. WIDER IMPLICATIONS OF THE FINDINGS S1P and its future analogs hold promise for preserving fertility by pharmacological means for patients undergoing chemotherapy. STUDY FUNDING/COMPETING INTEREST(S) This research is supported by NIH's NICHD and NCI (5R01HD053112-06 and 5R21HD061259-02) and the Flemish Foundation for Scientific Research (FWO-Vlaanderen, grant number FWO G0.065.11N10). The authors have no conflicts of interest to disclose. PMID:24221908

Tributyltin impaired reproductive success in female zebrafish through disrupting oogenesis, reproductive behaviors and serotonin synthesis.

PubMed

Xiao, Wei-Yang; Li, Ying-Wen; Chen, Qi-Liang; Liu, Zhi-Hao

2018-07-01

Tributyltin (TBT), an organotin acting as aromatase (Cyp19a1) inhibitor, has been found to disrupt gametogenesis and reproductive behaviors in several fish species. However, few studies addressing the mechanisms underlying the impaired gametogenesis and reproduction have been reported. In this study, female adults of zebrafish (Danio rerio) were continuously exposed to two nominal concentrations of TBT (100 and 500 ng/L, actual concentrations: 90.8 ± 1.3 ng/L and 470.3 ± 2.7 ng/L, respectively) for 28 days. After exposures, TBT decreased the total egg number, reduced the hatchability and elevated the mortality of the larvae. Decreased gonadosomatic index (GSI) and altered percentages of follicles in different developmental stages (increased early-stage follicles and reduced mid/late-stage follicles) were also observed in the ovary of TBT-treated fish. TBT also lowered the plasma level of 17β-estradiol and suppressed the expressions of cyp19a1a in the ovary. In treated fish, up-regulated expressions of aldhla2, sycp3 and dmc1 were present in the ovary, indicating an enhanced level of meiosis. The mRNA level of vtg1 was dramatically suppressed in the liver of TBT-treated fish, suggesting an insufficient synthesis of Vtg protein, consistent with the decreased percentage of mid/late-stage follicles in the ovaries. Moreover, TBT significantly suppressed the reproductive behaviors of the female fish (duration of both sexes simultaneously in spawning area, the frequency of meeting and the visit in spawning area) and down-regulated the mRNA levels of genes involved in the regulation of reproductive behaviors (cyp19a1b, gnrh-3 and kiss 2) in the brain. In addition, TBT significantly suppressed the expressions of serotonin-related genes, such as tph2 (encoding serotonin synthase), pet1 (marker of serotonin neuron) and kiss 1 (the modulator of serotonin synthesis), suggesting that TBT might disrupt the non-reproductive behaviors of zebrafish. The present study demonstrated that TBT may impair the reproductive success of zebrafish females probably through disrupting oogenesis, disturbing reproductive behaviors and altering serotonin synthesis. The present study greatly extends our understanding on the reproductive toxicity of TBT on fish. Copyright © 2018 Elsevier B.V. All rights reserved.

Immune cells in the normal ovary and spontaneous ovarian tumors in the laying hen (Gallus domesticus) model of human ovarian cancer.

PubMed

Bradaric, Michael J; Penumatsa, Krishna; Barua, Animesh; Edassery, Seby L; Yu, Yi; Abramowicz, Jacques S; Bahr, Janice M; Luborsky, Judith L

2013-01-01

Spontaneous ovarian cancer in chickens resembles human tumors both histologically and biochemically. The goal was to determine if there are differences in lymphocyte content between normal ovaries and ovarian tumors in chickens as a basis for further studies to understand the role of immunity in human ovarian cancer progression. Hens were selected using grey scale and color Doppler ultrasound to determine if they had normal or tumor morphology. Cells were isolated from ovaries (n = 6 hens) and lymphocyte numbers were determined by flow cytometry using antibodies to avian CD4 and CD8 T and B (Bu1a) cells. Ovarian sections from another set of hens (n = 26) were assessed to verify tumor type and stage and to count CD4, CD8 and Bu1a immunostained cells by morphometric analysis. T and B cells were more numerous in ovarian tumors than in normal ovaries by flow cytometry and immunohistochemistry. There were less CD4+ cells than CD8+ and Bu1a+ cells in normal ovaries or ovarian tumors. CD8+ cells were the dominant T cell sub-type in both ovarian stroma and in ovarian follicles compared to CD4+ cells. Bu1a+ cells were consistently found in the stroma of normal ovaries and ovarian tumors but were not associated with follicles. The number of immune cells was highest in late stage serous tumors compared to endometrioid and mucinous tumors. The results suggest that similar to human ovarian cancer there are comparatively more immune cells in chicken ovarian tumors than in normal ovaries, and the highest immune cell content occurs in serous tumors. Thus, this study establishes a foundation for further study of tumor immune responses in a spontaneous model of ovarian cancer which will facilitate studies of the role of immunity in early ovarian cancer progression and use of the hen in pre-clinical vaccine trials.

Immune Cells in the Normal Ovary and Spontaneous Ovarian Tumors in the Laying Hen (Gallus domesticus) Model of Human Ovarian Cancer

PubMed Central

Bradaric, Michael J.; Penumatsa, Krishna; Barua, Animesh; Edassery, Seby L.; Yu, Yi; Abramowicz, Jacques S.; Bahr, Janice M.; Luborsky, Judith L.

2013-01-01

Background Spontaneous ovarian cancer in chickens resembles human tumors both histologically and biochemically. The goal was to determine if there are differences in lymphocyte content between normal ovaries and ovarian tumors in chickens as a basis for further studies to understand the role of immunity in human ovarian cancer progression. Methods Hens were selected using grey scale and color Doppler ultrasound to determine if they had normal or tumor morphology. Cells were isolated from ovaries (n = 6 hens) and lymphocyte numbers were determined by flow cytometry using antibodies to avian CD4 and CD8 T and B (Bu1a) cells. Ovarian sections from another set of hens (n = 26) were assessed to verify tumor type and stage and to count CD4, CD8 and Bu1a immunostained cells by morphometric analysis. Results T and B cells were more numerous in ovarian tumors than in normal ovaries by flow cytometry and immunohistochemistry. There were less CD4+ cells than CD8+ and Bu1a+ cells in normal ovaries or ovarian tumors. CD8+ cells were the dominant T cell sub-type in both ovarian stroma and in ovarian follicles compared to CD4+ cells. Bu1a+ cells were consistently found in the stroma of normal ovaries and ovarian tumors but were not associated with follicles. The number of immune cells was highest in late stage serous tumors compared to endometrioid and mucinous tumors. Conclusions The results suggest that similar to human ovarian cancer there are comparatively more immune cells in chicken ovarian tumors than in normal ovaries, and the highest immune cell content occurs in serous tumors. Thus, this study establishes a foundation for further study of tumor immune responses in a spontaneous model of ovarian cancer which will facilitate studies of the role of immunity in early ovarian cancer progression and use of the hen in pre-clinical vaccine trials. PMID:24040191

The impact of cow nutrient status during the second and third trimesters on age at puberty, antral follicle count, and fertility of daughters

USDA-ARS?s Scientific Manuscript database

Fluctuating feed resources to beef cows across the production cycle is a proven method for decreasing input costs; however, limiting nutrients during late gestation have been demonstrated to decrease ovarian follicle numbers in female offspring in some studies. We hypothesize that limiting nutrients...

Refining insulin concentrations in culture medium containing growth factors BMP15 and GDF9: An in vitro study of the effects on follicle development of goats.

PubMed

Dipaz-Berrocal, D J; Sá, N A R; Guerreiro, D D; Celestino, J J H; Leiva-Revilla, J; Alves, B G; Alves, K A; Santos, R R; Cibin, F W S; Rodrigues, A P R; Figueiredo, J R

2017-10-01

The aim of the present study was to investigate the influence of two insulin concentrations (10ng/mL and 10μg/mL) combined or in the absence of BMP15 and/or GDF9, on the in vitro survival and development of preantral follicles of goat ovarian tissue. Ovarian slices from the same goat ovary pair were randomly assigned to a non-cultured control treatment or to be in vitro cultured for 1 or 7days in α-MEM containing 10ng/mL (Low) or 10μg/mL (High) of insulin in the absence or presence of BMP15 and/or GDF9. With the low insulin treatment, there was a greater percentage of normal follicles than with the high insulin treatment. The addition of BMP15 alone or in association with GDF9 to the medium containing low insulin resulted in a lesser percentage of normal follicles (P<0.05). The addition of BMP15 and GDF9 separately or in combination with the high insulin concentration enhanced the percentage of normal follicles. On day 7 of culture, the use of medium containing low insulin alone or high insulin supplemented with BMP15 and BMP15+GDF9 resulted in a greater percentage of secondary follicles than the non-cultured control, although follicles cultured with low insulin were smaller than those from the control group and had greater rates of oxidative stress. In conclusion, in the presence of physiological concentrations of insulin (10ng/mL), medium supplementation with GDF9 and BMP15 alone or in combination is unnecessary for normal follicle development in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantification, morphology, and viability of equine preantral follicles obtained via the Biopsy Pick-Up method.

PubMed

Haag, K T; Magalhães-Padilha, D M; Fonseca, G R; Wischral, A; Gastal, M O; King, S S; Jones, K L; Figueiredo, J R; Gastal, E L

2013-03-01

A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. A total of 33 ovarian biopsy procedures were performed on 18 mares during the breeding season. Mares were 5 to 21 years old and biopsies were performed during the estrous and/or diestrous phase, as confirmed by transrectal ultrasonography. Follicles were mechanically isolated using a tissue chopper, counted, and classified as normal or abnormal and primordial or primary. Viability of isolated follicles was determined by Trypan Blue dye. A total of 256 biopsy attempts were made resulting in 185 successful tissue sample collections (72% success rate). The mean weight of ovarian tissue collected per procedure was 25.0 ± 1.6 mg. Overall, 620 preantral follicles were collected and isolated (95% primordial and 5% primary). The mean (±SEM) number of follicles isolated per biopsy procedure was 18.8 ± 1.9. Primordial and primary follicles had an average diameter of 31.3 ± 6.2 and 41.1 ± 6.6 μm, respectively. Viability rate was higher (P < 0.001) for primordial follicles (91%) compared with primary follicles (50%). Primordial follicles tended (P < 0.06) to have a higher rate of morphological normality (96%) compared with primary follicles (80%). The total number of follicles isolated, amount of tissue harvested, and number of follicles per mg of tissue did not differ (P > 0.05) according to phase of the estrous cycle. Younger mares (5 to 7 years old) had more (P < 0.05) follicles isolated per procedure than older mares (14 to 21 years old). The length of the interovulatory interval was not affected (P > 0.05) by any biopsy procedure, and there were no adverse effects on cyclicity or general reproductive health. In conclusion, the BPU method provided large numbers of normal and viable preantral follicles for the study of early follicular development in mares. The BPU method might be used in the future to obtain preantral follicles for in vitro culture to enable the use of numerous oocytes present within the equine ovary. This could allow for the preservation of genetic material or large-scale embryo production. Copyright © 2013 Elsevier Inc. All rights reserved.

Transcriptome profiling of the theca interna in transition from small to large antral ovarian follicles.

PubMed

Hatzirodos, Nicholas; Hummitzsch, Katja; Irving-Rodgers, Helen F; Rodgers, Raymond J

2014-01-01

The theca interna layer of the ovarian follicle forms during the antral stage of follicle development and lies adjacent to and directly outside the follicular basal lamina. It supplies androgens and communicates with the granulosa cells and the oocyte by extracellular signaling. To better understand developmental changes in the theca interna, we undertook transcriptome profiling of the theca interna from small (3-5 mm, n = 10) and large (9-12 mm, n = 5) healthy antral bovine follicles, representing a calculated >7-fold increase in the amount of thecal tissue. Principal Component Analysis and hierarchical classification of the signal intensity plots for the arrays showed no clustering of the theca interna samples into groups depending on follicle size or subcategories of small follicles. From the over 23,000 probe sets analysed, only 76 were differentially expressed between large and small healthy follicles. Some of the differentially expressed genes were associated with processes such as myoblast differentiation, protein ubiquitination, nitric oxide and transforming growth factor β signaling. The most significant pathway affected from our analyses was found to be Wnt signaling, which was suppressed in large follicles via down-regulation of WNT2B and up-regulation of the inhibitor FRZB. These changes in the transcriptional profile could have been due to changes in cellular function or alternatively since the theca interna is composed of a number of different cell types it could have been due to any systematic change in the volume density of any particular cell type. However, our study suggests that the transcriptional profile of the theca interna is relatively stable during antral follicle development unlike that of granulosa cells observed previously. Thus both the cellular composition and cellular behavior of the theca interna and its contribution to follicular development appear to be relatively constant throughout the follicle growth phase examined.

Transcriptome Profiling of the Theca Interna in Transition from Small to Large Antral Ovarian Follicles

PubMed Central

Hatzirodos, Nicholas; Hummitzsch, Katja; Irving-Rodgers, Helen F.; Rodgers, Raymond J.

2014-01-01

The theca interna layer of the ovarian follicle forms during the antral stage of follicle development and lies adjacent to and directly outside the follicular basal lamina. It supplies androgens and communicates with the granulosa cells and the oocyte by extracellular signaling. To better understand developmental changes in the theca interna, we undertook transcriptome profiling of the theca interna from small (3–5 mm, n = 10) and large (9–12 mm, n = 5) healthy antral bovine follicles, representing a calculated >7-fold increase in the amount of thecal tissue. Principal Component Analysis and hierarchical classification of the signal intensity plots for the arrays showed no clustering of the theca interna samples into groups depending on follicle size or subcategories of small follicles. From the over 23,000 probe sets analysed, only 76 were differentially expressed between large and small healthy follicles. Some of the differentially expressed genes were associated with processes such as myoblast differentiation, protein ubiquitination, nitric oxide and transforming growth factor β signaling. The most significant pathway affected from our analyses was found to be Wnt signaling, which was suppressed in large follicles via down-regulation of WNT2B and up-regulation of the inhibitor FRZB. These changes in the transcriptional profile could have been due to changes in cellular function or alternatively since the theca interna is composed of a number of different cell types it could have been due to any systematic change in the volume density of any particular cell type. However, our study suggests that the transcriptional profile of the theca interna is relatively stable during antral follicle development unlike that of granulosa cells observed previously. Thus both the cellular composition and cellular behavior of the theca interna and its contribution to follicular development appear to be relatively constant throughout the follicle growth phase examined. PMID:24830430

RHOG-DOCK1-RAC1 Signaling Axis Is Perturbed in DHEA-Induced Polycystic Ovary in Rat Model.

PubMed

Ubba, Vaibhave; Soni, Upendra Kumar; Chadchan, Sangappa; Maurya, Vineet Kumar; Kumar, Vijay; Maurya, Ruchika; Chaturvedi, Himanshu; Singh, Rajender; Dwivedi, Anila; Jha, Rajesh Kumar

2017-05-01

The function of RHOG, a RAC1 activator, was explored in the ovary during ovarian follicular development and pathological conditions. With the help of immunoblotting and immunolocalization, we determined the expression and localization of RHOG in normal (estrous cycle) and polycystic ovaries using Sprague Dawley (SD) rat model. Employing polymerase chain reaction and flow cytometry, we analyzed the transcript and expression levels of downstream molecules of RHOG, DOCK1, and RAC1 in the polycystic ovarian syndrome (PCOS) ovary along with normal antral follicular theca and granulosa cells after dehydroepiandrosterone (DHEA) supplementation. The effect of RHOG knockdown on DOCK1, VAV, and RAC1 expression was evaluated in the human ovarian cells (SKOV3), theca cells, and granulosa cells from SD rats with the help of flow cytometry. Oocyte at secondary follicles along with stromal cells showed optimal expression of RHOG. Immunoblotting of RHOG revealed its maximum expression at diestrus and proestrus, which was downregulated at estrus stage. Mild immunostaining of RHOG was also present in the theca and granulosa cells of the secondary and antral follicles. Polycystic ovary exhibited weak immunostaining for RHOG and that was corroborated by immunoblotting-based investigations. RHOG effectors DOCK1 and ELMO1 were found reduced in the ovary in PCOS condition/DHEA. RHOG silencing reduced the expression of DOCK1 and RAC1 in the theca and granulosa cells from SD rat antral follicles and that was mirrored in the human ovarian cells. Collectively, RHOG can mediate signaling through downstream effectors DOCK1 and RAC1 during ovarian follicular development (theca and granulosa cells and oocyte), but DHEA downregulated them in the PCOS ovary.

[Explore microcosmic connection between autophagy mechanism and follicular development based on "kidney governing reproduction" theory].

PubMed

Bai, Jun; Wu, Ke-Ming; Gao, Ran-Ran

2018-03-01

In the theory of traditional Chinese medicine(TCM) that "kidney storing essence and governing reproduction", reproductive essence is an important part of the kidney essence and acts as the original material of offspring embryos. Sperm, oocyte and zygote should be all included in the range of reproductive essence. Ovum is the essence of reproduction from inborn. The follicles maturation depends on the quality of oocyte and the vigor of kidney essence. Meanwhile, discharge of mature ovum relies on the stimulation and promotion by kidney Qi. Autophagy almost exists in different cells stages and all various of mammalian cells. Many studies have found that autophagy not only participates in the formation of follicles, but also in every phase of the follicles development, and is involved in the occurrence and development of ovarian diseases. Recently, more and more scholars believe that autophagy is a new field to explore the microcosmic relationship between autophagy and TCM. Kidney-nourishing TCM could promote follicular growth and improve variety clinical symptoms by inhibiting the apoptosis of ovarian granulosa cells and reducing follicular atresia. Meanwhile, apoptosis of ovarian granulosa cells is closely related to autophagy of ovarian granulosa cells. In order to provide some theoretical foundation for kidney-nourishing therapy's promoting effect on follicular growth and improving effect on ovarian function, also to further explore the molecular mechanism of kidney-nourishing medicine in promoting follicular development, this paper would explain the microcosmic relationship between autophagy and follicular development based on the theory of "kidney governing reproduction". All of these would be of great significance to prevent and intervene the diseases of reproductive system timely and effectively. Copyright© by the Chinese Pharmaceutical Association.

[Fish ovarian fluid contains protease inhibitors].

PubMed

Minin, A A; Ozerova, S G

2015-01-01

Studies of the conditions under which fish egg is activated spontaneously without the sperm showed that the egg retains the ability for fertilization in the ovarian (coelomic) fluid, which surrounds it in the gonad cavity after ovulation. Earlier, we showed that, in artificial media, the spontaneous activation is suppressed by protease inhibitors. In this study, we investigated the presence of natural protease inhibitors in the ovarian fluid and showed that the ovarian fluid of zebrafish and loach contains protease inhibitors, in particular, type I serpin a, a protein inhibitor of trypsin proteases.

Serum anti-Müllerian hormone concentration in women with polycystic ovary syndrome and type 1 diabetes mellitus.

PubMed

Łebkowska, Agnieszka; Adamska, Agnieszka; Karczewska-Kupczewska, Monika; Nikołajuk, Agnieszka; Otziomek, Elżbieta; Milewski, Robert; Górska, Maria; Wołczyński, Sławomir; Kowalska, Irina

2016-05-01

A single prior study conducted in Chilean women has shown that women with type 1 diabetes mellitus (T1DM) and polycystic ovary syndrome (PCOS) have a normal serum anti-Müllerian hormone (AMH) concentrations despite polycystic ovarian morphology. As it is not clear why women with PCOS+T1DM would not have an elevated concentrations of AMH, we hypothesize that women with T1DM and PCOS have a similar hormonal profile and serum AMH levels as is observed in classic PCOS. We studied 89 women: 37 with T1DM (16 with PCOS+T1DM, 21 with T1DM/no-PCOS), 36 with PCOS (PCOS) and 16 healthy women (control group) matched for age and body mass index (BMI). A clinical examination, determination of serum AMH and sex hormones, and an ultrasonographic evaluation of the ovaries were performed for all study participants. Serum AMH concentrations were significantly higher in women with PCOS+T1DM than in those with T1DM/no-PCOS (p<0.001) and was not different between both PCOS groups (PCOS vs PCOS+T1DM). Ovarian volume and ovarian follicle count did not differ between women with PCOS+T1DM and PCOS. The number of ovarian follicles was higher in patients with PCOS+T1DM and PCOS versus the control (p=0.007, p<0.001) and versus cases of T1DM/no-PCOS (p<0.001, p<0.001, respectively). Cross-sectionally, AMH concentrations correlated positively with luteinizing hormone (LH) (r=0.4; p<0.001), testosterone (r=0.2, p=0.02), ovarian volume (r=0.4, p<0.001) and follicle count (r=0.7, p<0.001). In both groups, PCOS+T1DM and PCOS, AMH was related to LH (r=0.5; p=0.036; r=0.3; p=0.031) and to ovarian follicle number (r=0.7; p<0.001; r=0.4; p=0.006). In multivariate logistic regression analysis, serum AMH was the only predictor of PCOS in T1DM women (OR=1.73; 95% CI 1.07-2.79, p=0.023). Women with T1DM and PCOS have a similar hormonal profile and serum AMH concentrations as observed in classic PCOS. Copyright © 2016 Elsevier Inc. All rights reserved.

Ovarian follicular development and the follicular fluid hormones and growth factors in normal women of advanced reproductive age.

PubMed

Klein, N A; Battaglia, D E; Miller, P B; Branigan, E F; Giudice, L C; Soules, M R

1996-05-01

Reproductive aging in women (a physiological decline in the function of the hypothalamic-pituitary-ovarian axis) is an infrequently investigated and poorly understood biological phenomenon. Although menstrual irregularity and anovulation are known to precede the menopause, normal women in their fifth decade experience a profound decrease in fertility while still experiencing regular menstrual cycles. To further our understanding of the physiological changes associated with reproductive aging, this study examined the spontaneous development and function of ovarian follicles in normal women, aged 40-45 yr. The subjects were women (n = 21), aged 40-45 yr, who had regular 25- to 35-day ovulatory menstrual cycles, were not infertile, had no medical problems, and met specific criteria for weight, diet, and exercise. The controls were normal women (n = 20), age 20-25 yr, who met the same criteria. The subjects were monitored with daily hormone measurements [LH, FSH, estradiol (E), progesterone (P), and inhibin] and pelvic sonograms from day 1 of their study cycle until the dominant ovarian follicle reached a mean diameter of 15 mm and/or a serum E level of 550 pmol/L or higher was attained. At that time, 10,000 U hCG were given, and a transvaginal sonographic follicle aspiration was performed 32 h later. The follicular fluid (FF) was collected, stored frozen at -70 C, and later analyzed for E, P, testosterone (T), androstenedione, inhibin, insulin-like growth factor I (IGF-I), and IGF-II. The number of cycle days to aspiration was lower (11.6 vs. 15.6 days; P < 0.001) and the early follicular phase mean FSH and mean E levels were higher (9.3 vs. 6.6 mIU/mL and 305 vs. 160 pmol/L; P < 0.01) in the older (O) group compared to the younger group. There was a strong trend toward higher FF mean E (2280 vs. 1931 nmol/L) and lower FF mean T (978 vs. 2361 pmol/L) levels in group O. The E/T ratio was significantly higher (5253 vs. 2408; P < 0.03) in group O. In group O, the mean FF P levels were increased as well (25.1 vs. 18.8 micromol/L; P < 0.01). The serum mean IGF-I (153 vs. 226 ng/mL; P < 0.001) and FF mean IGF-I (113 vs. 158 ng/mL; P < 0.02) levels were significantly decreased in group O. There were no differences between groups in serum or FF IGF-II or inhibin levels. Whether reproductive aging is an intrinsic ovarian process or the ovary is simply responding to exogenous influences, the ovary in general and its follicles in particular are the primary site of the effects of aging. Ovarian follicles in older ovulatory women have some unique features: 1) the follicles are the same size as those in younger women, but form more rapidly; 2) secretion of E and inhibin is not compromised; 3) the concentrations of steroids in the FF are indicative of a healthier follicle, i.e. increased P levels and higher estrogen to androgen ratio; and 4) serum and FF levels of IGF-I are decreased, but there are no differences in IGF-II levels.

Etoposide damages female germ cells in the developing ovary.

PubMed

Stefansdottir, Agnes; Johnston, Zoe C; Powles-Glover, Nicola; Anderson, Richard A; Adams, Ian R; Spears, Norah

2016-08-11

As with many anti-cancer drugs, the topoisomerase II inhibitor etoposide is considered safe for administration to women in the second and third trimesters of pregnancy, but assessment of effects on the developing fetus have been limited. The purpose of this research was to examine the effect of etoposide on germ cells in the developing ovary. Mouse ovary tissue culture was used as the experimental model, thus allowing us to examine effects of etoposide on all stages of germ cell development in the same way, in vitro. Fetal ovaries from embryonic day 13.5 CD1 mice or neonatal ovaries from postnatal day 0 CD1 mice were cultured with 50-150 ng ml(-1) or 50-200 ng ml(-1) etoposide respectively, concentrations that are low relative to that in patient serum. When fetal ovaries were treated prior to follicle formation, etoposide resulted in dose-dependent damage, with 150 ng ml(-1) inducing a near-complete absence of healthy follicles. In contrast, treatment of neonatal ovaries, after follicle formation, had no effect on follicle numbers and only a minor effect on follicle health, even at 200 ng ml(-1). The sensitivity of female germ cells to etoposide coincided with topoisomerase IIα expression: in the developing ovary of both mouse and human, topoisomerase IIα was expressed in germ cells only prior to follicle formation. Exposure of pre-follicular ovaries, in which topoisomerase IIα expression was germ cell-specific, resulted in a near-complete elimination of germ cells prior to follicle formation, with the remaining germ cells going on to form unhealthy follicles by the end of culture. In contrast, exposure to follicle-enclosed oocytes, which no longer expressed topoisomerase IIα in the germ cells, had no effect on total follicle numbers or health, the only effect seen specific to transitional follicles. Results indicate the potential for adverse effects on fetal ovarian development if etoposide is administered to pregnant women when germ cells are not yet enclosed within ovarian follicles, a process that starts at approximately 17 weeks gestation and is only complete towards the end of pregnancy.

Acute 7,12-dimethylbenz[a]anthracene exposure causes differential concentration-dependent follicle depletion and gene expression in neonatal rat ovaries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Madden, Jill A.; Hoyer, Patricia B.; Devine, Patrick J.

2014-05-01

Chronic exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA), generated during combustion of organic matter including cigarette smoke, depletes all ovarian follicle types in the mouse and rat, and in vitro models mimic this effect. To investigate the mechanisms involved in follicular depletion during acute DMBA exposure, two concentrations of DMBA at which follicle depletion has (75 nM) and has not (12.5 nM) been observed were investigated. Postnatal day four F344 rat ovaries were maintained in culture for four days before a single exposure to vehicle control (1% DMSO; CT) or DMBA (12 nM; low-concentration or 75 nM; high-concentration). Aftermore » four or eight additional days of culture, DMBA-induced follicle depletion was evaluated via follicle enumeration. Relative to control, DMBA did not affect follicle numbers after 4 days of exposure, but induced large primary follicle loss at both concentrations after 8 days; while, the low-concentration DMBA also caused secondary follicle depletion. Neither concentration affected primordial or small primary follicle number. RNA was isolated and quantitative RT-PCR performed prior to follicle loss to measure mRNA levels of genes involved in xenobiotic metabolism (Cyp2e1, Gstmu, Gstpi, Ephx1), autophagy (Atg7, Becn1), oxidative stress response (Sod1, Sod2) and the phosphatidylinositol 3-kinase (PI3K) pathway (Kitlg, cKit, Akt1) 1, 2 and 4 days after exposure. With the exception of Atg7 and cKit, DMBA increased (P < 0.05) expression of all genes investigated. Also, BECN1 and pAKT{sup Thr308} protein levels were increased while cKIT was decreased by DMBA exposure. Taken together, these results suggest an increase in DMBA bioactivation, add to the mechanistic understanding of DMBA-induced ovotoxicity and raise concern regarding female low concentration DMBA exposures. - Highlights: • Acute DMBA exposures induce large primary and/or secondary follicle loss. • Acute DMBA exposure did not impact primordial and small primary follicle number. • Altered ovarian gene expression was observed due to DMBA exposure.« less

Effects of hypergravity on ovarian-hypophyseal function in antepartum and postpartum rats

NASA Technical Reports Server (NTRS)

Burden, Hubert W.; Zary, Joan T.; Hodson, Charles A.; Gregory, Heather L.; Baer, Lisa A.; Ronca, April E.

2003-01-01

BACKGROUND: Rats exposed to microgravity during the post-implantation phase of pregnancy had minimal alterations in ovarian and hypophyseal parameters during the antepartum and postpartum periods. In the current study, a similar parallel experimental design was employed to ascertain the effects of hypergravity on ovarian and hypophyseal function. HYPOTHESIS: We hypothesized that hypergravity exposure during the post-implantation stage of pregnancy would not alter antepartum and postpartum ovarian and hypophyseal function. METHODS: Pregnant rats were assigned to hypergravity (1.5 G, 1.75 G, or 2.0 G), rotational control, or stationary control groups (n = 10 each group) beginning on gestation day 11 and ending on day 20. Hypophyseal and ovarian analyses were conducted on 5 of the animals from each group at day 20. The remaining animals in each group were allowed to go to term and the same analyses were conducted 3 h postpartum. RESULTS: Hypergravity at all levels decreased the percent body mass gain from gestation day 11 to 20 (p < 0.05); however, the wet weight of the pituitaries and ovaries was not changed. There was no effect of hypergravity on the number of healthy or atretic antral follicles of any size at gestation day 20 or postpartum. The number of corpora lutea of pregnancy was decreased in all hypergravity groups, but the number of live fetuses at gestation day 20 or pups at term was not altered. Plasma concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, and progesterone were not changed at gestation day 20 or postpartum. Pituitary content of LH, FSH, and prolactin was not altered by hypergravity at gestation day 20, but LH content was significantly increased (p < 0.05) at 1.5 and 1.75 G postpartum. CONCLUSIONS: We conclude that hypergravity, up to and including 2.0 G, is compatible with maintenance of pregnancy and has minimal effects on hypophyseal parameters. Ovarian follicles are not altered by hypergravity, but corpora lutea may regress at a more rapid rate.

Potential role of follicle-stimulating hormone (FSH) and transforming growth factor (TGFβ1) in the regulation of ovarian angiogenesis.

PubMed

Kuo, Shih-Wei; Ke, Ferng-Chun; Chang, Geen-Dong; Lee, Ming-Ting; Hwang, Jiuan-Jiuan

2011-06-01

Angiogenesis occurs during ovarian follicle development and luteinization. Pituitary secreted FSH was reported to stimulate the expression of endothelial mitogen VEGF in granulosa cells. And, intraovarian cytokine transforming growth factor (TGF)β1 is known to facilitate FSH-induced differentiation of ovarian granulosa cells. This intrigues us to investigate the potential role of FSH and TGFβ1 regulation of granulosa cell function in relation to ovarian angiogenesis. Granulosa cells were isolated from gonadotropin-primed immature rats and treated once with FSH and/or TGFβ1 for 48 h, and the angiogenic potential of conditioned media (granulosa cell culture conditioned media; GCCM) was determined using an in vitro assay with aortic ring embedded in collagen gel and immunoblotting. FSH and TGFβ1 increased the secreted angiogenic activity in granulosa cells (FSH + TGFβ1 > FSH ≈ TGFβ1 >control) that was partly attributed to the increased secretion of pro-angiogenic factors VEGF and PDGF-B. This is further supported by the evidence that pre-treatment with inhibitor of VEGF receptor-2 (Ki8751) or PDGF receptor (AG1296) throughout or only during the first 2-day aortic ring culture period suppressed microvessel growth in GCCM-treated groups, and also inhibited the FSH + TGFβ1-GCCM-stimulated release of matrix remodeling-associated gelatinase activities. Interestingly, pre-treatment of AG1296 at late stage suppressed GCCM-induced microvessel growth and stability with demise of endothelial and mural cells. Together, we provide original findings that both FSH and TGFβ1 increased the secretion of VEGF and PDGF-B, and that in turn up-regulated the angiogenic activity in rat ovarian granulosa cells. This implicates that FSH and TGFβ1 play important roles in regulation of ovarian angiogenesis during follicle development. Copyright © 2010 Wiley-Liss, Inc.

Ginsenoside Rg1 improves fertility and reduces ovarian pathological damages in premature ovarian failure model of mice.

PubMed

He, Lianli; Ling, Li; Wei, Tianqin; Wang, Yaping; Xiong, Zhengai

2017-04-01

This study aims to investigate the effect as well as mechanism of ginsenoside Rg1 (Rg1) on premature ovarian failure (POF) induced by d-galactose (d-gal) in mice. C57BL/6 female mice were divided into four groups randomly, which were the saline group, the d-gal group, the d-gal + Rg1 group, and the Rg1 group. Body weight was recorded. Overall ovarian function including estrous cycles, sex hormone secretion, ovarian follicle development, and ovarian morphology was analyzed by H&E staining and ELISA. Effect of Rg1 on aging was determined by analyzing the activities of oxidation-associated biomarkers, pro-inflammatory cytokine secretion, expression of senescence-associated proteins, and fertility. Compared with the d-gal group, in Rg1 + d-gal group, body weight was increased significantly, estrous cycle block was released, and fertility and the morphology of ovaries were restored. And, Rg1 treatment after d-gal administration significantly reduced senescence-associated protein expression, increased the activity of total superoxide dismutase and glutathione peroxidase from bovine erythrocyte, and induced higher follicle stimulating hormone receptor protein expression. Additionally, the expression levels of malondialdehyde, interleukin-1β, tumor necrosis factor-α, and interleukin-6 were significantly decreased. Together, Rg1 improves mouse fertility and reduces ovarian pathological damage in d-gal-induced POF model possibly through enhancing anti-inflammatory and antioxidant capacities and reducing expression of senescence signal pathway proteins. Impact statement Ginsenoside Rg1 (Rg1) is a kind of natural estrogen and it has antioxidation and antiaging effects. However, whether Rg1 has effects on premature ovarian failure (POF) is still not clear. In this study, aging model induced by d-galactose was used to mimic POF. The effect and possible mechanism of Rg1 on ovary aging was investigated. We found that Rg1 treatment up-regulated the expression of follicle stimulating hormone receptor and down-regulated senescence-associated protein expression in granule cells of POF mice. Particularly, Rg1 improved fertility ability and reduced ovarian pathological damages by its antioxidative and anti-inflammation capacity. Thus, Rg1 enhances the antiaging ability of ovary and fertility ability of POF mice through enhancing the anti-inflammatory and antioxidant capacities of ovary.

GnRH agonist leuprolide acetate does not confer any protection against ovarian damage induced by chemotherapy and radiation in vitro.

PubMed

Bildik, Gamze; Akin, Nazlı; Senbabaoglu, Filiz; Sahin, Gizem Nur; Karahuseyinoglu, Sercin; Ince, Umit; Taskiran, Cagatay; Selek, Ugur; Yakin, Kayhan; Guzel, Yilmaz; Ayhan, Cem; Alper, Ebru; Cetiner, Mustafa; Balaban, Basak; Mandel, Nil Molinas; Esen, Tarık; Iwase, Akira; Urman, Bulent; Oktem, Ozgur

2015-12-01

Is there any in vitro evidence for or against ovarian protection by co-administration of a GnRH agonist with chemotherapy in human? The co-administration of GnRH agonist leuprolide acetate with cytotoxic chemotherapy agents does not preserve ovarian reserve in vitro. Randomized controlled trials of the co-administration of gonadotrophin-releasing hormone (GnRH) agonists with adjuvant chemotherapy to preserve ovarian function have shown contradictory results. This fact, together with the lack of a proven molecular mechanism of action for ovarian protection with GnRH agonist (GnRHa) places this approach as a fertility preservation strategy under scrutiny. We therefore aimed in this study to provide in vitro evidence for or against the role of GnRHa in the prevention of chemotherapy-induced damage in human ovary. This translational research study of ex vivo and in vitro models of human ovary and granulosa cells was conducted in a university hospital between 2013 and 2015. Ovarian cortical pieces (n = 15, age 14-37) and mitotic non-luteinized (COV434 and HGrC1) and non-mitotic luteinized human granulosa cells (HLGC) expressing GnRH receptor were used for the experiments. The samples were treated with cyclophosphamide, cisplatin, paclitaxel, 5-FU, or TAC combination regimen (docetaxel, adriamycin and cyclophosphamide) with and without GnRHa leuprolide acetate for 24 h. DNA damage, apoptosis, follicle reserve, hormone markers of ovarian function and reserve (estradiol (E2), progesterone (P) and anti-mullerian hormone (AMH)) and the expression of anti-apoptotic genes (bcl-2, bcl-xL, bcl-2L2, Mcl-1, BIRC-2 and XIAP) were compared among control, chemotherapy and chemotherapy + GnRHa groups. The greatest magnitude of cytotoxicity was observed in the samples treated with cyclophosphamide, cisplatin and TAC regimen. Exposure to these drugs resulted in DNA damage, apoptosis and massive follicle loss along with a concurrent decline in the steroidogenic activity of the samples. GnRHa co-administered with chemotherapy agents stimulated its receptors and raised intracellular cAMP levels. But it neither activated anti-apoptotic pathways nor prevented follicle loss, DNA damage and apoptosis induced by these drugs. Our findings do not conclusively rule out the possibility that GnRHa may offer protection, if any, through some other mechanisms in vivo. GnRH agonist treatment with chemotherapy does not prevent or ameliorate ovarian damage and follicle loss in vitro. These data can be useful when consulting a young patient who may wish to receive GnRH treatment with chemotherapy to protect her ovaries from chemotherapy-induced damage. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A new monozoic tapeworm, Parabreviscolex niepini n. g., n. sp. (Cestoda: Caryophyllidea), from schizothoracine fishes (Cyprinidae: Schizothoracinae) in Tibet, China.

PubMed

Xi, Bing-Wen; Oros, Mikuláš; Chen, Kai; Xie, Jun

2018-02-01

A new monozoic cestode, Parabreviscolex niepini n. gen. and n. sp. (Cestoda: Caryophyllidea), is described from the type-host Schizopygopsis younghusbandi Regan, 1905 (Cyprinidae: Schizothoracinae) and Schizothorax waltoni Regan, 1905 (Cyprinidae: Schizothoracinae) in the Yarlung Tsangpo River, the upper tributary of the Brahmaputra River on the Tibetan Plateau. The new genus is placed in the Capingentidae because the vitellarium is situated partly in the medullary and cortical parenchyma, i.e., neither completely external nor internal to inner longitudinal muscles. Parabreviscolex n. gen. is characterized by possessing an afossate and cuneiform scolex; numerous vitelline follicles and testes present immediately after the scolex, and spread backward near the cirrus sac; the uterus does not loop anterior to the cirrus sac; genital pores separate, opening to the common genital atrium; the pre-ovarian vitelline follicles lateral and median, post-ovarian vitelline follicles present; ovary H-shaped, compact, and ovarian arms long, anteriorly reaching the cirrus sac. Homology search by the basic local alignment search tool (BLAST) showed that the partial 18S rDNA and complete mtDNA cox-1 sequences obtained in this report were not consistent with any sequences available in GenBank, and molecular phylogenetic analyses revealed Parabreviscolex formed a separated long branch within the caryophyllideans from cyprinids.

Intra-uterine insemination: pregnancy rate in relation to number, size of pre-ovulatory follicles and day of insemination.

PubMed

Karuppaswamy, J; Smedley, Mamin; Carter, Lindsay

2009-03-01

The objective of the study was to analyse the pregnancy rate in intra-uterine insemination (IUI) in relation to pre-ovulatory follicular number, size and day of insemination. A retrospective analysis of 216 completed IUI cycles was used in an attempt to identify significant variables predictive of treatment success. Couples with unexplained infertility and male factor infertility underwent IUI with or without ovarian stimulation. The mean number of IUI cycles per patient was 4.1, the overall pregnancy rate was 27.3% per patient, and the pregnancy rate per cycle was 6.9%. The pregnancy rate was 4.4% when one follicle was produced, whereas with more than two follicles, the rate increased to 21.2%. Hormonal stimulation using clomiphene citrate and/or human menopausal gonadotrophin/follicle stimulating hormone yielded a significant higher pregnancy rate compared to IUI in natural cycles (10.3% versus 3.3%). Although not statistically significant, the pregnancy rate decreased with advancing age of woman. The results suggest that IUI is a useful method of assisted conception in unexplained infertility and higher pregnancy rates can be achieved with good patient selection and ovarian stimulation.

Manipulation of follicle development to ensure optimal oocyte quality and conception rates in cattle.

PubMed

Baruselli, P S; Sá Filho, M F; Ferreira, R M; Sales, J N S; Gimenes, L U; Vieira, L M; Mendanha, M F; Bó, G A

2012-08-01

Over the last several decades, a number of therapies have been developed that manipulate ovarian follicle growth to improve oocyte quality and conception rates in cattle. Various strategies have been proposed to improve the responses to reproductive biotechnologies following timed artificial insemination (TAI), superovulation (SOV) or ovum pickup (OPU) programmes. During TAI protocols, final follicular growth and size of the ovulatory follicle are key factors that may significantly influence oocyte quality, ovulation, the uterine environment and consequently pregnancy outcomes. Progesterone concentrations during SOV protocols influence follicular growth, oocyte quality and embryo quality; therefore, several adjustments to SOV protocols have been proposed depending on the animal category and breed. In addition, the success of in vitro embryo production is directly related to the number and quality of cumulus oocyte complexes harvested by OPU. Control of follicle development has a significant impact on the OPU outcome. This article discusses a number of key points related to the manipulation of ovarian follicular growth to maximize oocyte quality and improve conception rates following TAI and embryo transfer of in vivo- and in vitro-derived embryos in cattle. © 2012 Blackwell Verlag GmbH.

Ablation of beta subunit of protein kinase CK2 in mouse oocytes causes follicle atresia and premature ovarian failure.

PubMed

Liang, Qiu-Xia; Wang, Zhen-Bo; Lin, Fei; Zhang, Chun-Hui; Sun, Hong-Mei; Zhou, Liang; Zhou, Qian; Schatten, Heide; Odile, Filhol-Cochet; Brigitte, Boldyreff; Sun, Qing-Yuan; Qian, Wei-Ping

2018-05-03

Premature ovarian failure (POF), a major cause of female infertility, is a complex disorder, but the molecular mechanisms underlying the disorder are only poorly understood. Here we report that protein kinase CK2 contributes to maintaining follicular survival through PI3K/AKT pathway and DNA damage response pathway. Targeted deletion of CK2β in mouse oocytes from the primordial follicle stage resulted in female infertility, which was attributed to POF incurring by massive follicle atresia. Downregulated PI3K/AKT signaling was found after CK2β deletion, indicated by reduced level of phosphorylated AKT (S473, T308, and S129) and altered AKT targets related to cell survival. Further studies discovered that CK2β-deficient oocytes showed enhanced γH2AX signals, indicative of accumulative unrepaired DSBs, which activated CHK2-dependant p53 and p63 signaling. The suppressed PI3K/AKT signaling and failed DNA damage response signaling probably contribute to large-scale oocyte loss and eventually POF. Our findings provide important new clues for elucidating the mechanisms underlying follicle atresia and POF.

Development of a Mouse Model of Menopausal Ovarian Cancer

PubMed Central

Smith, Elizabeth R.; Wang, Ying; Xu, Xiang-Xi

2014-01-01

Despite significant understanding of the genetic mutations involved in ovarian epithelial cancer and advances in genomic approaches for expression and mutation profiling of tumor tissues, several key questions in ovarian cancer biology remain enigmatic: the mechanism for the well-established impact of reproductive factors on ovarian cancer risk remains obscure; cell of origin of ovarian cancer continue to be debated; and the precursor lesion, sequence, or events in progression remain to be defined. Suitable mouse models should complement the analysis of human tumor tissues and may provide clues to these questions currently perplexing ovarian cancer biology. A potentially useful model is the germ cell-deficient Wv (white spotting variant) mutant mouse line, which may be used to study the impact of menopausal physiology on the increased risk of ovarian cancer. The Wv mice harbor a point mutation in c-Kit that reduces the receptor tyrosine kinase activity to about 1–5% (it is not a null mutation). Homozygous Wv mutant females have a reduced ovarian germ cell reservoir at birth and the follicles are rapidly depleted upon reaching reproductive maturity, but other biological phenotypes are minimal and the mice have a normal life span. The loss of ovarian function precipitates changes in hormonal and metabolic activity that model features of menopause in humans. As a consequence of follicle depletion, the Wv ovaries develop ovarian tubular adenomas, a benign epithelial tumor corresponding to surface epithelial invaginations and papillomatosis that mark human ovarian aging. Ongoing work will test the possibility of converting the benign epithelial tubular adenomas into neoplastic tumors by addition of an oncogenic mutation, such as of Tp53, to model the genotype and biology of serous ovarian cancer. Model based on the Wv mice may have the potential to gain biological and etiological insights into ovarian cancer development and prevention. PMID:24616881

Long-term effects of methamphetamine exposure in adolescent mice on the future ovarian reserve in adulthood.

PubMed

Wang, Lan; Qu, Guoqiang; Dong, Xiyuan; Huang, Kai; Kumar, Molly; Ji, Licheng; Wang, Ya; Yao, Junning; Yang, Shulin; Wu, Ruxing; Zhang, Hanwang

2016-02-03

Currently, there is an increasing prevalence of adolescent exposure to methamphetamine (MA). However, there is a paucity of information concerning the long-term impact of early exposure to MA upon female fertility and ovarian reserve. The aim of this study was to investigate the effect of long-term MA exposure in adolescents on their ovarian reserve in adulthood. Adolescent mice received intraperitoneal injections of MA (5mg/kg, three times per week) or saline from the 21st postnatal day for an 8 week period. Morphological, histological, biochemical, hormonal and ethological parameters were evaluated. An impaired ovarian reserve and vitality was found in the group treated with MA, manifesting in morphological-apparent mitochondrial damage, an activated apoptosis pathway in the ovarian tissue, a downward expression of ovarian anti-Mullerian hormone (AMH), a decreased number of primordial and growing follicles, an increased number of atretic follicles, and a depressed secretion of AMH, estradiol and progesterone from granulosa cells. However, no significant difference was noticed regarding the estrous cycle, the mating ability and the fertility outcome in the reproductive age of the mice after a period of non-medication. The present results confirmed that a long term exposure to methamphetamine in adolescent mice does have an adverse impact on their ovarian reserve, which indicates that such an early abuse of MA might influence the fertility lifespan of the female mouse. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Cox-1 Suppression and Follicle Depletion in the Etiology of Menopause- Associated Ovarian Cancer

DTIC Science & Technology

2008-04-01

follicular function (2). Most oocytes are progressively lost by atresia, or apoptosis (3), and the ovary may develop a number of atrophic features...the risk of ovarian epithelial cancers, by far the most predominant form (14), and cause growth inhibition and apoptosis in ovarian cancer cell lines...1990;4:390-400. 8. Mintz B. Embryological development of primordial germ-cells in the mouse: influence of a new mutation, Wj. J Embryol Exp

COX-1 Suppression and Follicle Depletion in the Etiology of Menopause-Associated Ovarian Cancer

DTIC Science & Technology

2008-10-01

form (8), and cause growth inhibition and apoptosis in ovarian cancer cell lines (9). However, the link between morphological inhibition and...of the c-kit receptor. Genes Dev 1990;4:390-400. 2. Mintz B. Embryological development of primordial germ-cells in the mouse: influence of a new...708–14. 12 Rodriguez GC, Walmer DK, Cline M, et al. Effect of progestin on the ovarian epithelium of macaques: cancer prevention through apoptosis

COX-1 Suppression and Follicle Depletion in the Etiology of Menopause-Associated Ovarian Cancer

DTIC Science & Technology

2009-10-01

Increased parity and oral contraceptive use are the clearest examples of factors that decrease ovarian cancer risk, both of which limit ovulation. Two...act by suppressing ovulation decrease the risk of ovarian cancer by about 40% after 3 years of use .10,11 Why pregnancy and oral contraceptives ...regular use of progestin-containing oral contraceptives is a protective factor, and the benefi t decreases after time.13 Gonadotropin stimulation in

Occurrence of ovarian follicular dominance during stimulation for IVM impacts usable blastocyst yield.

PubMed

Romero, Sergio; Pella, Ricardo; Escudero, Francisco; Pérez, Ygor; García, Mario; Orihuela, Patricia

2018-03-01

To evaluate the influence of ovarian follicular dominance on the outcome of oocyte in-vitro maturation. This retrospective cohort study included 21 patients with polycystic ovaries or polycystic ovary syndrome (Rotterdam criteria, 2004) subjected to 24 invitro maturation (IVM) cycles between October 2015 and January 2017. Patients undergoing IVM received minimal gonadotropin stimulation starting on day 2 or 3 of the cycle; ovum pick-up typically occurred on days 6 to 8. No hCG-trigger shot was given. Following 30h of IVM, mature oocytes were inseminated by ICSI and the resulting embryos cultured up to the blastocyst stage. Ovarian follicular dominance was observed in nine of the 24 IVM cycles. Oocyte IVM yielded an overall maturation rate of 69.3±23.8%, and no difference was observed when the groups with or without a dominant follicle were assessed independently. The rates of fertilization and usable blastocysts per fertilized oocyte, mature oocyte (Metaphase II) or cumulus-oocyte-complex were nearly three times higher (28.7±22.5%) in the group without ovarian follicular dominance. No differences were found in the clinical pregnancy rates attained by the individuals with or without a dominant follicle after 21 vitrified-warmed blastocyst transfer cycles. Occurrence of ovarian follicular dominance during hormonal stimulation for in-vitro maturation negatively impacted embryological outcomes. Strategies devised to limit the appearance of ovarian follicular dominance must be further explored.

Ovarian reserve and subsequent ART outcomes following methotrexate therapy for ectopic pregnancy and pregnancy of unknown location

PubMed Central

Hill, Micah J.; Cooper, Janelle C.; Levy, Gary; Alford, Connie; Richter, Kevin S.; DeCherney, Alan H.; Katz, Charles; Levens, Eric D.; Wolff, Erin F.

2013-01-01

Objective It is unclear whether the stimulated state of the ovaries as part of ART results in an increased vulnerability to the effects of methotrexate. The objective of this study was to assess ovarian reserve following methotrexate treatment for ectopic pregnancy or pregnancy of unknown location after ART. Design Retrospective cohort study. Setting Large ART practice. Patients Methotrexate or surgery following ART. Interventions None. Main Outcome Measures Follicle stimulating hormone (FSH), antral follicle count (AFC), and oocyte yield were compared between subjects treated with methotrexate and surgery. Secondary outcomes were clinical pregnancy and live birth. Results There were 153 patients in the methotrexate group and 36 patients in the surgery group. Neither group demonstrated differences in ovarian reserve or oocyte yield comparing before and after treatment values. The change in ovarian reserve and oocyte yield after treatment were similar between the two groups. The number of doses of methotrexate was not correlated with changes in ovarian reserve, indicating no dose-dependent effect. Time between treatment and repeat ART was not correlated with outcomes. Live birth in subsequent cycles was similar in the two groups. Conclusions Ovarian reserve and subsequent ART cycle outcomes were reassuring following methotrexate and surgical management of ectopic pregnancy. An adverse impact of methotrexate was not detected in this large fertility cohort as has been previously described. PMID:24269042

Acute Doxorubicin Insult in the Mouse Ovary Is Cell- and Follicle-Type Dependent

PubMed Central

Roti Roti, Elon C.; Leisman, Scott K.; Abbott, David H.; Salih, Sana M.

2012-01-01

Primary ovarian insufficiency (POI) is one of the many unintended consequences of chemotherapy faced by the growing number of female cancer survivors. While ovarian repercussions of chemotherapy have long been recognized, the acute insult phase and primary sites of damage are not well-studied, hampering efforts to design effective intervention therapies to protect the ovary. Utilizing doxorubicin (DXR) as a model chemotherapy agent, we defined the acute timeline for drug accumulation, induced DNA damage, and subsequent cellular and follicular demise in the mouse ovary. DXR accumulated first in the core ovarian stroma cells, then redistributed outwards into the cortex and follicles in a time-dependent manner, without further increase in total ovarian drug levels after four hours post-injection. Consistent with early drug accumulation and intimate interactions with the blood supply, stroma cell-enriched populations exhibited an earlier DNA damage response (measurable at 2 hours) than granulosa cells (measurable at 4 hours), as quantified by the comet assay. Granulosa cell-enriched populations were more sensitive however, responding with greater levels of DNA damage. The oocyte DNA damage response was delayed, and not measurable above background until 10–12 hours post-DXR injection. By 8 hours post-DXR injection and prior to the oocyte DNA damage response, the number of primary, secondary, and antral follicles exhibiting TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive granulosa cells plateaued, indicating late-stage apoptosis and suggesting damage to the oocytes is subsequent to somatic cell failure. Primordial follicles accumulate significant DXR by 4 hours post-injection, but do not exhibit TUNEL-positive granulosa cells until 48 hours post-injection, indicating delayed demise. Taken together, the data suggest effective intervention therapies designed to protect the ovary from chemotherapy accumulation and induced insult in the ovary must act almost immediately to prevent acute insult as significant damage was seen in stroma cells within the first two hours. PMID:22876313

Carryover Effects of Acute DEHP Exposure on Ovarian Function and Oocyte Developmental Competence in Lactating Cows

PubMed Central

Kalo, Dorit; Hadas, Ron; Furman, Ori; Ben-Ari, Julius; Maor, Yehoshua; Patterson, Donald G.; Tomey, Cynthia; Roth, Zvi

2015-01-01

We examined acute exposure of Holstein cows to di(2-ethylhexyl) phthalate (DEHP) and its carryover effects on ovarian function and oocyte developmental competence. Synchronized cows were tube-fed with water or 100 mg/kg DEHP per day for 3 days. Blood, urine and milk samples were collected before, during and after DEHP exposure to examine its clearance pattern. Ovarian follicular dynamics was monitored through an entire estrous cycle by ultrasonographic scanning. Follicular fluids were aspirated from the preovulatory follicles on days 0 and 29 of the experiment and analyzed for phthalate metabolites and estradiol concentration. The aspirated follicular fluid was used as maturation medium for in-vitro embryo production. Findings revealed that DEHP impairs the pattern of follicular development, with a prominent effect on dominant follicles. The diameter and growth rate of the first- and second-wave dominant follicles were lower (P < 0.05) in the DEHP-treated group. Estradiol concentration in the follicular fluid was lower in the DEHP-treated group than in controls, and associated with a higher number of follicular pathologies (follicle diameter >25 mm). The pattern of growth and regression of the corpus luteum differed between groups, with a lower volume in the DEHP-treated group (P < 0.05). The follicular fluid aspirated from the DEHP-treated group, but not the controls, contained 23 nM mono(2-ethylhexyl) phthalate. Culturing of cumulus oocyte complexes in the follicular fluid aspirated from DEHP-treated cows reduced the proportion of oocytes progressing to the MII stage, and the proportions of 2- to 4-cell-stage embryos (P < 0.04) and 7-day blastocysts (P < 0.06). The results describe the risk associated with acute exposure to DEHP and its deleterious carryover effects on ovarian function, nuclear maturation and oocyte developmental competence. PMID:26154164

Phenotypic variation in anti-Mullerian hormone (AMH) production per follicle in women with polycystic ovary syndrome (PCOS) and isolated polycystic ovarian morphology (PCOM): an observational cross-sectional study.

PubMed

Bhide, Priya; Kulkarni, Abhijit; Dilgil, Merve; Dhir, Puja; Shah, Amit; Gudi, Anil; Homburg, Roy

2017-10-01

This observational study compares the ratio of serum anti-Mullerian hormone (AMH) to the total antral follicle count (AFC) (as a marker of AMH production per follicle) in the various phenotypes of women with polycystic ovary syndrome (PCOS) and isolated polycystic ovarian morphology (PCOM). Two hundred and sixty-two women were recruited. Women with PCOS were divided into four phenotypes based on the diagnostic inclusion criteria of oligo-anovulation (OA), hyperandrogenism (HA) and polycystic ovarian morphology (PCOM). These included Group A (OA + HA + PCOM), Group B (OA + HA), Group C (HA + PCOM) and Group D (OA + PCOM). A ratio of serum AMH to total AFC was calculated and expressed as the AMH/AFC ratio which was compared in the phenotypes of PCOS and isolated PCOM. The median AMH/AFC ratios in PCOS-A, PCOS-D, PCOS-C and PCOM were 1.5, 1.6, 1.2 and 1.1, respectively. There were significant differences in the groups compared [F(3, 238) = 6.14, p = 0.000)]. The ratios were significantly higher in the oligo-anovulatory phenotypes PCOS-A and PCOS-D than the PCOM (p = 0.004 and 0.002, respectively). There was no significant difference in the ratio between ovulatory phenotype PCOS-C and PCOM (p = 0.59). The role of androgens and LH in per-follicle AMH production remains limited. The findings support the hypothesis of a key role for AMH in the mechanism of anovulation in PCOS.

Freezing solution containing dimethylsulfoxide and fetal calf serum maintains survival and ultrastructure of goat preantral follicles after cryopreservation and in vitro culture of ovarian tissue.

PubMed

Castro, Simone Vieira; de Carvalho, Adeline Andrade; da Silva, Cleidson Manoel Gomes; Faustino, Luciana Rocha; Campello, Cláudio Cabral; Lucci, Carolina Madeira; Báo, Sônia Nair; de Figueiredo, José Ricardo; Rodrigues, Ana Paula Ribeiro

2011-11-01

Goat ovarian cortex fragments were subjected to slow freezing in the presence of various solutions containing intracellular cryoprotectants, including 1.0 M ethylene glycol (EG), propanediol (PROH), or dimethyl sulfoxide (DMSO), with or without sucrose and/or fetal calf serum (FCS). Histological examination revealed that only the DMSO-containing solutions were able to maintain a follicular ultrastructure similar to the morphology observed in the fresh control. Therefore, fragments previously cryopreserved in DMSO solutions (with and without sucrose and/or FCS) were cultured in vitro for 48 h and then subjected to viability, histological, and ultrastructural analysis. No significant differences were observed among the percentages of morphologically normal follicles in cryopreserved ovarian tissue before in vitro culture (DMSO: 62.5%; DMSO + sucrose: 68.3%; DMSO + FCS: 60.0%; DMSO + sucrose + FCS: 60.0%) and after culture (DMSO: 60.8%; DMSO + sucrose: 64.2%; DMSO + FCS: 70.8%; DMSO + sucrose + FCS: 55.0%). Following in vitro culture, the viability analysis showed that only the freezing solution containing DMSO and FCS (75.6%) maintained a percentage of viable follicles similar to that observed after culture without cryopreservation (89.3%). As determined by ultrastructural analysis, morphologically normal preantral follicles were detected in the fresh control and in fragments cultured before and after cryopreservation with DMSO and FCS. Thus, a freezing solution containing DMSO and FCS, under the experimental conditions tested here, guaranteed the maintenance of viability and follicular ultrastructure after short-term in vitro culture.

Developmental programming: postnatal estradiol amplifies ovarian follicular defects induced by fetal exposure to excess testosterone and dihydrotestosterone in sheep.

PubMed

Veiga-Lopez, A; Wurst, A K; Steckler, T L; Ye, W; Padmanabhan, V

2014-04-01

Excess of prenatal testosterone (T) induces reproductive defects including follicular persistence. Comparative studies with T and dihydrotestosterone (DHT) have suggested that follicular persistence is programmed via estrogenic actions of T. This study addresses the androgenic and estrogenic contributions in programming follicular persistence. Because humans are exposed to estrogenic environmental steroids from various sources throughout their life span and postnatal insults may also induce organizational and/or activational changes, we tested whether continuous postnatal exposure to estradiol (E) will amplify effects of prenatal steroids on ovarian function. Pregnant sheep were treated with T, DHT, E, or ED (E and DHT) from days 30 to 90 of gestation. Postnatally, a subset of the vehicle (C), T, and DHT females received an E implant. Transrectal ultrasonography was performed in the first breeding season during a synchronized cycle to monitor ovarian follicular dynamics. As expected, number of ≥8 mm follicles was higher in the T versus C group. Postnatal E reduced the number of 4 to 8 mm follicles in the DHT group. Percentage of females bearing luteinized follicles and the number of luteinized follicles differed among prenatal groups. Postnatal E increased the incidence of subluteal cycles in the prenatal T-treated females. Findings from this study confirm previous findings of divergences in programming effects of prenatal androgens and estrogens. They also indicate that some aspects of follicular dynamics are subject to postnatal modulation as well as support the existence of an extended organizational period or the need for a second insult to uncover the previously programmed event.

Early ovarian follicular development in prepubertal Wistar rats acutely exposed to androgens.

PubMed

Paixão, L; Velez, L M; Santos, B R; Tusset, C; Lecke, S B; Motta, A B; Spritzer, P M

2016-08-01

Androgens may directly modulate early ovarian follicular development in preantral stages and androgen excess before puberty may disrupt this physiological process. Therefore, the aim of this study was to investigate the dynamics of follicular morphology and circulating androgen and estradiol levels in prepubertal Wistar rats acutely exposed to androgens. Prepubertal female Wistar rats were distributed into three groups: control, equine chorionic gonadotropin (eCG) intervention and eCG plus dehydroepiandrosterone (DHEA) intervention (eCG+DHEA). Serum DHEA, testosterone and estradiol levels were determined, and ovarian morphology and morphometry were assessed. The eCG+DHEA group presented increased serum estradiol and testosterone levels as compared with the control group (P<0.01), and higher serum DHEA concentration v. the eCG-only and control groups (P<0.01). In addition, the eCG+DHEA group had a higher number of, and larger-sized, primary and secondary follicles as compared with the control group (P<0.05). The eCG group presented intermediate values for number and size of primary and secondary follicles, without significant differences as compared with the other two groups. The number of antral follicles was higher in the eCG+DHEA and eCG groups v. controls (P<0.05). The number of primordial, atretic and cystic follicles were similar in all groups. In conclusion, the present experimental model using an acute eCG+DHEA intervention was useful to investigate events involved in initial follicular development under hyperandrogenic conditions, and could provide a reliable tool to study defective follicular development with possible deleterious reproductive consequences later in life.

Transcriptome and gene expression profile of ovarian follicle tissue of the triatomine bug Rhodnius prolixus

PubMed Central

Medeiros, Marcelo N.; Logullo, Raquel; Ramos, Isabela B.; Sorgine, Marcos H. F.; Paiva-Silva, Gabriela O.; Mesquita, Rafael D.; Machado, Ednildo Alcantara; Coutinho, Maria Alice; Masuda, Hatisaburo; Capurro, Margareth L.; Ribeiro, José M.C.; Cardoso Braz, Glória Regina; Oliveira, Pedro L

2013-01-01

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types. PMID:21736942

Alginate beads as a tool to handle, cryopreserve and culture isolated human primordial/primary follicles.

PubMed

Camboni, A; Van Langendonckt, A; Donnez, J; Vanacker, J; Dolmans, M M; Amorim, C A

2013-08-01

One major concern of grafting cryopreserved ovarian tissue to restore fertility in cancer patients is the possibility of reintroducing tumor cells. Cryopreservation of isolated primordial/primary follicles (PFs) may circumvent this problem. The aim of our work was to compare dimethyl sulfoxide (ME2SO) and ethylene glycol (EG) as cryoprotectants (CPAs) for slow-freezing of isolated human PFs in alginate. Ovarian biopsies from four women were processed for follicle isolation. PFs were embedded in alginate (5-15 per group). Follicles were frozen-thawed using 1.4M ME2SO or 1.5M EG as CPAs. Fresh and cryopreserved isolated follicles were in vitro cultured (IVC) for 7 days. At different time periods (after isolation, cryopreservation and IVC), follicles were evaluated with live/dead assay (using fluorescent probes) and diameter measurement. Follicle viability was calculated according to the percentage of dead follicular cells and the presence of a live/dead oocyte. A total of 841 PFs were isolated, embedded in alginate and cryopreserved with ME2SO (n=424) or EG (n=259), or used as controls (n=158). After 7 days of IVC, a significant increase in follicle size was observed in the fresh and ME2SO groups, but not in the EG group. The percentage of totally viable PFs was not significantly different before or after seven days of culture in fresh (100% and 82%) or ME2SO (93.2% and 85.1%) tissue. The EG group showed significantly lower viability before (63.9%) and after IVC (66.2%) than the fresh and ME2SO groups. Our results show that 1.4M ME2SO yields better preservation of isolated PF viability after thawing and 7 days of IVC than 1.5M EG. Alginate constitutes an easy, safe hydrogel matrix to handle and cryopreserve isolated human follicles using ME2SO as a CPA. Copyright © 2013 Elsevier Inc. All rights reserved.

What threshold values of antral follicle count and serum AMH levels should be considered for oocyte cryopreservation after in vitro maturation?

PubMed

Sonigo, C; Simon, C; Boubaya, M; Benoit, A; Sifer, C; Sermondade, N; Grynberg, M

2016-07-01

What threshold values of ultrasonographic antral follicle count (AFC) and serum anti-Müllerian hormone (AMH) levels should be considered for ensuring the cryopreservation of sufficient number of in vitro matured (IVM) oocytes, in cancer patients seeking fertility preservation (FP)? AFC and serum AMH values >20 follicles and 3.7 ng/ml, respectively, are required for obtaining at least 10 IVM oocytes for cryopreservation. IVM of cumulus oocyte complexes (COCs) followed by oocyte cryopreservation has emerged recently as an option for urgent FP. Recent data have reported that, in healthy patients, 8-20 cryopreserved oocytes after ovarian stimulation would maximize the chance of obtaining a live birth. Although both AFC and AMH have been reported as predictive factors of IVM success in infertile patients with polycystic ovary syndrome (PCOS), there is a dramatic lack of data regarding the values of these parameters in oncological patients as candidates for FP. From January 2009 to April 2015, we prospectively studied 340 cancer patients, aged 18-41 years, as candidates for oocyte cryopreservation following IVM. All patients had AFC and AMH measurements, 48-72 h before oocyte retrieval, regardless of the phase of the cycle. COCs were recovered under ultrasound guidance 36 h after hCG priming. Logistic regression allowed the determination of threshold values of AFC and AMH, for obtaining at least 8, 10 or 15 matures oocytes frozen after the IVM procedure. Similar analyses were performed for a final number of mature oocytes ≤2. Among the 340 cancer patients included, 300 were diagnosed with breast cancers, 14 had hematological malignancies and 26 underwent the procedure for others indications. Overall, the mean age of the population was 31.8 ± 4.5 years. Mean AFC and serum AMH levels were 21.7 ± 13.3 follicles and 4.4 ± 3.8 ng/ml, respectively. IVM was performed in equal proportions during the follicular or luteal phase of the cycle (49 and 51%, respectively). Statistical analysis showed that AFC and AMH values above 28 follicles and 3.9 ng/ml, 20 follicles and 3.7 ng/ml and 19 follicles and 3.5 ng/ml are required, respectively, for obtaining at least 15, 10 or 8 frozen IVM oocytes with a sensitivity ranging from 0.82 to 0.90. On the contrary, ≤2 IVM oocytes were cryopreserved when AFC and AMH were <19 follicles and 3.0 ng/ml, respectively. Although the potential of cryopreserved IVM oocytes from cancer patients remains unknown, data obtained from infertile PCOS women have shown a dramatically reduced competence of these oocytes when compared with that of oocytes recovered after ovarian stimulation. As a consequence, the optimal number of IVM oocytes frozen in candidates for FP is currently unpredictable. Cryopreservation of oocytes after IVM should be considered in the FP strategy when ovarian stimulation is unfeasible, in particular when markers of the follicular ovarian status are at a relatively high range. Further investigation is needed to objectively assess the real potential of these IVM oocytes after cryopreservation. Therefore, even when a good COCs yield is expected, we should systematically encourage IVM in combination with ovarian tissue cryopreservation. No external funding was obtained for the present study. The authors have no conflict of interest to declare. Not applicable. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Microscopic morphology and apoptosis of ovarian tissue after cryopreservation using a vitrification method in post-hatching turkey poults, Meleagris gallopavo

USDA-ARS?s Scientific Manuscript database

1. Microscopic morphology of ovarian tissue in post-hatching turkey poults at various ages was investigated. 2. Hematoxylin and eosin staining were used and the diameter of the oocytes and follicles were measured using microphotography. 3. Immediately after hatching, oocytes in one-day turkey pou...

Litter size influences lamb weight and ovarian reserve but not reproductive tract development or expression of genes involved in early follicular development

USDA-ARS?s Scientific Manuscript database

In ruminants, development of the female reproductive tract starts before birth, and primordial follicles in the ovaries activate during the third trimester of gestation. Lambs born as twins have lower birth weights and smaller ovarian reserves than lambs born as singletons. We hypothesized that ew...

Gene expression profiling of bovine ovarian follicular and luteal cells provides insight into cellular identities and functions

USDA-ARS?s Scientific Manuscript database

After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these fou...

Premature Ovarian Failure in French Canadian Leigh Syndrome.

PubMed

Ghaddhab, Chiraz; Morin, Charles; Brunel-Guitton, Catherine; Mitchell, Grant A; Van Vliet, Guy; Huot, Céline

2017-05-01

In all surviving girls with Leigh syndrome, French Canadian variety, a mitochondrial disease, we detected premature ovarian failure, manifested as absent or arrested breast development, lack of menarche, high follicle-stimulating hormone, a prepubertal uterus, and small ovaries. Pubertal onset and progression should be evaluated in girls with mitochondrial diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

RNA SYNTHESIS IN THE MOUSE OOCYTE

PubMed Central

Moore, G. P. M.; Lintern-Moore, Sue; Peters, Hannah; Faber, M.

1974-01-01

RNA synthesis in the oocyte and granulosa cell nuclei of growing follicles has been studied in the mouse ovary. The RNA precursor [3H]uridine was administered intraperitoneally to adult mice and the amount of label incorporated into ovarian RNA was quantitated autoradiographically using grain-counting procedures. Uridine incorporation into the nucleus is low in oocytes of small, resting follicles but increases during follicle growth and reaches a peak prior to the beginning of antrum formation. Thereafter uptake rapidly declines and is very low in the oocytes of maturing follicles. Uridine incorporation into granulosa cell nuclei, in contrast to that found in the oocyte, increases gradually during most of the period of follicle growth. Qualitative studies of the activity of endogenous, DNA-dependent RNA polymerases have also been made in fixed oocytes isolated from follicles at different stages of growth. Polymerase activity is demonstrable in the nucleolus and nucleoplasm of oocytes from growing follicles, but is absent from maturing oocytes of large follicles. PMID:4813213

Alternative treatment of ovarian cysts with Tribulus terrestris extract: a rat model.

PubMed

Dehghan, A; Esfandiari, A; Bigdeli, S Momeni

2012-02-01

Tribulus terrestris has long been used in traditional medicine to treat impotency and improve sexual functions in man. The aim of this study was to evaluate the efficiency of T. terrestris extract in the treatment of polycystic ovary (PCO) in Wistar rat. Estradiol valerate was injected to 15 mature Wistar rats to induce PCO. Rats were randomly divided into three groups (control, low-dose and high-dose groups) of five each and received 0, 5 and 10 mg of T. terrestris extract, respectively.Treatments began on days 50 and 61 after estradiol injection; at the same time, vaginal smear was prepared. The ovaries were removed on day 62, and histological sections were prepared accordingly. The number and diameter of corpora lutea, thickness of the theca interna layer and the number of all follicles were evaluated in both ovaries. In comparison with the control group, the number of corpora lutea and primary and secondary follicles significantly increased following T. terrestris treatment; however, the number of ovarian cysts significantly decreased. It can be concluded that T. terrestris have a luteinizing effect on ovarian cysts, which may relate to its gonadotropin-like activity; also, a high dose of the extract can efficiently remove ovarian cysts and resume ovarian activity. © 2011 Blackwell Verlag GmbH.

Assessment of ovarian reserve in euthyroid adolescents with Hashimoto thyroiditis.

PubMed

Pirgon, Ozgur; Sivrice, Cigdem; Demirtas, Hakan; Dundar, Bumin

2016-01-01

We aimed to investigate the ovarian function and reserve in euthyroid adolescents (TSH < 2.5 mIU/L) diagnosed with Hashimoto thyroiditis (HT). This case-control study included 30 adolescent girls (mean age 15.1 ± 1.4 years) newly diagnosed as HT with presence of high thyroid antibodies with gland heterogeneity in ultrasound and age-matched 30 healthy female subjects. Anti-ovarian antibody (AOAb), LH/FSH ratio, estradiol, anti-mullerian hormone (AMH), inhibin-B, total testosterone, antral follicle count, ovarian volumes and uterine length were measured. The clinical, laboratory, and ultrasound data of the HT and control groups were compared. There were no significant differences between the girls with HT and healthy controls in relation to LH/FSH ratio, estradiol and inhibin-B levels. AOAb (p = 0.02), AMH (p = 0.007) and total testosterone levels were higher in HT group than the control group (p = 0.03). AOAb level was found to be positively correlated with LH/FSH ratio (p = 0.03), AMH (p = 0.01) and inhibin-B (p < 0.001) in HT group. This study demonstrated that the adolescent girls diagnosed with autoimmune thyroiditis had normal ovarian reserve based on measurements of AMH, inhibin B, FSH, LH/FSH ratio, estradiol and antral follicle counts.

Ovario-protective effects of genistein against cyclophosphamide toxicity in rats: Role of anti-müllerian hormone and oestradiol.

PubMed

Saleh, Dalia O; Mansour, Dina F

2016-10-15

Cyclophosphamide (CP), the commonly used chemotherapeutic agent in cancer treatment, is proven to cause ovarian toxicity and infertility in women. In the present study, we investigated the protective effect of genistein (GEN), a phytoestrogen found in the soy protein, against CP-induced ovarian toxicity in rats. Forty female adult rats were allocated into five groups. A normal control group received the vehicle; another group was injected with a single acute intraperitoneal dose of CP (200mg/kg). Three other groups were pretreated with GEN (0.5, 1 or 2mg/kg; s.c.) for 14 days. Sera and ovaries were obtained 48h after CP treatment. Serum levels of anti-müllerian hormone (AMH) and oestradiol (E2) were detected as well as the ovarian level of reduced glutathione (GSH), activity of superoxide dismutase (SOD), level of malondialdehyde (MDA) and interleukin 1β (IL-1β) were evaluated. Histopathological examination and immunohistochemical detection of inducible nitric oxide synthetase (iNOS) were conducted. Results of the present study revealed that CP-induced severe ovarian toxicity via decreasing serum levels of AMH and E2 and elevating oxidative stress and inflammation in ovarian tissues. Histologically, CP caused increase in primordial follicles with less graafian follicles and corpora lutea in ovarian tissues as well as severe induction of iNOS. GEN inhibited the severe decrease in serum AMH and E2 with alleviation of oxidative stress and inflammation significantly compared to CP-treated group. GEN improved ovarian histology and immunostaining of ovarian iNOS disrupted by CP. Finally, it can be concluded that GEN exerted protective effects against CP-induced ovarian toxicity. Copyright © 2016 Elsevier B.V. All rights reserved.

Relationship between Numerous Mast Cells and Early Follicular Development in Neonatal MRL/MpJ Mouse Ovaries

PubMed Central

Nakamura, Teppei; Otsuka, Saori; Ichii, Osamu; Sakata, Yuko; Nagasaki, Ken-Ichi; Hashimoto, Yoshiharu; Kon, Yasuhiro

2013-01-01

In the neonatal mouse ovary, clusters of oocytes called nests break into smaller cysts and subsequently form individual follicles. During this period, we found numerous mast cells in the ovary of MRL/MpJ mice and investigated their appearance and morphology with follicular development. The ovarian mast cells, which were already present at postnatal day 0, tended to localize adjacent to the surface epithelium. Among 11 different mouse strains, MRL/MpJ mice possessed the greatest number of ovarian mast cells. Ovarian mast cells were also found in DBA/1, BALB/c, NZW, and DBA/2 mice but rarely in C57BL/6, NZB, AKR, C3H/He, CBA, and ICR mice. The ovarian mast cells expressed connective tissue mast cell markers, although mast cells around the surface epithelium also expressed a mucosal mast cell marker in MRL/MpJ mice. Some ovarian mast cells migrated into the oocyte nests and directly contacted the compressed and degenerated oocytes. In MRL/MpJ mice, the number of oocytes in the nest was significantly lower than in the other strains, and the number of oocytes showed a positive correlation with the number of ovarian mast cells. The gene expression of a mast cell marker also correlated with the expression of an oocyte nest marker, suggesting a link between the appearance of ovarian ? 4mast cells and early follicular development. Furthermore, the expression of follicle developmental markers was significantly higher in MRL/MpJ mice than in C57BL/6 mice. These results indicate that the appearance of ovarian mast cells is a unique phenotype of neonatal MRL/MpJ mice, and that ovarian mast cells participate in early follicular development, especially nest breakdown. PMID:24124609

Knockout of RNA Binding Protein MSI2 Impairs Follicle Development in the Mouse Ovary: Characterization of MSI1 and MSI2 during Folliculogenesis

PubMed Central

Sutherland, Jessie M.; Sobinoff, Alexander P.; Gunter, Kara M.; Fraser, Barbara A.; Pye, Victoria; Bernstein, Ilana R.; Boon, Evan; Siddall, Nicole A.; De Andres, Luisa I.; Hime, Gary R.; Holt, Janet E.; Graf, Thomas; McLaughlin, Eileen A.

2015-01-01

Characterizing the mechanisms underlying follicle development in the ovary is crucial to understanding female fertility and is an area of increasing research interest. The RNA binding protein Musashi is essential for post-transcriptional regulation of oocyte maturation in Xenopus and is expressed during ovarian development in Drosophila. In mammals Musashi is important for spermatogenesis and male fertility, but its role in the ovary has yet to be characterized. In this study we determined the expression of mammalian Musashi proteins MSI1 and MSI2 during mouse folliculogenesis, and through the use of a MSI2-specific knockout mouse model we identified that MSI2 is essential for normal follicle development. Time-course characterization of MSI1 and MSI2 revealed distinct differences in steady-state mRNA levels and protein expression/localization at important developmental time-points during folliculogenesis. Using a gene-trap mouse model that inactivates Msi2, we observed a significant decrease in ovarian mass, and change in follicle-stage composition due to developmental blocking of antral stage follicles and pre-antral follicle loss through atresia. We also confirmed that hormonally stimulated Msi2-deficient mice produce significantly fewer MII oocytes (60.9% less than controls, p < 0.05). Furthermore, the majority of these oocytes are of poor viability (62.2% non-viable/apoptotic, p < 0.05), which causes a reduction in female fertility evidenced by decreased litter size in Msi2-deficient animals (33.1% reduction to controls, p < 0.05). Our findings indicate that MSI1 and MSI2 display distinct expression profiles during mammalian folliculogenesis and that MSI2 is required for pre-antral follicle development. PMID:26131972

Changes in homologous and heterologous gap junction contacts during maturation-inducing hormone-dependent meiotic resumption in ovarian follicles of Atlantic croaker

USGS Publications Warehouse

Bolamba, D.; Patino, R.; Yoshizaki, G.; Thomas, P.

2003-01-01

Homologous (granulosa cell-granulosa cell) gap junction (GJ) contacts increase in ovarian follicles of Atlantic croaker (Micropogonias undulatus) during the early (first) stage of maturation, but their profile during the second stage [i.e., during maturation-inducing hormone (MIH)-mediated meiotic resumption] is unknown. The profile of homologous GJ contacts during the second stage of maturation in croaker follicles was examined in this study and compared to that of heterologous (granulosa cell-oocyte) GJ, for which changes have been previously documented. Follicles were incubated with human chorionic gonadotropin to induce maturational competence (first stage), and then with MIH to induce meiotic resumption. The follicles were collected for examination immediately before and after different durations of MIH exposure until the oocyte had reached the stage of germinal vesicle breakdown (GVBD; index of meiotic resumption). Ultrathin sections were observed by transmission electron microscopy, and homologous and heterologous GJ contacts were quantified along a 100-??m segment of granulosa cell-zona radiata complex per follicle (three follicles/time/fish, n=3 fish). Relatively high numbers of both types of GJ were observed before and after the first few hours of MIH exposure (up to the stage of oil droplet coalescence). GJ numbers declined during partial yolk globule coalescence (at or near GVBD) and were just under 50% of starting values after the completion of GVBD (P<0.05). These results confirm earlier observations that GVBD temporally correlates with declining heterologous GJ contacts, and for the first time in teleosts show that there is a parallel decline in homologous GJ. The significance of the changes in homologous and heterologous GJ is uncertain and deserves further study. ?? 2003 Elsevier Science (USA). All rights reserved.

Effects of excess metabolizable protein on ovarian function and circulating amino acids of beef cows: 1. Excessive supply from corn gluten meal or soybean meal.

PubMed

Geppert, T C; Meyer, A M; Perry, G A; Gunn, P J

2017-04-01

In the dairy industry, excess dietary CP is consistently correlated with decreased conception rates. However, the source from which excess CP is derived and how it affects reproductive function in beef cattle is largely undefined. The objective of this experiment was to determine the effects of feeding excess metabolizable protein (MP) from feedstuffs differing in rumen degradability on ovulatory follicular dynamics, subsequent corpus luteum (CL) development, steroid hormone production and circulating amino acids (AA) in beef cows. Non-pregnant, non-lactating mature beef cows (n=18) were assigned to 1 of 2 isonitrogenous diets (150% of MP requirements) designed to maintain similar BW and body condition score (BCS) between treatments. Diets consisted of ad libitum corn stalks supplemented with corn gluten meal (moderate rumen undegradable protein (RUP); CGM) or soybean meal (low RUP; SBM). After a 20-day supplement adaptation period, cows were synchronized for ovulation. After 10 days of synchronization, gonadotropin releasing hormone (GnRH) was administered to reset ovarian follicular growth. Starting at GnRH administration and daily thereafter until spontaneous ovulation, transrectal ultrasonography was used to diagram ovarian follicular growth, and blood samples were collected for hormone, metabolite and AA analyses. After 7 days of visual detection of estrus, CL size was determined via ultrasound. Data were analyzed using the MIXED procedures of SAS. As designed, cow BW and BCS were not different (P⩾0.33). Ovulatory follicular wavelength, antral follicle count, ovulatory follicle size at dominance and duration of dominance were not different (P>0.13) between treatments. Cows supplemented with CGM had greater post-dominance ovulatory follicle growth, larger dominant follicles at spontaneous luteolysis, shorter proestrus, and larger ovulatory follicles (P⩽0.03) than SBM cows. No differences (P⩾0.44) in peak estradiol, ratio of estradiol to ovulatory follicle volume, or plasma urea nitrogen were observed. While CL volume and the ratio of progesterone to CL volume were not affected by treatment (P⩾0.24), CGM treated cows tended to have decreased (P=0.07) circulating progesterone 7 days post-estrus compared with SBM cows. Although total circulating plasma AA concentration did not differ (P=0.70) between treatments, CGM cows had greater phenylalanine (P=0.03) and tended to have greater leucine concentrations (P=0.07) than SBM cows. In summary, these data illustrate that excess MP when supplemented to cows consuming a low quality forage may differentially impact ovarian function depending on ruminal degradability of the protein source.

Relationship Between 17-Hydroxyprogesterone Responses to Human Chorionic Gonadotropin and Markers of Ovarian Follicle Morphology in Women With Polycystic Ovary Syndrome

PubMed Central

Maas, Kevin H.; Chuan, Sandy S.; Cook-Andersen, Heidi; Su, H. Irene; Duleba, A.

2015-01-01

Context: Women with polycystic ovary syndrome (PCOS) have increased 17-hydroxyprogesterone (17-OHP) responses to gonadotropin stimulation although individual variability is substantial, as reflected by exaggerated as well as normal responses. The relationship between 17-OHP responses to gonadotropin stimulation and markers of ovarian function has not been assessed. Objective: To determine whether 17-OHP responses are associated with antral follicle count (AFC), anti-Mullerian hormone (AMH), or inhibin B (Inh B) levels in PCOS and normal women. Design: Prospective study. Setting: Research center at an academic medical center. Participants: Women with PCOS (n = 18) and normal controls (n = 18). Interventions: Blood samples were obtained before and 24 hours after administration of 25 μg recombinant-human chorionic gonadotropin. Ovarian imaging was conducted with three-dimensional pelvic ultrasound. Main Outcome Measures: Basal and stimulated levels of 17-OHP, androgens, estrogen, AMH, Inh B, and AFC. Results: In women with PCOS, 17-OHP responses were heterogeneous and inversely correlated with AMH and Inh B levels, but not AFC. In a subgroup of PCOS women with exaggerated 17-OHP responses, AMH levels were equivalent to that of normal women. In PCOS women with normal 17-OHP responses, AMH levels were markedly elevated. Conclusion: Based on heterogeneous 17-OHP responses to human chorionic gonadotropin in women with PCOS, AMH levels are inversely linked to ovarian androgen production while positively correlated with AFC. These findings suggest that in PCOS, AMH production may reflect redistribution of the follicle population or regulation by intraovarian mechanisms. PMID:25313914

Study on the zona pellucida 4 (ZP4) gene sequence and its expression in the ovaries of patients with polycystic ovary syndrome.

PubMed

Meczekalski, B; Nawrot, R; Nowak, W; Czyzyk, A; Kedzia, H; Gozdzicka-Jozefiak, A

2015-07-01

Polycystic ovary syndrome (PCOS) is a common endocrine disorder of unknown pathology, involving reproductive and metabolic abnormalities. Oocyte-specific genes are a group of genes expressed exclusively in ovarian tissue; therefore, they can play an important role in ovarian pathologies such as PCOS. The zona pellucida 4 (ZP4) gene encodes glycoprotein which is a part of the extracellular matrix of oocyte. We analyzed 87 patients with PCOS, which were divided into four groups depending on their phenotype. In each patient, we performed profound clinical and biochemical analysis, including the measurement of serum androgens. The ovarian tissue samples were used to perform a real-time polymerase chain reaction and immunohistochemical staining using anti-ZP4 monoclonal antibodies. The ZP4 gene was sequenced from peripheral lymphocytes. The expression of ZP4 was present in early antral follicles and was stronger in mature follicles. The subgroup of patients with eumenorrhea and without hyperandrogenism presented the highest expression of ZP4 in ovarian tissue. In one case, we found a mutation of the ZP4 gene. No correlations were found between the ZP4 expression level and biochemical or clinical indices. Data from this and animal studies suggest a possible relationship between androgens and ZP4 expression. ZP4 expression is highest among patients with PCOS and a regular cycle, and this is a consequence of the presence of mature follicles in this group. In some patients with PCOS and infertility, ZP4 mutation can be found.

Protective effects of sildenafil citrate administration on cisplatin-induced ovarian damage in rats.

PubMed

Taskin, Mine Islimye; Yay, Arzu; Adali, Ertan; Balcioglu, Esra; Inceboz, Umit

2015-04-01

The aim of this study is to evaluate the effects of sildenafil citrate on cisplatin-induced ovarian toxicity. Thirty-two female rats were divided into four groups. Group 1: saline control; group 2: cisplatin; group 3: sildenafil citrate; and group 4: cisplatin plus sildenafil citrate group. In groups 2 and 4, the rats were injected with 5 mg/kg cisplatin intraperitoneally (i.p.). In groups 3 and 4, the rats were injected with 1.4 mg/kg sildenafil citrate i.p. The ovaries were removed two weeks later in all groups. Histopathologic examination, follicle counting and classification were performed. The expression of anti-Müllerian hormone (AMH) was detected immunohistochemically in the ovarian tissues. Sildenafil alleviated cisplatin-induced histopathological changes in the ovarian tissue. Primordial, secondary and tertiary follicles were diminished in group 2 compared with group 1 (p < 0.05). Pretreatment with sildenafil citrate preserved primordial follicle count in group 4 compared with group 2, and the difference was statistically significant (p < 0.05). According to our results, immunoreactivity intensity of AMH was lower in group 2 compared with group 1 (92.4 ± 3.97 versus 88.8 ± 1.77) but not significantly, whereas immunoreactivity intensity of AMH was higher in group 4 compared with group 2 (88.8 ± 1.77 versus 94.1 ± 2.36; p < 0.05). Our results demonstrated that pretreatment with sildenafil citrate is beneficial for protecting the ovaries from cisplatin-induced damage. Sildenafil citrate can be a choice for fertility preservation.

Anovulation and ovulation induction

PubMed Central

Katsikis, I; Kita, M; Karkanaki, A; Prapas, N; Panidis, D

2006-01-01

Conventional treatment of normogonadotropic anovulatory infertility is ovulation induction using the antiestrogen clomiphene citrate, followed by follicle-stimulating hormone. Multiple follicle development, associated with ovarian hyperstimulation, and multiple pregnancy remain the major complications. Cumulative singleton and multiple pregnancy rate data after different induction treatments are needed. Newer ovulation induction interventions, such as insulin-sensitizing drugs, aromatase inhibitors and laparoscopic ovarian electrocoagulation, should be compared with conventional treatments. Ovulation induction efficiency might improve if patient subgroups with altered chances for success or complications with new or conventional techniques could be identified, using multivariate prediction models based on initial screening characteristics. This would make ovulation induction more cost-effective, safe and convenient, enabling doctors to advise patients on the most effective and patient-tailored treatment strategy. PMID:20351807

The role of cryopreservation for women prior to treatment of malignancy.

PubMed

Donnez, Jacques; Dolmans, Marie-Madeleine; Martinez-Madrid, Belen; Demylle, Dominique; Van Langendonckt, Anne

2005-08-01

The purpose of this review is to investigate recent advances in xenografting, as well as in orthotopic and heterotopic autotransplantation of human cryopreserved ovarian tissue. The first livebirth after orthotopic transplantation of cryopreserved ovarian tissue was reported recently. We discuss this case and other cases of reimplantation of cryopreserved ovarian tissue, bearing in mind that many questions remain. Finally, we report the latest developments in research on the transplantation of an intact ovary and the reimplantation of isolated follicles.

Estrus behavior, ovarian dynamics, and progesterone secretion in Criollo cattle during estrous cycles with two and three follicular waves.

PubMed

Quezada-Casasola, Andrés; Avendaño-Reyes, Leonel; Macías-Cruz, Ulises; Ramírez-Godínez, José Alejandro; Correa-Calderón, Abelardo

2014-04-01

In beef and dairy cattle, the number of follicular waves affects endocrine, ovarian, and behavioral events during a normal estrous cycle. However, in Mexican-native Criollo cattle, a shortly and recently domesticated breed, the association between wave patterns and follicular development has not been studied. The objective of this study was to evaluate the effect of number of follicular waves in an estrous cycle on development of anovulatory and ovulatory follicles, corpus luteum (CL) development and functionality, as well as estrual behavior in Criollo cows. Ovarian follicular activities of 22 cycling multiparous Criollo cows were recorded daily by transrectal ultrasound examinations during a complete estrous cycle. Additionally, blood samples were collected daily to determine serum progesterone concentrations. Only two- (n = 17, 77.3%) and three-wave follicular (n = 5, 22.7%) patterns were observed. Duration of estrus, length of estrous cycle, and length of follicular and luteal phases were similar (P > 0.05) between cycles of two and three waves. Two-wave cows ovulated earlier (P < 0.05) after detection of estrus than three-wave cows. Detected day and maximum diameter of first anovulatory follicle were not affected (P > 0.05) by number of waves. Growth rate of first dominant follicle was higher (P < 0.05) in three-wave cycles. Onset of regression of the first dominant follicle was earlier (P < 0.01) in cycles with three waves than in those with two waves. In two-wave cycles, ovulatory follicles were detected earlier (P < 0.01) and had lower (P < 0.01) growth rate than in three-wave cycles. Development (i.e., maximum diameter and volume) and functionality (minimum and maximum progesterone concentration) of CL were similar (P > 0.05) between two- and three-wave patterns. In conclusion, Criollo cows have two or three follicular waves per estrous cycle, which alters partially ovulatory follicle development and ovulation time after detection of estrus. Length of estrous cycle, as well as CL development and functionality, was not affected by number of follicular waves.

Effects of Space Flight on Ovarian-Hypophyseal Function in Postpartum Rats

NASA Technical Reports Server (NTRS)

Burden, H. W.; Zary, J.; Lawrence, I. E.; Jonnalagadda, P.; Davis, M.; Hodson, C. A.

1997-01-01

The effect of space flight in a National Aeronautics and Space Administration (NASA) shuttle was studied in pregnant rats. Rats were launched on day 9 of gestation and recovered on day 20 of gestation. On day 20 of gestation, rats were unilaterally hysterectomized and subsequently allowed to go to term and deliver vaginally. There was no effect of space flight on pituitary and ovary mass postpartum. In addition, space flight did not alter healthy and atretic ovarian antral follicle populations, fetal wastage in utero, plasma concentrations of progesterone and luteinizing hormone (LH) or pituitary content of follicle stimulating hormone (FSH). Space flight significantly increased plasma concentrations of FSH and decreased pituitary content of LH at the postpartum sampling time. Collectively, these data show that space flight, initiated during the postimplantation period of pregnancy, and concluded before parturition, is compatible with maintenance of pregnancy and has minimal effects on postpartum hypophyseal parameters; however, none of the ovarian parameters examined was altered by space flight.

Gene expression of matrix metalloproteinases and LH receptors in mare follicular development.

PubMed

Bastos, H B A; Kretzmann, N A; Santos, G O; Esmeraldino, A T; Rechsteiner, S Fiala; Mattos, R C; Neves, A P

2014-11-01

The period from the emergence of a dominant follicle until its formation requires tissue remodeling. Enzymes promoting collagen lysis, such as matrix metalloproteinases (MMPs), are fundamental for the process of extracellular matrix remodeling, which allows changes in ovarian tissue architecture during follicular growth. It has been suggested that the production of these enzymes may be affected by the rise in circulating concentrations of LH, which acts on the ovarian surface epithelium (OSE). The aim of this study was to determine the expression of MMP-1, MMP-2, and LH receptor (LHR) in the ovulation fossa and in the central portion of the equine ovary during follicular deviation and dominance. Ovaries of 12 cyclic mares were selected and subsequently divided into two groups: development (DEV) group and dominant (DOM) group. The DEV group consisted of ovaries from six animals whose follicles were less than 28 mm in diameter (follicular deviation), and the DOM group consisted of ovaries from six animals whose follicles measured 28 mm or more in diameter (dominant follicles). The latter group was divided into two subgroups: the group of ovaries with a dominant follicle (DOM-D) and the group of contralateral ovaries (DOM-C). Our results showed that mRNA for MMP-1, MMP-2, and LHR was present in the equine ovary during follicle development, in the ovulation fossa, and in the central portion of the ovary. MMP-1 and LHR gene expression was greater (P < 0.05) for the DOM-D group compared with the DOM-C group. In the DOM-D group, MMP-1, MMP-2, and LHR gene expression was greater (P < 0.05) in the ovarian stroma compared with the ovulation fossa. Using immunohistochemistry, OSE from the DOM group showed increased expression compared with the DEV group (P < 0.05). In conclusion, we demonstrated that MMP-1 and MMP-2 might be fundamental for events related to tissue remodeling, which occurs during follicular development until the formation of the dominant follicle. We also demonstrated the relationship between the gene expression of MMPs and the gene and protein expression of LHR, suggesting that LHR in the OSE might be an important factor to initiate the signaling cascade that culminates with the production of MMPs. Copyright © 2014 Elsevier Inc. All rights reserved.

Intracellular lipid dysregulation interferes with leukocyte function in the ovaries of meat-type hens under unrestricted feed intake.

PubMed

Liu, Zu-Chen; Su, Chia-Ming; Xie, Yi-Lun; Chang, Chai-Ju; Chen, Jiang-Young; Wu, Shu-Wei; Chen, Yu-Hui; Walzem, Rosemary L; Huang, San-Yuan; Chen, Shuen-Ei

2016-04-01

Meat-type Red-feather country hens fed ad libitum (AD-hens) exhibit obesity-associated morbidities and a number of ovarian irregularities. Leukocyte participations in ovarian activities are unstudied in AD-hens. In contrast to feed-restricted hens (R-hens), ovulatory process of the F1 follicle appeared delayed in AD-hens in association with reduced F1 follicle progesterone content, gelatinase A (MMP-2) and collagenase-3 (MMP-13) activities coincident with elevated IL-1β and no production (P<0.05), and increased leukocyte infiltration of inflamed necrotic follicle walls. Extracts of AD-hen F1 follicle walls induced greater leukocyte migration than extracts from F1 follicle wall extracts of R-hens (P<0.05). Co-cultures of granulosa cells with increasing numbers of leukocytes from either AD-hens or R-hens exhibited dose dependent reductions in progesterone production and increases in cell death. AD-hen leukocytes were less proapoptotic than their R counterparts (P<0.05). Granulosa MMP-13 and MMP-2 activities were also suppressed in the co-cultures with heterophils or monocytes in a dose-dependent manner (P<0.05). AD heterophils and R monocytes had a greater inhibitory effect on MMP activities in the co-cultures than their respective counterparts (P<0.05). Both basal and LPS-induced IL-1β secretion and MMP-22 or MMP-2 activities in freshly isolated AD-hen leukocytes were reduced (P<0.05). Exposure of AD or R leukocytes to 0.5mM palmitate impaired IL-1β secretion and MMP-22 or MMP-2 activity. Inhibition of ceramide synthesis with FB1 and ROS production with n-MPG scavenging rescued MMP activity and IL-1β production in palmitate treated heterophils, but exacerbated monocyte suppression. These latter findings suggest that intracellular lipid dysregulation in leukocytes contributes to ovarian dysfunction in AD-hens. Copyright © 2016 Elsevier B.V. All rights reserved.

Intraindividual right-left comparison of sonographic features in polycystic ovary syndrome (PCOS) diagnosis.

PubMed

Köninger, Angela; Koch, Laura; Edimiris, Philippos; Nießen, Stefanie; Kasimir-Bauer, Sabine; Kimmig, Rainer; Strowitzki, Thomas; Schmidt, Börge

2014-10-01

Sonographic features of polycystic ovaries consist of elevated antral follicle count or ovarian volume of at least one ovary. The aim of this prospective cross-sectional study was to estimate intraindividual differences in sonographic measurements between the both ovaries of PCOS patients and controls and clinical consequences. Both ovaries of 85 PCOS patients and 48 controls were scanned transvaginally and agreement of sonographic measurements was analyzed using the Bland-Altman method. Concordance correlation coefficients (CCC) were computed. Mean differences between right and left ovaries were 0.24 (95% confidence interval [95% CI]: -0.32-0.80) follicles for AFC and 1.14 (95% CI: 0.34-1.92)ml for OV in the whole study population, 0.14 (95% CI: -0.68-0.96) follicles for AFC and 1.48 (95% CI: 0.39-2.58)ml for OV in PCOS patients, 0.42 (95% CI: -0.19-1.02) follicles for AFC and 0.53 (95% CI: -0.50-1.56)ml for OV in controls. Rather wide limits of agreement and low CCCs (<0.7 for all estimates) indicated poor agreement between the ovaries for both sonographic measurements. Width between lower and upper limits of agreement was higher for PCOS patients than for controls. 23.5% of the PCOS patients showed polycystic ovarian morphology (PCOM) only in one ovary, resulting in 9.4% potentially missed PCOS diagnosis according to the Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Substantial differences in antral follicle count and ovarian volume between the right and left ovary were observed. In approximately 10% of the PCOS patients in our study only the examination of both ovaries has led to a reliable diagnosis of PCOS. In clinical practice it is recommended to scan both ovaries for a reliable diagnosis of abnormal sonographic findings in PCOM and PCOS diagnosis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles.

PubMed

Shuhaibar, Leia C; Egbert, Jeremy R; Norris, Rachael P; Lampe, Paul D; Nikolaev, Viacheslav O; Thunemann, Martin; Wen, Lai; Feil, Robert; Jaffe, Laurinda A

2015-04-28

Meiosis in mammalian oocytes is paused until luteinizing hormone (LH) activates receptors in the mural granulosa cells of the ovarian follicle. Prior work has established the central role of cyclic GMP (cGMP) from the granulosa cells in maintaining meiotic arrest, but it is not clear how binding of LH to receptors that are located up to 10 cell layers away from the oocyte lowers oocyte cGMP and restarts meiosis. Here, by visualizing intercellular trafficking of cGMP in real-time in live follicles from mice expressing a FRET sensor, we show that diffusion of cGMP through gap junctions is responsible not only for maintaining meiotic arrest, but also for rapid transmission of the signal that reinitiates meiosis from the follicle surface to the oocyte. Before LH exposure, the cGMP concentration throughout the follicle is at a uniformly high level of ∼2-4 μM. Then, within 1 min of LH application, cGMP begins to decrease in the peripheral granulosa cells. As a consequence, cGMP from the oocyte diffuses into the sink provided by the large granulosa cell volume, such that by 20 min the cGMP concentration in the follicle is uniformly low, ∼100 nM. The decrease in cGMP in the oocyte relieves the inhibition of the meiotic cell cycle. This direct demonstration that a physiological signal initiated by a stimulus in one region of an intact tissue can travel across many layers of cells via cyclic nucleotide diffusion through gap junctions could provide a general mechanism for diverse cellular processes.

Follicle Online: an integrated database of follicle assembly, development and ovulation.

PubMed

Hua, Juan; Xu, Bo; Yang, Yifan; Ban, Rongjun; Iqbal, Furhan; Cooke, Howard J; Zhang, Yuanwei; Shi, Qinghua

2015-01-01

Folliculogenesis is an important part of ovarian function as it provides the oocytes for female reproductive life. Characterizing genes/proteins involved in folliculogenesis is fundamental for understanding the mechanisms associated with this biological function and to cure the diseases associated with folliculogenesis. A large number of genes/proteins associated with folliculogenesis have been identified from different species. However, no dedicated public resource is currently available for folliculogenesis-related genes/proteins that are validated by experiments. Here, we are reporting a database 'Follicle Online' that provides the experimentally validated gene/protein map of the folliculogenesis in a number of species. Follicle Online is a web-based database system for storing and retrieving folliculogenesis-related experimental data. It provides detailed information for 580 genes/proteins (from 23 model organisms, including Homo sapiens, Mus musculus, Rattus norvegicus, Mesocricetus auratus, Bos Taurus, Drosophila and Xenopus laevis) that have been reported to be involved in folliculogenesis, POF (premature ovarian failure) and PCOS (polycystic ovary syndrome). The literature was manually curated from more than 43,000 published articles (till 1 March 2014). The Follicle Online database is implemented in PHP + MySQL + JavaScript and this user-friendly web application provides access to the stored data. In summary, we have developed a centralized database that provides users with comprehensive information about genes/proteins involved in folliculogenesis. This database can be accessed freely and all the stored data can be viewed without any registration. Database URL: http://mcg.ustc.edu.cn/sdap1/follicle/index.php © The Author(s) 2015. Published by Oxford University Press.

Evaluation of preventive effect of shilajit on radiation-induced apoptosis on ovaries.

PubMed

Kececi, Mete; Akpolat, Meryem; Gulle, Kanat; Gencer, Ercan; Sahbaz, Ahmet

2016-06-01

Canc er is the second leading cause of death in children in developed countries and most of childhood malignancies can be treated with chemo-radiotherapy. Although radiation therapy is a successful treatment modality in cancer patients, it has various adverse effects. Especially the gonads are very sensitive and prone to radiation-related damage. Radiation impairs the ovaries by triggering apoptosis of follicular cells and chromosomal damage and oxidative stress. Shilajit, a traditional medicinal agent in India, Russia, and other parts of the world, contains various antioxidant agents and has ovogenic effects. To evaluate the ability of shilajit to prevent radiation-induced ovarian damage. Forty Wistar albino female rats were divided into four groups as: Control group, shilajit group, radiation only group, and radiation + shilajit group. Four days after radiation exposure, the rats were sacrificed and the ovaries were removed and evaluated immuno-histopathologically. There was a statistically significant difference in follicle counts (primordial, primary, preantral, antral, and atretic follicles) between the groups (p < 0.001). Almost all follicles at all stages were atretic in the radiation only group whereas normal-looking primordial follicles were detected in the radiation + shilajit group. In radiation + shilajit group, p53, Bax and caspase 3 expression was less intense than that in the radiation only group follicles. This is the first reported study evaluating the effects of shilajit on radiation-related ovarian damage prevention. Shilajit decreased the expression of p53, Bax, and caspase 3, thereby blocking the apoptotic pathways. Shilajit was found to be especially protective of primordial follicles.

Follicle Online: an integrated database of follicle assembly, development and ovulation

PubMed Central

Hua, Juan; Xu, Bo; Yang, Yifan; Ban, Rongjun; Iqbal, Furhan; Zhang, Yuanwei; Shi, Qinghua

2015-01-01

Folliculogenesis is an important part of ovarian function as it provides the oocytes for female reproductive life. Characterizing genes/proteins involved in folliculogenesis is fundamental for understanding the mechanisms associated with this biological function and to cure the diseases associated with folliculogenesis. A large number of genes/proteins associated with folliculogenesis have been identified from different species. However, no dedicated public resource is currently available for folliculogenesis-related genes/proteins that are validated by experiments. Here, we are reporting a database ‘Follicle Online’ that provides the experimentally validated gene/protein map of the folliculogenesis in a number of species. Follicle Online is a web-based database system for storing and retrieving folliculogenesis-related experimental data. It provides detailed information for 580 genes/proteins (from 23 model organisms, including Homo sapiens, Mus musculus, Rattus norvegicus, Mesocricetus auratus, Bos Taurus, Drosophila and Xenopus laevis) that have been reported to be involved in folliculogenesis, POF (premature ovarian failure) and PCOS (polycystic ovary syndrome). The literature was manually curated from more than 43 000 published articles (till 1 March 2014). The Follicle Online database is implemented in PHP + MySQL + JavaScript and this user-friendly web application provides access to the stored data. In summary, we have developed a centralized database that provides users with comprehensive information about genes/proteins involved in folliculogenesis. This database can be accessed freely and all the stored data can be viewed without any registration. Database URL: http://mcg.ustc.edu.cn/sdap1/follicle/index.php PMID:25931457

Genome-scale gene expression characteristics define the follicular initiation and developmental rules during folliculogenesis.

PubMed

Shi, Kerong; He, Feng; Yuan, Xuefeng; Zhao, Yaofeng; Deng, Xuemei; Hu, Xiaoxiang; Li, Ning

2013-08-01

The ovarian follicle supplies a unique dynamic system for gametes that ensures the propagation of the species. During folliculogenesis, the vast majority of the germ cells are lost or inactivated because of ovarian follicle atresia, resulting in diminished reproductive potency and potential infertility. Understanding the underlying molecular mechanism of folliculogenesis rules is essential. Primordial (P), preantral (M), and large antral (L) porcine follicles were used to reveal their genome-wide gene expression profiles. Results indicate that primordial follicles (P) process a diverse gene expression pattern compared to growing follicles (M and L). The 5,548 differentially expressed genes display a similar expression mode in M and L, with a correlation coefficient of 0.892. The number of regulated (both up and down) genes in M is more than that in L. Also, their regulation folds in M (2-364-fold) are much more acute than in L (2-75-fold). Differentially expressed gene groups with different regulation patterns in certain follicular stages are identified and presumed to be closely related following follicular developmental rules. Interestingly, functional annotation analysis revealed that these gene groups feature distinct biological processes or molecular functions. Moreover, representative candidate genes from these gene groups have had their RNA or protein expressions within follicles confirmed. Our study emphasized genome-scale gene expression characteristics, which provide novel entry points for understanding the folliculogenesis rules on the molecular level, such as follicular initiation, atresia, and dominance. Transcriptional regulatory circuitries in certain follicular stages are expected to be found among the identified differentially expressed gene groups.

Effect of stem cell transplantation of premature ovarian failure in animal models and patients: A meta-analysis and case report.

PubMed

Chen, Lei; Guo, Shilei; Wei, Cui; Li, Honglan; Wang, Haiya; Xu, Yan

2018-05-01

Stem cell transplantation has been considered a promising therapeutic approach for premature ovarian failure (POF). However, to date, no quantitative data analysis of stem cell therapy for POF has been performed. Therefore, the present study performed a meta-analysis to assess the efficacy of stem cell transplantation in improving ovarian function in animal models of POF. In addition, a case report of a patient with POF subjected to stem cell treatment was included to demonstrate that stem cell therapy also contributes to the recovery of ovarian function in patients. Published studies were identified by a systematic review of the PubMed, Embase, and Cochrane's library databases, and references cited in associated reviews were also considered. Data regarding follicle-stimulating hormone (FSH), estradiol (E2), ovarian weight, follicle count, the number of pregnancies and other parameters, including delivery route and cell type, were extracted. Pooled analysis, sensitivity analyses, subgroup analyses and meta-regression were performed. In the case of POF, transvaginal ultrasound (TVS), abdominal ultrasound (TAS) and color Doppler flow imaging (CDFI) were performed to observe the endometrial morphology and blood flow signals in the patient. Overall, pooled results from 16 pre-clinical studies demonstrated that stem cell-based therapy significantly improved FSH levels [standardized mean difference (SMD)=-1.330; 95% confidence interval (CI), -(2.095-0.565); P=0.001], E2 levels (SMD=2.334; 95% CI, 1.350-3.319; P<0.001), ovarian weight (SMD=1.310; 95% CI, 0.157-2.463; P=0.026), follicle count (SMD=1.871; 95% CI, 1.226-2.516; P<0.001), and the number of pregnancies (risk ratio=1.715, 95% CI, 1.213-2.424; P=0.002). The results of TVS and TAS demonstrated improved ovarian size and endometrial thickness in the patient with POF after MSC treatment. Of note, a rich blood flow signal in the endometrium was observed on CDFI. It appeared that stem cell-based therapy may be an effective method for the resumption of ovarian function in a patient and in animal models of POF; however, large-scale and high-quality future studies are required to confirm the present findings due to heterogeneity.

Maternal age and ovarian stimulation independently affect oocyte mtDNA copy number and cumulus cell gene expression in bovine clones.

PubMed

Cree, Lynsey M; Hammond, Elizabeth R; Shelling, Andrew N; Berg, Martin C; Peek, John C; Green, Mark P

2015-06-01

Does maternal ageing and ovarian stimulation alter mitochondrial DNA (mtDNA) copy number and gene expression of oocytes and cumulus cells from a novel bovine model for human IVF? Oocytes collected from females with identical nuclear genetics show decreased mtDNA copy number and increased expression of an endoplasmic reticulum (ER) stress gene with repect to ovarian stimulation, whilst differences in the expression of genes involved in mitochondrial function, antioxidant protection and apoptosis were evident in relation to maternal ageing and the degree of ovarian stimulation in cumulus cells. Oocyte quality declines with advancing maternal age; however, the underlying mechanism, as well as the effects of ovarian stimulation are poorly understood. Human studies investigating these effects are often limited by differences in age and ovarian stimulation regimens within a patient cohort, as well as genetic and environmental variability. A novel bovine cross-sectional maternal age model for human IVF was undertaken. Follicles were aspirated from young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian clones following multiple unstimulated, mild and standard ovarian stimulation cycles. These bovine cloned females were generated by the process of somatic cell nuclear transfer (SCNT) from the same founder and represent a homogeneous population with reduced genetic and environmental variability. Maternal age and ovarian stimulation effects were investigated in relation to mtDNA copy number, and the expression of 19 genes involved in mitochondrial function, antioxidant protection, oocyte-cumulus cell signalling and follicle development in both oocytes and cumulus cells. Young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian bovine clones were maintained as one herd. Stimulation cycles were based on the long GnRH agonist down-regulation regimen used in human fertility clinics. Follicle growth rates, numbers and diameters were monitored by ultrasonography and aspirated when the lead follicles were >14 mm in diameter. Follicle characteristics were analysed using a mixed model procedure. Quantitative PCR (qPCR) was used to determine mtDNA copy number and reverse transcriptase-qPCR (RT-qPCR) was used to measure gene expression in oocytes and cumulus cells. Method of ovarian stimulation (P = 0.04), but not maternal age (P > 0.1), was associated with a lower mtDNA copy number in oocytes. Neither factor affected mtDNA copy number in cumulus cells. In oocytes, maternal age had no effect on gene expression; however, ovarian stimulation in older females increased the expression of GRP78 (P = 0.02), a gene involved in ER stress. In cumulus cells, increasing maternal age was associated with the higher expression of genes involved in mitochondrial maintenance (TXN2 P = 0.008 and TFAM P = 0.03), whereas ovarian stimulation decreased the expression of genes involved in mitochondrial oxidative stress and apoptosis (TXN2 P = 0.002, PRDX3 P = 0.03 and BAX P = 0.03). The low number of oocyte and cumulus cell samples collected from the unstimulated cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed because these were used for mtDNA and gene expression quantification. Delineation of the independent effects of maternal age and ovarian stimulation regimen on mtDNA copy number gene expression in oocytes and cumulus cells was enabled by the removal of genetic and environmental variability in this bovine model for human IVF. Therefore, these extend upon previous knowledge and findings provide relevant insights that are applicable for improving human ovarian stimulation regimens. Funding was provided by Fertility Associates and the University of Auckland. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that could be perceived as prejudicing the impartiality of the reported research. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Androstenedione response to recombinant human FSH is the most valid predictor of the number of selected follicles in polycystic ovarian syndrome: (a case-control study).

PubMed

Ozyurek, Eser Sefik; Yoldemir, Tevfik; Artar, Gokhan

2017-05-12

We aimed to test the hypothesis that the correlation of the changes in the blood Androstenedione (A 4 ) levels to the number of selected follicles during ovulation induction with low-dose recombinant human follicle stimulating hormone (rhFSH) is as strong as the correlation to changes in the blood Estradiol (E 2 ) levels in polycystic ovary syndrome (PCOS). Prospective Case-control study conducted from October 2014 to January 2016. 61 non-PCOS control (Group I) and 46 PCOS (Group II) patients treated with the chronic low-dose step up protocosl with rhFSH. A 4 , E 2 , progesterone blood levels and follicular growth were monitored.. Univariate and hierarchical multivariable analysis were performed for age, BMI, HOMA-IR, A 4 and E 2 (with the number of selected follicles as the dependent variable in both groups). ROC analysis was performed to define threshold values for the significant determinants of the number of selected follicles to predict cyle cancellations due to excessive ovarian response. The control group (Group I) was comprised of 61 cycles from a group of primary infertile non-PCOS patients, and the study group (Group II) of 46 cycles of PCOS patients. The analysis revealed that the strongest independent predictor of the total number of selected follicles in Group I was the E 2 (AUC) (B = 0.0006[0.0003-0.001]; P < 0.001); whereas for Group II, it was the A 4 (AUC) (B = 0.114[0.04-0.25]; P = 0.01). Optimum thresholds for the A 4 related parameters were defined to predict excessive response within Group II were 88.7%, 3.1 ng/mL and 5.4 ng*days for the percentage increase in A 4 , the maximum A 4 value and area under the curve values for A 4 , respectively. A 4 response to low-dose rhFSH in PCOS has a stronger association with the number of follicles selected than the E 2 reponse. A 4 response preceding the E 2 response is essential for progressive follicle development. Monitoring A 4 rather than E 2 may be more preemptive to define the initial ovarian response and accurate titration of the rhFSH doses. The study was registered as a prospective case-control study in the ClinicalTrials.gov registry with the identifier NCT02329483 .

Characterization of ovarian follicular dynamics in dromedary camels (Camelus dromedarius).

PubMed

Manjunatha, B M; Pratap, N; Al-Bulushi, Samir; Hago, B E

2012-09-15

Ovarian follicular dynamics was monitored by transrectal ultrasonography, for a period of 60 to 90 days, and its correlation with plasma estradiol-17β (E2) and progesterone (P4) were studied in seventeen, multiparous, non-lactating, 12 to 20-year-old dromedary camels. The average number of follicles recruited (12.77 ± 0.93) in each wave between animals varied (P < 0.001). The number of follicles recruited during different follicular waves was highly repeatable (0.95) within individual animals. The growth and mature phase periods of the dominant follicle (DF) were 6.10 ± 0.15 and 10.20 ± 0.47 days, respectively with a linear growth rate of 1.17 ± 0.02 mm/day between Day 0 and 10 of the follicular wave. There was an inverse relationship between the diameter of the largest DF and number of follicles (r = -0.95, P < 0.001). The DF development did not regularly alternate between the ovaries and the incidence of codominance was 45%. The mean maximum diameter of DF during its mature phase was 27.30 ± 0.78 mm and oversized follicle was 38.43 ± 1.41 mm. In 73.3% waves, the DF continued its growth for a period of 10.64 ± 1.53 days even after losing its dominance and developed into oversized follicle. The duration of the regression phase of DF and oversized follicle were 24.71 ± 3.79 and 18.50 ± 2.23 days. The mean duration of a complete follicular wave was 47.11 ± 2.94 days with an interwave interval (IWI) of 16.36 ± 0.37 days. The IWI within an individual was repeatable (0.88) and between the animals was variable (P < 0.001). Plasma E2 concentration profiles showed a wave like pattern. The peak plasma E2 concentrations were attained approximately 12 days after beginning of the growth phase, when the largest DF grew to a diameter of 18.7 mm. Plasma concentration of P4 was below 1.0 ng/mL in 85% of waves and above 1.0 ng/mL in 15% of the waves for a period of 3 to 6 days in the absence of spontaneous ovulation. It is concluded that ovarian follicular development and plasma E2 concentrations occurs in a wave like pattern in dromedary camels and the IWI and follicle numbers recruited per wave are variable between the animals and repeatable within an individual animal. Copyright © 2012 Elsevier Inc. All rights reserved.

Changes in ovarian reserve and ovarian blood flow in patients with polycystic ovary syndrome following laparoscopic ovarian drilling.

PubMed

Kamal, Nasser; Sanad, Zakaria; Elkelani, Osama; Rezk, Mohamed; Shawky, Mohamed; Sharaf, Abd-Elbar

2018-04-10

This prospective cohort study was conducted on 80 patients with clomiphene citrate (CC)-resistant polycystic ovary syndrome undergoing laparoscopic ovarian drilling (LOD). Pre- and post-LOD ovarian reserve parameters (anti-Mullerian hormone: AMH, ovarian volume: OV, and antral follicle count: AFC) and ovarian stromal blood flow indices (Vascularization index: VI, flow index: FI, and vascularization flow index: VFI) were measured to explore the effect of LOD and to find out the correlation between serum AMH and different clinical, hormonal, and ultrasonic variables. There was a highly significant reduction of the serum AMH (p < .001) after LOD with significant reduction in OV, AFC and vascular indices (VI, FI and VFI) of the right and left ovaries (p < .05). LOD significantly reduced ovarian reserve parameters (AMH, OV and AFC) and ovarian stromal blood flow indices (VI, FI and VFI) with no observed correlation between AMH levels and Doppler indices.

Effect of detorsion alone and in combination with enoxaparin therapy on ovarian reserve and serum antimüllerian hormone levels in a rat ovarian torsion model.

PubMed

Kaya, Cihan; Turgut, Hürriyet; Cengiz, Hüseyin; Turan, Ayşenur; Ekin, Murat; Yaşar, Levent

2014-09-01

To investigate the effect of enoxaparin on ovarian reserve and serum antimüllerian hormone (AMH) levels in a rat ovarian torsion model. Experimental study. Experimental surgery laboratory in a training and research hospital. Fourteen female Wistar Hannover rats. 1) Control group received no special treatment other than abdominal exposure; 2) detorsion-only group received bilateral adnexal torsion (3-hour ischemia), and then after 3-hour torsion period, detorsion (reperfusion) was performed; and 3) detorsion-enoxaparin group received 0.5 mg/kg enoxaparin subcutaneously 2 hours before the same surgery as the detorsion-only group and a second 0.5 mg/kg dose of enoxaparin 24 hours after the first surgeries. Apart from the surgeries, preoperative and postoperative 1-mL blood samples were drawn from the right jugular vein of each rat. Preoperative and postoperative serum AMH levels, histopathologic damage scores, and follicle counts in the ovarian tissue of the rats. Vascular congestion and hemorrhage scores were higher in the detorsion-enoxaparin group than in the detorsion-only and control groups. The number of small antral follicles was smaller in the detorsion-only group than in the control group. The difference in the pre- and postoperative AMH levels was higher in the detorsion-only group than in the control and detorsion-enoxaparin groups. The combination of enoxaparin therapy with conventional ovarian detorsion is more effective in protecting the ovarian reserve than detorsion alone. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Orthotopic Transplantation of Cryopreserved Mouse Ovaries and Gonadotrophin Releasing Hormone Analogues in the Restoration of Function following Chemotherapy-Induced Ovarian Damage

PubMed Central

Li, Qing; Szatmary, Peter; Liu, Yanyang; Ding, Zhenyu; Zhou, Jin; Sun, Yi; Luo, Feng

2015-01-01

Therapy advances are constantly improving survival rates of cancer patients, however the toxic effects of chemotherapy drugs can seriously affect patients’ quality of life. In women, fertility and premature ovarian endocrine dysfunction are of particular concern. It is urgently we find methods to preserve or reconstruct ovarian function for these women. This study compares GnRHa treatment with ovarian tissue cryopreservation and orthotopic transplantation in a chemotherapy-induced ovarian damage murine model. 56 inbred Lewis rats were divided into 4 treatment groups: Saline control (group I); cyclophosphamide only (group II); cyclophosphamide plus GnRHa (group III); cyclophosphamide and grafting of thawed cryopreserved ovaries (group IV). Body weight, estrous cycle recovery time, ovarian weight, morphology and follicle count, as well as breeding and fertility were compared among groups. Only group IV was able to restore to normal body weight by the end of the observation period and resumed normal estrous cycles in a shorter time compared to other treatment groups. There was a decrease in primordial follicles in all treatment groups, but group III had the greatest reduction. Although, there was no difference in pregnancy, only one animal littered normal pups in group II, none littered in group III and four littered in group IV. Thus, cryopreservation and orthotopic transplantation of ovarian tissue can restore the fertility of rats subjected to chemotherapy in a manner that is superior to GnRHa treatment. We also observed increased rates of hepatic, splenic and pulmonary haemorrhage in group III, suggesting there may be synergistic toxicity of GnRHa and cyclophosphamide. PMID:25811681

Antimüllerian hormone levels and antral follicle counts are not reduced compared with community controls in patients with rigorously defined unexplained infertility.

PubMed

Greenwood, Eleni A; Cedars, Marcelle I; Santoro, Nanette; Eisenberg, Esther; Kao, Chia-Ning; Haisenleder, Daniel J; Diamond, Michael P; Huddleston, Heather G

2017-12-01

To test the hypothesis that women with unexplained infertility demonstrate evidence of diminished ovarian reserve when compared with a population of community controls. Cross-sectional study. Multicenter university-based clinical practices. Study participants included 277 healthy, normo-ovulatory female partners with rigorously defined unexplained infertility randomly selected from a multicenter trial (Assessment of Multiple Intrauterine Gestations from Ovarian Stimulation). Controls included 226 healthy, normo-ovulatory women not seeking treatment for fertility from a community-based cohort (Ovarian Aging study). Serum antimüllerian hormone (AMH) assay at a central laboratory, FSH, fasting serum metabolic testing, transvaginal ultrasonography for antral follicle counts (AFCs), anthropometric measurements. Average AMH, AFC, and AMH/AFC were compared between infertile and control women by age. Analyses of covariance compared these outcomes while controlling for confounders, including age, race, body mass index, smoking history, and study site. In our models, AMH, AFC, and AMH/AFC ovarian reserve indices did not differ between infertile women and community-based controls, after controlling for age, race, body mass index, smoking history, and study site. Currently utilized predictors of ovarian reserve do not discriminate women with rigorously defined unexplained infertility from healthy community-based women of similar demographic characteristics. Contrary to our hypothesis, among women with FSH in the normal range (≤12 IU/L), women with unexplained infertility did not show evidence of decreased ovarian reserve as measured by AMH and AFC. Ovarian reserve markers in isolation may not serve as predictors of future fertility. Copyright © 2017 American Society for Reproductive Medicine. All rights reserved.

Evaluation of bovine (Bos indicus) ovarian potential for in vitro embryo production in the Adamawa plateau (Cameroon)

PubMed Central

Kouamo, J.; Dawaye, S.M.; Zoli, A.P.; Bah, G.S.

2014-01-01

An abattoir study was conducted to evaluate the ovarian potential of 201 local zebu cattle from Ngaoundere, Adamawa region (Cameroon) for in vitro embryo production (IVEP). The ovaries were excised, submerged in normal saline solution (0.9%) and transported to the laboratory for a detailed evaluation. Follicles on each ovary were counted, their diameters (Φ) measured and were grouped into 3 categories: small (Φ < 3 mm), medium (3 ≥ Φ ≤ 8 mm) and large (Φ > 8 mm). Each ovary was then sliced into a petri dish; the oocytes were recovered in Dulbecco’s phosphate buffered saline, examined under a stereoscope (x10) and graded into four groups based on the morphology of cumulus oophorus cells and cytoplasmic changes of the oocytes. Grade I (GI): oocytes with more than 4 layers of bunch of compact cumulus cells mass with evenly granulated cytoplasm; grade II (GII): oocyte with at least 2-4 layers of compact cumulus cell mass with evenly granulated cytoplasm; grade III (GIII): oocyte with at least one layer of compact cumulus cell mass with evenly granulated cytoplasm; grade IV (GIV): denuded oocyte with no cumulus cells or incomplete layer of cumulus cell or expanded cells and having dark or unevenly granulated cytoplasm. The effects of both ovarian (ovarian localization, corpus luteum, size and weight of ovary) and non-ovarian factors (breed, age, body condition score (BCS) and pregnancy status of cow) on the follicular population and oocyte recovery rate were determined. There were an average of 16.75±0.83 follicles per ovary. The small, medium and large follicles were 8.39±0.60, 8.14±0.43 and 0.21±0.02 respectively. Oocyte recovery was 10.97±0.43 per ovary (65%). Oocytes graded I, II, III and IV were 3.53±0.19 (32.21%), 2.72±0.15 (24.82%), 2.24±0.15 (20.43%) and 2.47±0.20 (22.54%) respectively. The oocyte quality index was 2.26. Younger non pregnant cows having BCS of 3 and large ovaries presented higher number of follicles and oocyte quality (P < 0.05) compared with other animals. Oocytes with quality (grade I and II) acceptable for IVEP constituted 57.15% of the harvest. This study indicated that factors such as age, pregnancy status, BCS and ovarian size must be taken into account to increase the potential of the ovary for IVEP. PMID:26623353

Effect of Antiprogesterone RU486 on VEGF Expression and Blood Vessel Remodeling on Ovarian Follicles before Ovulation

PubMed Central

Berardinelli, Paolo; Russo, Valentina; Bernabò, Nicola; Di Giacinto, Oriana; Mattioli, Mauro; Barboni, Barbara

2014-01-01

Background The success of ovarian follicle growth and ovulation is strictly related to the development of an adequate blood vessel network required to sustain the proliferative and endocrine functions of the follicular cells. Even if the Vascular Endothelial Growth Factor (VEGF) drives angiogenesis before ovulation, the local role exerted by Progesterone (P4) remains to be clarified, in particular when its concentration rapidly increases before ovulation. Aim This in vivo study was designed to clarify the effect promoted by a P4 receptor antagonist, RU486, on VEGF expression and follicular angiogenesis before ovulation, in particular, during the transition from pre to periovulatory follicles induced by human Chorionic Gonadotropins (hCG) administration. Material and Methods Preovulatory follicle growth and ovulation were pharmacologically induced in prepubertal gilts by combining equine Chorionic Gonadotropins (eCG) and hCG used in the presence or absence of RU486. The effects on VEGF expression were analyzed using biochemical and immunohistochemical studies, either on granulosa or on theca layers of follicles isolated few hours before ovulation. This angiogenic factor was also correlated to follicular morphology and to blood vessels architecture. Results and Conclusions VEGF production, blood vessel network and follicle remodeling were impaired by RU486 treatment, even if the cause-effect correlation remains to be clarified. The P4 antagonist strongly down-regulated theca VEGF expression, thus, preventing most of the angiogenic follicle response induced by hCG. RU486-treated follicles displayed a reduced vascular area, a lower rate of endothelial cell proliferation and a reduced recruitment of perivascular mural cells. These data provide important insights on the biological role of RU486 and, indirectly, on steroid hormones during periovulatory follicular phase. In addition, an in vivo model is proposed to evaluate how periovulatory follicular angiogenesis may affect the functionality of the corpus luteum (CL) and the success of pregnancy. PMID:24756033

Rapamycin Prevents cyclophosphamide-induced Over-activation of Primordial Follicle pool through PI3K/Akt/mTOR Signaling Pathway in vivo.

PubMed

Zhou, Linyan; Xie, Yanqiu; Li, Song; Liang, Yihua; Qiu, Qi; Lin, Haiyan; Zhang, Qingxue

2017-08-16

Primordial follicular depletion has thought to be a common adverse effect of chemotherapy especially for female of reproductive age. The study aimed to evaluate the protective effect of rapamycin on the primordial follicles and its potential mechanism for patients receiving chemotherapy. 8-week old BALB/c female mice were randomly assigned into four groups (control; rapamycin; cyclophosphamide; and rapamycin combined with cyclophosphamide). Hematoxylin staining, immunohistochemical, TUNEL, western blotting and ELISA were employed to assess inter-group differences using Student's t-test and Mann-Whitney test. Cyclophosphamide depleted the follicular reserve and induced the phosphorylation of the key proteins of PI3K/Akt/mTOR pathway in mice in a dose-dependent manner. Co-treatment with rapamycin significantly reduced primordial follicle loss at all cyclophosphamide dose groups and prevent the follicle growth wave caused by cyclophosphamide treatment (P < 0.05). TUNEL staining showed that no apoptosis occured in the primordial follicles in all groups and fewer apoptosis in large growing follicles were observed in ovaries from rapamycin + cyclophosphamide group compared to that received cyclophosphamide alone. Serum anti-Müllerian hormone (AMH) was significantly reduced in cyclophosphamide alone group, in contrast to the normal level in rapamycin + cyclophosphamide group. Compared to p-Akt/Akt and p-mtor/mtor, p-rps6/rps6 was significantly decreased in rapamycin + cyclophosphamide group (P < 0.05), indicating that rapamycin attenuated the increased level of phosphorylation of rpS6 after cyclophosphamide treatment. Rapamycin can prevent the primordial follicle activation induced by cyclophosphamide through PI3K/Akt/mTOR signaling pathway and thus plays a role in preserving the follicle pool. These results suggest that rapamycin may be an effective protection for ovarian function during chemotherapy, which means a new nonsurgical application for protection of ovarian reserve and prevention of POF.

A delay differential equation model of follicle waves in women.

PubMed

Panza, Nicole M; Wright, Andrew A; Selgrade, James F

2016-01-01

This article presents a mathematical model for hormonal regulation of the menstrual cycle which predicts the occurrence of follicle waves in normally cycling women. Several follicles of ovulatory size that develop sequentially during one menstrual cycle are referred to as follicle waves. The model consists of 13 nonlinear, delay differential equations with 51 parameters. Model simulations exhibit a unique stable periodic cycle and this menstrual cycle accurately approximates blood levels of ovarian and pituitary hormones found in the biological literature. Numerical experiments illustrate that the number of follicle waves corresponds to the number of rises in pituitary follicle stimulating hormone. Modifications of the model equations result in simulations which predict the possibility of two ovulations at different times during the same menstrual cycle and, hence, the occurrence of dizygotic twins via a phenomenon referred to as superfecundation. Sensitive parameters are identified and bifurcations in model behaviour with respect to parameter changes are discussed. Studying follicle waves may be helpful for improving female fertility and for understanding some aspects of female reproductive ageing.

Ovarian reserve in women of late reproductive age by the method of treatment of PCOS.

PubMed

Beltadze, Ketevan; Barbakadze, Ludmila

2015-05-01

The prevalence of polycystic ovarian syndrome (PCOS) particularly is increased in adolescents. Very few longitudinal follow-up for assessment of ovarian reserve in women of late reproductive age with previously confirmed PCOS have been conducted, especially after its diagnosis and treatment in adolescence. The aim of the present study was to compare of the ovarian reserve of the women of late reproductive age by the method of treatment of PCOS in adolescence. This cross sectional study in an unselected population was conducted from January to June 2014. A total of 123 women of late reproductive age were included. They had been diagnosed with PCOS between 1984 and 1990 when they were 13-18 yr. From these, first group of the study was consisted of 67 participants who underwent conservative treatment with antiandrogens and combined oral contraceptives and second group of the study was consisted of 56 participants after surgery (34-bilateral ovarian drilling and 22- ovarian wedge resection). At the time of investigation patients were 35-45 yr. The participants were collected via analysis of histories at primary diagnosis of PCOS in adolescence and at the time of the investigation analyses of reproductive hormones were conducted. Data were compared between the groups. After conservative treatment PCOS women had higher levels of anti- mullerian hormone and lower follicle-stimulating hormone levels (p=0.02 and p=0.04, respectively). The number of antral follicles and mean ovarian volume were significantly greater also, than in women who underwent surgical treatment (p=0.03 and p=0.04, respectively). Our data suggest that PCOS patients who underwent conservative treatment have the better ovarian reserve than women who underwent surgical treatment of PCOS in adolescence.

Lysyl oxidase interacts with AGE signalling to modulate collagen synthesis in polycystic ovarian tissue

PubMed Central

Papachroni, Katerina K; Piperi, Christina; Levidou, Georgia; Korkolopoulou, Penelope; Pawelczyk, Leszek; Diamanti-Kandarakis, Evanthia; Papavassiliou, Athanasios G

2010-01-01

Abstract Connective tissue components – collagen types I, III and IV – surrounding the ovarian follicles undergo drastic changes during ovulation. Abnormal collagen synthesis and increased volume and density of ovarian stroma characterize the polycystic ovary syndrome (PCOS). During the ovulatory process, collagen synthesis is regulated by prolyl hydroxylase and lysyl oxidase (LOX) activity in ovarian follicles. LOX catalyzes collagen and elastin cross-linking and plays essential role in coordinating the control of ovarian extracellular matrix (ECM) during follicular development. We have recently shown accumulation of advanced glycation end products (AGEs), molecules that stimulate ECM production and abnormal collagen cross-linking, in ovarian tissue. However, the possible link between LOX and AGEs-induced signalling in collagen production and stroma formation in ovarian tissue from PCOS remains elusive. The present study investigates the hypothesis of AGE signalling pathway interaction with LOX gene activity in polycystic ovarian (PCO) tissue. We show an increased distribution and co-localization of LOX, collagen type IV and AGE molecules in the PCO tissue compared to control, as well as augmented expression of AGE signalling mediators/effectors, phospho(p)-ERK, phospho(p)-c-Jun and nuclear factor κB (NF-κB) in pathological tissue. Moreover, we demonstrate binding of AGE-induced transcription factors, NF-κB and activator protein-1 (AP-1) on LOX promoter, indicating a possible involvement of AGEs in LOX gene regulation, which may account for the documented increase in LOX mRNA and protein levels compared to control. These findings suggest that deposition of excess collagen in PCO tissue that induces cystogenesis may, in part, be due to AGE-mediated stimulation of LOX activity. PMID:19583806

Occurrence of ovarian follicular dominance during stimulation for IVM impacts usable blastocyst yield

PubMed Central

Romero, Sergio; Pella, Ricardo; Escudero, Francisco; Pérez, Ygor; García, Mario; Orihuela, Patricia

2018-01-01

Objective To evaluate the influence of ovarian follicular dominance on the outcome of oocyte in-vitro maturation. Methods This retrospective cohort study included 21 patients with polycystic ovaries or polycystic ovary syndrome (Rotterdam criteria, 2004) subjected to 24 in-vitro maturation (IVM) cycles between October 2015 and January 2017. Patients undergoing IVM received minimal gonadotropin stimulation starting on day 2 or 3 of the cycle; ovum pick-up typically occurred on days 6 to 8. No hCG-trigger shot was given. Following 30h of IVM, mature oocytes were inseminated by ICSI and the resulting embryos cultured up to the blastocyst stage. Results Ovarian follicular dominance was observed in nine of the 24 IVM cycles. Oocyte IVM yielded an overall maturation rate of 69.3±23.8%, and no difference was observed when the groups with or without a dominant follicle were assessed independently. The rates of fertilization and usable blastocysts per fertilized oocyte, mature oocyte (Metaphase II) or cumulus-oocyte-complex were nearly three times higher (28.7±22.5%) in the group without ovarian follicular dominance. No differences were found in the clinical pregnancy rates attained by the individuals with or without a dominant follicle after 21 vitrified-warmed blastocyst transfer cycles. Conclusion Occurrence of ovarian follicular dominance during hormonal stimulation for in-vitro maturation negatively impacted embryological outcomes. Strategies devised to limit the appearance of ovarian follicular dominance must be further explored. PMID:29338139

Follicular Fluid redox involvement for ovarian follicle growth.

PubMed

Freitas, Cláudia; Neto, Ana Catarina; Matos, Liliana; Silva, Elisabete; Ribeiro, Ângela; Silva-Carvalho, João Luís; Almeida, Henrique

2017-07-12

As the human ovarian follicle enlarges in the course of a regular cycle or following controlled ovarian stimulation, the changes in its structure reveal the oocyte environment composed of cumulus oophorus cells and the follicular fluid (FF).In contrast to the dynamic nature of cells, the fluid compartment appears as a reservoir rich in biomolecules. In some aspects, it is similar to the plasma, but it also exhibits differences that likely relate to its specific localization around the oocyte. The chemical composition indicates that the follicular fluid is able to detect and buffer excessive amounts of reactive oxygen species, employing a variety of antioxidants, some of them components of the intracellular milieu.An important part is played by albumin through specific cysteine residues. But the fluid contains other molecules whose cysteine residues may be involved in sensing and buffering the local oxidative conditions. How these molecules are recruited and regulated to intervene such process is unknown but it is a critical issue in reproduction.In fact, important proteins in the FF, that regulate follicle growth and oocyte quality, exhibit cysteine residues at specific points, whose untoward oxidation would result in functional loss. Therefore, preservation of controlled oxidative conditions in the FF is a requirement for the fine-tuned oocyte maturation process. In contrast, its disturbance enhances the susceptibility to the establishment of reproductive disorders that would require the intervention of reproductive medicine technology.

Alterations in gonadotropin secretion and ovarian function in prepubertal gilts by elevated environmental temperature.

PubMed

Flowers, B; Day, B N

1990-03-01

The effect of chronic exposure to elevated environmental temperature on gonadotropin secretion and ovarian function was studied in prepubertal gilts. Gilts were maintained under control (15.6 degrees C) or elevated temperature (33.3 degrees C) conditions from 150 to 180 days of age. Endocrine and ovarian responses to bilateral (BLO), unilateral (ULO), and sham ovariectomy were evaluated between 175 and 180 days of age. During the 96-h sampling period after BLO, plasma concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were suppressed in heat-stressed females. Similarly, elevated temperatures abolished the transient rise in FSH and subsequent follicular growth normally associated with ULO. In contrast, environmental treatment had no effect on the secretion of FSH and LH after sham ovariectomy, yet the number of small follicles was lower in gilts exposed to elevated temperatures than in females maintained under control conditions. These results indicate that a chronic exposure to elevated environmental temperature during pubertal development diminished the ability of the hypothalamo-hypophyseal axis to secrete FSH and LH, which had physiological consequences on follicular growth. When provided an appropriate stimulus (ULO), an acute period of FSH secretion and subsequent development of follicles failed to occur in females exposed to elevated temperatures. Consequently, we propose that delayed puberty in gilts during periods of elevated environmental temperatures is due, in part, to a diminished capacity for gonadotropin secretion.

Anti-Müllerian hormone levels and antral follicle count in women enrolled in in vitro fertilization cycles: relationship to lifestyle factors, chronological age and reproductive history.

PubMed

Nardo, Luciano G; Christodoulou, Dimitra; Gould, Della; Roberts, Steve A; Fitzgerald, Cheryl T; Laing, Ian

2007-01-01

The aims of this prospective study were to investigate the relationship between anti-Müllerian hormone (AMH) and antral follicle count (AFC), and to determine whether these markers of ovarian reserve correlate with lifestyle factors, ethnicity, chronological age and reproductive history. Participants were 136 normo-ovulatory women undergoing infertility work-up within 3 months of their first ovarian stimulation cycle for in vitro fertilization. On day 3 of a spontaneous menstrual cycle, a blood sample for measurement of plasma AMH levels was taken and a transvaginal ultrasound scan to determine the AFC (follicles measuring 2-5 mm in diameter) was performed. Information about smoking, body mass index, alcohol consumption, ethnic origin, chronological age, age at menarche, years since menarche and gravidity were recorded using a case report form. The main outcome measures were plasma AMH concentrations and total number of small antral follicles (AFC). Median plasma levels of AMH were 2.0 ng/ml (interquartile range 1.1-3.6) and AFC was 10 (interquartile range 7-15). A positive correlation between AMH and AFC (r = 0.54, p < 0.0001) was found. AMH and AFC correlated negatively with age (r = -0.30, p < 0.001 and r = -0.27, p = 0.001 respectively) and number of years since menarche (r = -0.23, p = 0.007 and r = -0.21, p = 0.015 respectively), but not with any of the other measures. Circulating AMH levels and AFC correlated with each other and declined significantly with age. There were only weak, non-significant, correlations with lifestyle factors and reproductive history. These putative markers could be used individually or together to assess the age-related decline of ovarian function in normo-ovulatory candidates for IVF.

Frozen and fresh ovarian tissue require different culture media to promote in vitro development of bovine preantral follicles.

PubMed

Castro, Simone Vieira; Carvalho, Adeline Andrade; Silva, Cleidson Manoel Gomes; Santos, Francielli Weber; Campello, Cláudio Cabral; de Figueiredo, José Ricardo; Rodrigues, Ana Paula Ribeiro

2014-10-01

The aim of this study was to evaluate the efficiency of different media in the in vitro culture of bovine preantral follicles that were used either fresh or following slow freezing treatment. Frozen and fresh noncultured or cultured ovarian fragments were processed for histological, viability, and cell proliferation analyses. For cryopreservation, a solution containing 1.5 M ethylene glycol was frozen in a programmable biological freezer. After thawing, a portion of the samples was destined for frozen controls. The remainder were cultured in vitro for 5 days in three media: α-MEM, McCoy, or M199. Samples from these culture media were collected on days 1 and 5 for quantification of reactive oxygen species (ROS) and for hormonal assays. In fresh-cultured tissues, the percentage of morphologically normal follicles was significantly higher when cultured in M199 compared to that in the other media. In frozen-cultured tissues, McCoy medium was significantly superior to the other media, and was the only treatment that helped in maintaining the viability similar to fresh and frozen controls. Upon quantification of the nucleolus organizer region, we observed greater proliferation of granulosa cells in the frozen-cultured tissues with McCoy medium, and lesser proliferation in fresh-cultured tissues only with α-MEM. In frozen-cultured tissues, ROS levels were highest at day 1 and progressively reduced during culture, independent of the media used. In conclusion, under the conditions used in this study, the M199 and McCoy media are recommended for the culture of follicles derived from fresh and frozen ovarian tissues, respectively.

Methoxychlor causes mitochondrial dysfunction and oxidative damage in the mouse ovary

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gupta, R.K.; Schuh, R.A.; Department of Anesthesiology, University of Maryland, Baltimore, MD

2006-11-01

Methoxychlor (MXC) is an organochlorine pesticide that reduces fertility in female rodents by causing ovarian atrophy, persistent estrous cyclicity, and antral follicle atresia (apoptotic cell death). Oxidative damage resulting from reactive oxygen species (ROS) generation has been demonstrated to lead to toxicant-induced cell death. Thus, this work tested the hypothesis that MXC causes oxidative damage to the mouse ovary and affects mitochondrial respiration in a manner that stimulates ROS production. For the in vitro experiments, mitochondria were collected from adult cycling mouse ovaries, treated with vehicle (dimethyl sulfoxide; DMSO) or MXC, and subjected to polarographic measurements of respiration. For themore » in vivo experiments, adult cycling CD-1 mice were dosed with either vehicle (sesame oil) or MXC for 20 days. After treatment, ovarian mitochondria were isolated and subjected to measurements of respiration and fluorimetric measurements of H{sub 2}O{sub 2} production. Some ovaries were also fixed and processed for immunohistochemistry using antibodies for ROS production markers: nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHG). Ovaries from in vivo experiments were also used to measure the mRNA expression and activity of antioxidants such as Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPX), and catalase (CAT). The results indicate that MXC significantly impairs mitochondrial respiration, increases production of H{sub 2}O{sub 2}, causes more staining for nitrotyrosine and 8-OHG in antral follicles, and decreases the expression and activity of SOD1, GPX, and CAT as compared to controls. Collectively, these data indicate that MXC inhibits mitochondrial respiration, causes ROS production, and decreases antioxidant expression and activity in the ovary, specifically in the antral follicles. Therefore, it is possible that MXC causes atresia of ovarian antral follicles by inducing oxidative stress through mitochondrial production of ROS.« less

Ovarian response to recombinant human follicle-stimulating hormone in luteinizing hormone-depleted women: examination of the two cell, two gonadotropin theory.

PubMed

Ben-Chetrit, A; Gotlieb, L; Wong, P Y; Casper, R F

1996-04-01

To evaluate the relative contribution of FSH to ovarian estrogen production. Nonrandomized, prospective study. University of Toronto teaching hospital reproductive biology unit. Five women who had been treated with depot GnRH agonist with hormonal add-back for 4 to 48 months and who were confirmed to be gonadotropin depleted by both bioassay and RIA. Subjects received 75 IU SC recombinant human FSH daily for 7 days followed by 150 IU daily for 7 days and 225 IU daily for the third week. Serum steroid determination and vaginal sonography for follicle size and endometrial thickness were performed serially and follicular fluid hormone levels were measured in two subjects. Bioactive LH and FSH activity were less than the detection limit of the assay (0.1 mIU/mL; conversion factor to SI units, 1.00 for LH and FSH) before recombinant FSH treatment in all five women. In all subjects, at least one preovulatory follicle developed by the end of two to three weeks. Endometrial thickness increased to between 7 and 9 mm in four women. Mean serum E2 in the five subjects increased from 17 pg/mL (range: 5 to 33 pg/mL; conversion factor to SI unit, 3.671) at baseline to 230 pg/mL (range: 37 to 489 pg/mL) at the end of the study. Follicular fluid E2 concentrations ranged from 44,296 to 69,367 pg/mL in the four follicles aspirated. Our results indicate that LH is not necessary for ovarian E2 production. We speculate that the granulosa cells, in the absence of detectable LH bioactivity, can use circulating adrenal androgens or constitutive or FSH-stimulated thecal androgens, to produce intrafollicular E2.

Microdose gonadotropin-releasing hormone agonist in the absence of exogenous gonadotropins is not sufficient to induce multiple follicle development.

PubMed

Chung, Karine; Fogle, Robin; Bendikson, Kristin; Christenson, Kamilee; Paulson, Richard

2011-01-01

Because the effectiveness of the "microdose flare" stimulation protocol often is attributed to the dramatic endogenous gonadotropin release induced by the GnRH agonist, the aim of this study was to determine whether use of microdose GnRH agonist alone could induce multiple ovarian follicle development in normal responders. Based on these data, the duration of gonadotropin rise is approximately 24 to 48 hours and is too brief to sustain continued multiple follicle growth. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Changes in sex steroid hormone levels reflect the reproductive status of captive female zebra sharks (Stegostoma fasciatum).

PubMed

Nozu, Ryo; Murakumo, Kiyomi; Yano, Nagisa; Furuyama, Rina; Matsumoto, Rui; Yanagisawa, Makio; Sato, Keiichi

2018-03-03

Captive breeding in aquaria is a useful means for ex situ preservation of threatened elasmobranch species. To promote captive breeding, it is important to determine the female reproductive status. However, information regarding reproductive status in female elasmobranchs is limited. Here, we used zebra sharks, Stegostoma fasciatum, as a model for elasmobranch reproduction in captivity. We investigated the relationships among changes in the sex steroid hormone levels, follicle size, and egg-laying period to develop indicators for the female reproductive status. We confirmed that mature female zebra sharks undergo an annual reproductive cycle. Additionally, we showed that the variations in sex steroid hormone levels correlated with reproductive status in mature female zebra sharks. Plasma estradiol-17ß (E2) concentrations increased two months before ovarian follicle development and decreased along with follicle regression. Interestingly, E2 levels were inversely correlated with water temperature (R = -0.901). Moreover, high levels of testosterone (T) correlated well with the laying period. These results strongly suggest that E2 is an indicator for ovarian follicle development, and that T is a useful indicator for both the onset and end of the egg-laying period in captive zebra sharks. Copyright © 2018 Elsevier Inc. All rights reserved.

The role of androgens in follicle maturation and ovulation induction: friend or foe of infertility treatment?

PubMed Central

2011-01-01

Background Effects of androgens on follicle maturation have been controversial for some time. Here, we review the potential of their applications in improving human ovulation induction, based on human and animal data, reported in the literature. Methods We reviewed the published literature for the years 2005-2011, using relevant key words, in PubMed, Medline and Cochrane reviews, and then performed secondary reviews of referenced articles, which previously had not been known or preceded the searched time period. A total of 217 publications were reviewed. Results Contrary to widely held opinion, recent data, mostly developed in the mouse, convincingly demonstrate essential contribution of androgens to normal follicle maturation and, therefore, female fertility. Androgens appear most engaged at preantral and antral stages, primarily affect granulosa cells, and exert effects via androgen receptors (AR) through transcriptional regulation but also in non-genomic ways, with ligand-activated AR modulating follicle stimulating hormone (FSH) activity in granulosa cells. While some androgens, like testosterone (T) and dehydroepiandrosterone (DHEA), appear effective in improving functional ovarian reserve (FOR) in women with diminished ovarian reserve (DOR), others may even exert opposite effects. Such differences in androgens may, at least partially, reflect different levels of agonism to AR. Discussion Selective androgens appear capable of improving early stages of folliculogenesis. They, therefore, may represent forerunners of a completely new class of ovulation-inducing medications, which, in contrast to gonadotropins, affect follicle maturation at much earlier stages. PMID:21849061

The importance of neuronal growth factors in the ovary.

PubMed

Streiter, S; Fisch, B; Sabbah, B; Ao, A; Abir, R

2016-01-01

The neurotrophin family consists of nerve growth factor (NGF), neurotrophin 3 (NT3) and neurotrophin 4/5 (NT4/5), in addition to brain-derived neurotrophic factor (BDNF) and the neuronal growth factors, glial cell line-derived neurotrophic factor (GDNF) and vasointestinal peptide (VIP). Although there are a few literature reviews, mainly of animal studies, on the importance of neurotrophins in the ovary, we aimed to provide a complete review of neurotrophins as well as neuronal growth factors and their important roles in normal and pathological processes in the ovary. Follicular assembly is probably stimulated by complementary effects of NGF, NT4/5 and BDNF and their receptors. The neurotrophins, GDNF and VIP and their receptors have all been identified in preantral and antral follicles of mammalian species, including humans. Transgenic mice with mutations in the genes encoding for Ngf, Nt4/5 and Bdnf and their tropomyosin-related kinase β receptor showed a reduction in preantral follicles and an abnormal ovarian morphology, whereas NGF, NT3, GDNF and VIP increased the in vitro activation of primordial follicles in rats and goats. Additionally, NGF, NT3 and GDNF promoted follicular cell proliferation; NGF, BDNF and VIP were shown to be involved in ovulation; VIP inhibited follicular apoptosis; NT4/5, BDNF and GDNF promoted oocyte maturation and NGF, NT3 and VIP stimulated steroidogenesis. NGF may also exert a stimulatory effect in ovarian cancer and polycystic ovarian syndrome (PCOS). Low levels of NGF and BDNF in follicular fluid may be associated with diminished ovarian reserve and high levels with endometriosis. More knowledge of the roles of neuronal growth factors in the ovary has important implications for the development of new therapeutic drugs (such as anti-NGF agents) for ovarian cancer and PCOS as well as various infertility problems, warranting further research. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The non-preventive effects of human menopausal gonadotropins on ovarian tissues in Nandrolone decanoate-treated female rats: A histochemical and ultra-structural study.

PubMed

Mesbah, Fakhroddin; Bordbar, Hossein; Talaei Khozani, Tahereh; Dehghani, Farzaneh; Mirkhani, Hossein

2018-03-01

The follicular growth and development may be affected by abused drugs. Nandrolone decanoate (ND) as an anabolic androgenic steroid can damage the morphological and functional features of the ovary and may lead to reproductive failure. This study was designed to evaluate the effects of synchronized and non-synchronized administration of Human Menopausal Gonadotropins (hMG) with ND on ovarian tissue and level of sex hormones in the adult female rat. Forty adult female Sprague Dawley rats were divided into eight groups. The five experimental groups received 3 and/or 10 mg/kg of ND synchronized and non-synchronized with 10 IU of hMG and hMG alone. The two shams and control groups received solvents of ND and hMG. The animals' serum levels of Follicle-stimulating hormone, Luteinizing hormone, progesterone and estrogen and the weight, volume and dimensions of the ovaries were measured. The ovaries were prepared for apoptosis assessment and morphological study. The ovarian volume and sex hormones in the experimental groups were decreased, but ovarian weight and dimensions didn't change. The rate of apoptosis was increased in the experimental groups as follows; a low and high dose of ND synchronized with hMG 48.80±18.70 and 65.20±14.20 respectively vs. Sham 1, 33.20±17.80, a low and high dose of ND non-synchronized with hMD 55.80±17.20 and 75.20±14.30 respectively vs. Sham 2, 31.60±32.40 groups, p˂0.01. Follicular and stromal cells were damaged in the experimental groups except for the hMG group. Administration of ND decreased the serum level of Luteinizing hormone, Follicle-stimulating hormone, progesterone and estrogen and damaged ovarian tissue irreversibly and irreparably and hMG cannot prevent the destruction of the follicles in the adult female rats. This can be a serious warning to women who abuse ND.

Ovarian Stem Cells—the Pros and Cons

PubMed Central

Evron, Ayelet; Blumenfeld, Zeev

2013-01-01

The potential for postnatal de novo oogenesis in mammals and in humans has become very controversial in the fields of reproductive science and biology. Historically, it has been thought that females of most mammalian species lose the ability to produce oocytes at birth. A contemporary understanding of stem cell biology together with novel experimental methods has challenged the model of a prenatal fixed ovarian primordial follicle pool that declines with age. Researchers have suggested replenishment of post-natal oocytes by germ-line stem cells (GSCs). According to this theory, GSCs produce oocytes and primordial follicles throughout the lifetime of the adult female. This review describes recent approaches supporting the revolutionary idea of de novo oogenesis in mammals and humans of reproductive-age and provides counter arguments from opponents of this novel and innovative concept. PMID:24453518

Ovarian Stem Cells-the Pros and Cons.

PubMed

Evron, Ayelet; Blumenfeld, Zeev

2013-03-20

The potential for postnatal de novo oogenesis in mammals and in humans has become very controversial in the fields of reproductive science and biology. Historically, it has been thought that females of most mammalian species lose the ability to produce oocytes at birth. A contemporary understanding of stem cell biology together with novel experimental methods has challenged the model of a prenatal fixed ovarian primordial follicle pool that declines with age. Researchers have suggested replenishment of post-natal oocytes by germ-line stem cells (GSCs). According to this theory, GSCs produce oocytes and primordial follicles throughout the lifetime of the adult female. This review describes recent approaches supporting the revolutionary idea of de novo oogenesis in mammals and humans of reproductive-age and provides counter arguments from opponents of this novel and innovative concept.

Effects of previous ovarian surgery for endometriosis on the outcome of assisted reproduction treatment.

PubMed

Geber, Selmo; Ferreira, Daniela Parreiras; Spyer Prates, Luis Felipe Víctor; Sales, Liana; Sampaio, Marcos

2002-01-01

Endometriosis affects 2-50% of women at reproductive age. Surgery is an option for treatment, but there is no convincing evidence that it promotes a significant improvement in fertility. Also, the removal of ovarian endometrioma might lead to a reduction in the follicular reserve and response to stimulation. Therefore, the aim of this study was to evaluate the effect of previous ovarian surgery for endometriosis on the ovarian response in assisted reproduction treatment cycles and its pregnancy outcome. A total of 61 women, with primary infertility and previously having undergone ovarian surgery for endometriosis, who had received 74 IVF/intracytoplasmic sperm injection (ICSI) cycles, were studied (study group). A further 74 patients with primary infertility who underwent 77 IVF/ICSI cycles within#10; the same period of time, at the same clinic and without previous ovarian surgery or endometriosis were studied as a control group. Patients were matched for age and treatment performed. Patients 35 years with previous ovarian surgery needed more ampoules for ovulation induction (P = 0.017) and had fewer follicles and oocytes than women in the control group (P = 0.001). Duration of folliculogenesis was similar in both groups, as was fertilization rate. A total of 10 patients achieved pregnancy in the study group (34.5%) and 14 (48.3%) in the control group. Although a lower pregnancy rate was observed in patients who had undergone previous ovarian surgery, this difference was not statistically significant (P = 0.424). In conclusion, ovarian surgery for the treatment of endometriosis reduces the ovarian outcome in IVF/ICSI cycles in women >35 years old, and might also decrease pregnancy rates. Therefore, for infertile patients, non-surgical treatment might be a better option to avoid reduction of the ovarian response.

Environmentally Induced Epigenetic Transgenerational Inheritance of Ovarian Disease

PubMed Central

Nilsson, Eric; Larsen, Ginger; Manikkam, Mohan; Guerrero-Bosagna, Carlos; Savenkova, Marina I.; Skinner, Michael K.

2012-01-01

The actions of environmental toxicants and relevant mixtures in promoting the epigenetic transgenerational inheritance of ovarian disease was investigated with the use of a fungicide, a pesticide mixture, a plastic mixture, dioxin and a hydrocarbon mixture. After transient exposure of an F0 gestating female rat during embryonic gonadal sex determination, the F1 and F3 generation progeny adult onset ovarian disease was assessed. Transgenerational disease phenotypes observed included an increase in cysts resembling human polycystic ovarian disease (PCO) and a decrease in the ovarian primordial follicle pool size resembling primary ovarian insufficiency (POI). The F3 generation granulosa cells were isolated and found to have a transgenerational effect on the transcriptome and epigenome (differential DNA methylation). Epigenetic biomarkers for environmental exposure and associated gene networks were identified. Epigenetic transgenerational inheritance of ovarian disease states was induced by all the different classes of environmental compounds, suggesting a role of environmental epigenetics in ovarian disease etiology. PMID:22570695

Continuous 900-megahertz electromagnetic field applied in middle and late-adolescence causes qualitative and quantitative changes in the ovarian morphology, tissue and blood biochemistry of the rat.

PubMed

Okatan, Derya Öztürk; Kaya, Haydar; Aliyazıcıoğlu, Yüksel; Demir, Selim; Çolakoğlu, Serdar; Odacı, Ersan

2018-02-01

The purpose of this study was to use histological and biochemical methods in order to evaluate changes taking place in the ovarian of rats exposed to the effect of a 900-megahertz (MHz) electromagnetic field (EMF) in middle and late adolescence. Twenty-four 34-d-old female Sprague-Dawley rats were assigned equally to control, sham and EMF groups. EMF group rats were exposed to the effect of a 900-MHz EMF for 1 h a day, at the same time every day between postnatal days 35 and 59, while inside an EMF cage. Sham group rats were kept inside the EMF cage for the same time between postnatal days 35 and 59 without being exposed to any EMF effect. At the end of the study, rats' ovarian were removed and blood specimens were taken. Right ovarium tissues were subjected to routine histological procedures and stained with hematoxylin and eosin, periodic acid shift and Masson's trichrome. Follicles were counted in ovarian sections stained with hematoxylin and eosin. The TUNEL method was used to evaluate apoptosis. Left ovarian tissue and blood specimens were investigated biochemically. Histopathological examination of EMF group ovarian tissue revealed thinning in the zona granulosa and theca layers, shrinking in granulosa cells, reduced mitotic activity and leukocyte infiltration in the follicles and stroma. Secondary follicle numbers in the EMF group were significantly lower than in the other groups. In terms of biochemistry, EMF and sham group superoxide dismutase, catalase and anti-Mullerian hormone levels and EMF group 3-nitrotyrosine values increased significantly compared to the control group. EMF and sham group serum catalase and 8-hydroxy-deoxiguanosine values increased significantly compared to the control group, and EMF group total oxidant status and oxidative stress index values were significantly higher compared to the sham and control groups. A total of 900-MHz EMF applied in middle and late adolescence may cause changes in the morphology and biochemistry of the rat ovarium.

Inhibition of cyclooxygenase-2 (COX-2) by meloxicam decreases the incidence of ovarian hyperstimulation syndrome in a rat model.

PubMed

Quintana, Ramiro; Kopcow, Laura; Marconi, Guillermo; Young, Edgardo; Yovanovich, Carola; Paz, Dante A

2008-10-01

To investigate the effects of selective cyclooxygenase-2 (COX-2) inhibition on the ovarian hyperstimulation syndrome (OHSS) in an experimental model. Controlled laboratory study. University-affiliated fertility center. Female Wistar rats. Female Wistar rats (22 days old) were divided into four groups: group 1 (control group; n = 10) received 0.1 mL of intraperitoneal (IP) saline from days 22-26; group 2 (mild-stimulated group; n = 10) received 10 IU of pregnant mare serum gonadotropin (PMSG) on day 24 and 10 IU of hCG 48 hours later (day 26); group 3 (OHSS group; n = 10) was given 10 IU of PMSG for 4 consecutive days from day 22 and 30 IU hCG on the fifth day to induce OHSS; group 4 was treated the same as group 3, but received 2 muL (15 mg/mL) of meloxicam 2 hours before the PMSG injection for 4 consecutive days, and 2 hours before the hCG injection on the fifth day. All groups were killed on day 26. Number of antral and luteinized follicles, ovarian weight, semiquantitative vascular endothelial growth factor (VEGF) and COX-2 immunohistochemistry. There were no differences in the ovarian weight between groups 1 and 2. Group 3 showed significantly increased ovarian weight that was suppressed, in group 4, by meloxicam. There was no difference in the number of antral follicles among the four groups. In the mild-stimulated and OHSS groups, the granulosa cells (GC) of preovulatory follicles and the stromal cells showed intense VEGF immunoreactivity. The ovaries from the meloxicam-treated group showed less immunoreactivity than the OHSS group, indicating diminished VEGF expression associated with meloxicam treatment. Group 3 (OHSS group) showed increased COX-2 immunoreactivity that was diminished in the meloxicam-treated group. Meloxicam treatment did not affect the hormone-induced increase in serum E(2) levels seen in OHSS rats. Our results in a rat model suggest that meloxicam has a beneficial effect on OHSS by reducing the increases in ovarian weight and VEGF expression associated with OHSS. These effects may be mediated by the COX-2 inhibitory capacity of meloxicam.

Influence of short-term energy supplementation on estrus, ovarian activity, and blood biochemistry in Ossimi ewes synchronized with fluorogestone acetate in the subtropics.

PubMed

Senosy, W; Mahmoud, G B; Abdel-Raheem, Sh M

2017-01-15

The objective of this study was to evaluate if short-term high-energy diet treatments have any overstimulatory effects on ovarian function and metabolic status in Ossimi ewes synchronized with progesterone sponge. Thirteen ewes were divided into high-energy (HEG; n = 7) and normal-energy or control (NEG; n = 6) groups. Progesterone sponges were placed intravaginally for 14 days during the winter breeding season (December-February). Four days before the removal of the sponges, a high-energy diet (130% of maintenance) was fed to HEG, whereas NEG was offered maintenance diet throughout the experiment. Ovarian performance and progesterone, estradiol, and blood metabolites were assessed daily starting from the day of removal of the sponges. Estrus period was longer in HEG (P < 0.05) when compared with NEG. Ovulation took place considerably longer with larger ovulatory follicles in HEG (P < 0.05). A marked increase in the level of total protein, albumin, glucose, and blood urea during the first 2 days following the removal of progesterone sponge was noticed in HEG when compared with NEG ewes. Eighty-five percentage (85.7%; 6/7) and 66.6% (4/6) had ovulation for the HEG and NEG, respectively. Dietary energy had a nonsignificant effect on the number of the recruited follicles, whereas a significant effect was observed for the diameter of the ovulatory follicle and ovulation rate (HEG, 2.3 ± 0.1 vs. NEG, 1.2 ± 0.3). It is concluded that short-term energy supplementation improves estrus expression and ovarian activity in fluorgestone acetate (FGA)-synchronized Ossimi ewes. Copyright © 2016 Elsevier Inc. All rights reserved.

Maternal sympathetic stress impairs follicular development and puberty of the offspring.

PubMed

Barra, Rafael; Cruz, Gonzalo; Mayerhofer, Artur; Paredes, Alfonso; Lara, Hernán E

2014-08-01

Chronic cold stress applied to adult rats activates ovarian sympathetic innervation and develops polycystic ovary (PCO) phenotype. The PCO syndrome in humans originates during early development and is expressed before or during puberty, which suggests that the condition derived from in utero exposure to neural- or metabolic-derived insults. We studied the effects of maternal sympathetic stress on the ovarian follicular development and on the onset of puberty of female offspring. Timed pregnant rats were exposed to chronic cold stress (4 °C, 3 h/daily from 1000 to 1300 h) during the entire pregnancy. Neonatal rats exposed to sympathetic stress during gestation had a lower number of primary, primordial, and secondary follicles in the ovary and a lower recruitment of primary and secondary follicles derived from the primordial follicular pool. The expression of the FSH receptor and response of the neonatal ovary to FSH were reduced. A decrease in nerve growth factor (NGF) mRNA was found without change in the low-affinity NGF receptor. The FSH-induced development of secondary follicles was decreased. At puberty, estradiol plasma levels decreased without changes in LH plasma levels. Puberty onset (as shown by the vaginal opening) was delayed. Ovarian norepinephrine (NE) was reduced; there was no change in its metabolite, 3-methoxy-4-hydroxyphenylglycol, in stressed rats and no change in NE turnover. The changes in ovarian NE in prepubertal rats stressed during gestation could represent a lower development of sympathetic nerves as a compensatory response to the chronically increased NE levels during gestation and hence participate in delaying reproductive performance in the rat. © 2014 Society for Reproduction and Fertility.

Identification of transcripts involved in meiosis and follicle formation during ovine ovary development.

PubMed

Baillet, Adrienne; Mandon-Pépin, Béatrice; Cabau, Cédric; Poumerol, Elodie; Pailhoux, Eric; Cotinot, Corinne

2008-09-23

The key steps in germ cell survival during ovarian development are the entry into meiosis of oogonies and the formation of primordial follicles, which then determine the reproductive lifespan of the ovary. In sheep, these steps occur during fetal life, between 55 and 80 days of gestation, respectively. The aim of this study was to identify differentially expressed ovarian genes during prophase I meiosis and early folliculogenesis in sheep. In order to elucidate the molecular events associated with early ovarian differentiation, we generated two ovary stage-specific subtracted cDNA libraries using SSH. Large-scale sequencing of these SSH libraries identified 6,080 ESTs representing 2,535 contigs. Clustering and assembly of these ESTs resulted in a total of 2,101 unique sequences depicted in 1,305 singleton (62.11%) and 796 contigs (37.9%) ESTs (clusters). BLASTX evaluation indicated that 99% of the ESTs were homologous to various known genes/proteins in a broad range of organisms, especially ovine, bovine and human species. The remaining 1% which exhibited any homology to known gene sequences was considered as novel. Detailed study of the expression patterns of some of these genes using RT-PCR revealed new promising candidates for ovary differentiation genes in sheep. We showed that the SSH approach was relevant to determining new mammalian genes which might be involved in oogenesis and early follicle development, and enabled the discovery of new potential oocyte and granulosa cell markers for future studies. These genes may have significant implications regarding our understanding of ovarian function in molecular terms, and for the development of innovative strategies to both promote and control fertility.

Follicular development and hormonal levels following highly purified or recombinant follicle-stimulating hormone administration in ovulatory women undergoing ovarian stimulation after pituitary suppression for in vitro fertilization: implications for implantation potential.

PubMed

Balasch, J; Fábregues, F; Creus, M; Peñarrubia, J; Vidal, E; Carmona, F; Puerto, B; Vanrell, J A

2000-01-01

The main goal in the present study was to compare follicular development and estradiol levels after ovarian stimulation in pituitary suppressed normally ovulating women undergoing IVF, using highly purified urinary follicle stimulating hormone (FSH) (u-FSH-HP) and recombinant FSH (rec-FSH). A secondary variable in our study was embryo implantation potential, which is closely related to appropriate follicular development and oocyte competence. For the main purpose of this study, 30 IVF patients (group 1) were treated during IVF consecutive cycles, using the same stimulation protocol, with u-FSH-HP in the first treatment study cycle and rec-FSH in the second one. As a control group (group 2) for implantation rates obtained in cycles treated with rec-FSH, 30 additional IVF patients were included who underwent a second IVF attempt again with u-FSH-HP. The total dose of FSH used and ovarian response obtained in terms of estradiol plasma levels and the total number of growing follicles on the day of human chronic gonadotropin (HCG) injection were similar in both treatment cycles in group 1 but better follicular dynamics and oocyte maturity were obtained with rec-FSH. The implantation rate was significantly higher in rec-FSH treated cycles in patients in group 1 than in control women (group 2). rec-FSH is more efficacious than u-FSH-HP when used in the same patient in inducing multiple follicular development in down-regulated cycles as indicated by ovarian performance and oocyte maturity. In addition, rec-FSH yields significantly higher implantation rates than u-FSH-HP when used in patients undergoing their second IVF attempt.

Supplementation with a recombinant human chorionic gonadotropin microdose leads to similar outcomes in ovarian stimulation with recombinant follicle-stimulating hormone using either a gonadotropin-releasing hormone agonist or antagonist for pituitary suppression.

PubMed

Cavagna, Mario; Maldonado, Luiz Guilherme Louzada; de Souza Bonetti, Tatiana Carvalho; de Almeida Ferreira Braga, Daniela Paes; Iaconelli, Assumpto; Borges, Edson

2010-06-01

To compare the outcomes of protocols for ovarian stimulation with recombinant hCG microdose, with GnRH agonists and antagonists for pituitary suppression. Prospective nonrandomized clinical trial. A private assisted reproduction center. We studied 182 patients undergoing intracytoplasmic sperm injection (ICSI) cycles, allocated into two groups: GnRH agonist group, in which patients received a GnRH agonist (n = 73), and a GnRH antagonist group, in which patients were administered a GnRH antagonist for pituitary suppression (n = 109). Pituitary suppression with GnRH agonist or GnRH antagonist. Ovarian stimulation carried out with recombinant FSH and supplemented with recombinant hCG microdose. Total dose of recombinant FSH and recombinant hCG administered; E(2) concentrations and endometrial width on the day of hCG trigger; number of follicles aspirated, oocytes and mature oocytes retrieved; fertilization, pregnancy (PR), implantation, and miscarriage rates. The total dose of recombinant FSH and recombinant hCG administered were similar between groups, as were the E(2) concentrations and endometrial width. The number of follicles aspirated, oocytes, and metaphase II oocytes collected were also comparable. There were no statistically significant differences in fertilization, PR, implantation, and miscarriage rates in the GnRH agonist and GnRH antagonist groups. When using recombinant hCG microdose supplementation for controlled ovarian stimulation (COS), there are no differences in laboratory or clinical outcomes with the use of either GnRH antagonist or agonist for pituitary suppression. Copyright (c) 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

The effects of in utero bisphenol A exposure on ovarian follicle numbers and steroidogenesis in the F1 and F2 generations of mice.

PubMed

Mahalingam, Sharada; Ther, Laura; Gao, Liying; Wang, Wei; Ziv-Gal, Ayelet; Flaws, Jodi A

2017-12-01

Bisphenol A (BPA) is a commonly used plasticizer. Previous studies show that in utero exposure to BPA affects reproductive outcomes in the F1-F3 generations of mice. However, its multigenerational effects on ovarian histology and steroidogenesis over the reproductive lifespan are unknown. Thus, we tested the hypothesis that BPA has multigenerational effects on follicle numbers and steroidogenesis. Mice were exposed in utero to vehicle control or BPA (0.5, 20, and 50μg/kg/day). Ovaries were collected for histological and gene expression analyses and sera were collected for hormone assays. In utero BPA exposure decreased preantral follicle numbers, cytochrome P450 aromatase mRNA levels, and estradiol levels in the F1 generation, whereas it decreased testosterone levels and altered steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage, 3β-hydroxysteroid dehydrogenase 1, and cytochrome P450 aromatase mRNA levels in the F2 generation. These data suggest that BPA has multigenerational effects on the ovary in mice. Copyright © 2017 Elsevier Inc. All rights reserved.

The effect of pinealectomy, melatonin and leptin hormones on ovarian follicular development in female Syrian hamsters (Mesocricetus auratus).

PubMed

Karakaş, A; Kaya, Aliye; Gündüz, B

2010-12-01

We studied the effects of melatonin and leptin hormones on ovarian follicular development in intact and pinealectomized female Syrian hamsters. We first monitored the oestrous cycle of the hamsters by the vaginal smear samples throughout a ten day period to start the injections simultaneously in all groups and performed saline, melatonin and leptin hormone injection groups for both control and pinealectomized hamsters. Then the injections were applied for four days starting the oestrus phase of the cycle and the ovaries were removed for preparation of histological analysis. We measured the diameters and the numbers of the follicles and we classified the follicles according to the number of the granulosa cell layer. Leptin hormone injection increased melatonin hormone injection decreased the number and the diameter of the follicles. The stimulating effect of the leptin hormone was more pronounced in the pinealectomized group. The results of the present study indicate that the removal of the pineal gland and leptin hormone administration are playing a stimulatory while melatonin hormone administration is playing an inhibitory role on the follicular development in female Syrian hamsters.

Reproductive parameters of the Pacific angel shark Squatina californica (Selachii: Squatinidae).

PubMed

Romero-Caicedo, A F; Galván-Magaña, F; Hernández-Herrera, A; Carrera-Fernández, M

2016-04-01

Reproductive characteristics of the Pacific angel shark, Squatina californica, were evaluated from 420 specimens obtained from the artisanal fishery in La Paz Bay, Gulf of California, Mexico. Females (99 cm, 6000 g) were larger than males (95 cm, 5000 g) in terms of both total length (L(T)) and body mass (M(T)). The overall sex ratio was significantly different from the expected 1:1, suggesting sexual segregation of mature individuals in La Paz Bay. Males had developed reproductive organs and calcified claspers from 72 cm L(T); the median size at maturity (LT50 ) was 75.6 cm. In females, only the left ovary was functional and mature ovarian follicles were present from 77 cm L(T); the estimated LT50 was 77.7 cm. For the 10 gravid females sampled, uterine fecundity was between two and 10 embryos. Mature, non-gravid females with small and large ovarian follicles appeared simultaneously with gravid females with follicles that did not exceed 1.9 cm diameter. © 2016 The Fisheries Society of the British Isles.

Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified-warmed ovarian tissue survival.

PubMed

Youm, Hye Won; Lee, Jung Ryeol; Lee, Jaewang; Jee, Byung Chul; Suh, Chang Suk; Kim, Seok Hyun

2014-04-01

What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation? The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification. Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified-warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented. A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC. The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of α-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified-warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level. The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4-58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6-92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation. The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation. We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.

[Anti-Müllerian hormone levels as a predictor of ovarian reserve in systemic lupus erythematosus patients: a review].

PubMed

Gasparin, Andrese Aline; Chakr, Rafael Mendonça da Silva; Brenol, Claiton Viegas; Palominos, Penélope Ester; Xavier, Ricardo Machado; Souza, Lucian; Brenol, João Carlos Tavares; Monticielo, Odirlei André

2015-01-01

The anti-Müllerian hormone (AMH) is secreted from granulosa cells of growing ovarian follicles and appears to be the best endocrine marker capable of estimating ovarian reserve. Systemic lupus erythematosus (SLE) is an autoimmune disease that predominantly affects women of reproductive age and may negatively affect their fertility due to disease activity and the treatments used. Recently, several studies assessed AMH levels to understand the real impact of SLE and its treatment on fertility. Copyright © 2014 Elsevier Editora Ltda. All rights reserved.

Promising Loci and Genes for Yolk and Ovary Weight in Chickens Revealed by a Genome-Wide Association Study.

PubMed

Sun, Congjiao; Lu, Jian; Yi, Guoqiang; Yuan, Jingwei; Duan, Zhongyi; Qu, Lujiang; Xu, Guiyun; Wang, Kehua; Yang, Ning

2015-01-01

Because it serves as the cytoplasm of the oocyte and provides a large amount of reserves, the egg yolk has biological significance for developing embryos. The ovary and its hierarchy of follicles are the main reproductive organs responsible for yolk deposition in chickens. However, the genetic architecture underlying the yolk and ovarian follicle weights remains elusive. Here, we measured the yolk weight (YW) at 11 age points from onset of egg laying to 72 weeks of age and measured the follicle weight (FW) and ovary weight (OW) at 73 weeks as part of a comprehensive genome-wide association study (GWAS) in 1,534 F2 hens derived from reciprocal crosses between White Leghorn (WL) and Dongxiang chickens (DX). For all ages, YWs exhibited moderate single nucleotide polymorphism (SNP)-based heritability estimates (0.25-0.38), while the estimates for FW (0.16) and OW (0.20) were relatively low. Independent univariate genome-wide screens for each trait identified 12, 3, and 31 novel significant associations with YW, FW, and OW, respectively. A list of candidate genes such as ZAR1, STARD13, ACER1b, ACSBG2, and DHRS12 were identified for having a plausible function in yolk and follicle development. These genes are important to the initiation of embryogenesis, lipid transport, lipoprotein synthesis, lipid droplet promotion, and steroid hormone metabolism, respectively. Our study provides for the first time a genome-wide association (GWA) analysis for follicle and ovary weight. Identification of the promising loci as well as potential candidate genes will greatly advance our understanding of the genetic basis underlying dynamic yolk weight and ovarian follicle development and has practical significance in breeding programs for the alteration of yolk weight at different age points.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sobinoff, A.P.; Pye, V.; Nixon, B.

Benzo(a)pyrene (BaP) is an ovotoxic constituent of cigarette smoke associated with pre-mature ovarian failure and decreased rates of conception in IVF patients. Although the overall effect of BaP on female fertility has been documented, the exact molecular mechanisms behind its ovotoxicity remain elusive. In this study we examined the effects of BaP exposure on the ovarian transcriptome, and observed the effects of in vivo exposure on oocyte dysfunction. Microarray analysis of BaP cultured neonatal ovaries revealed a complex mechanism of ovotoxicity involving a small cohort of genes associated with follicular growth, cell cycle progression, and cell death. Histomorphological and immunohistochemicalmore » analysis supported these results, with BaP exposure causing increased primordial follicle activation and developing follicle atresia in vitro and in vivo. Functional analysis of oocytes obtained from adult Swiss mice treated neonatally revealed significantly increased levels of mitochondrial ROS/lipid peroxidation, and severely reduced sperm-egg binding and fusion in both low (1.5 mg/kg/daily) and high (3 mg/kg/daily) dose treatments. Our results reveal a complex mechanism of BaP induced ovotoxicity involving developing follicle atresia and accelerated primordial follicle activation, and suggest short term neonatal BaP exposure causes mitochondrial leakage resulting in reduced oolemma fluidity and impaired fertilisation in adulthood. This study highlights BaP as a key compound which may be partially responsible for the documented effects of cigarette smoke on follicular development and sub-fertility. -- Highlights: ► BaP exposure up-regulates canonical pathways linked with follicular growth/atresia. ► BaP causes primordial follicle activation and developing follicle atresia. ► BaP causes oocyte mitochondrial ROS and lipid peroxidation, impairing fertilisation. ► Short term neonatal BaP exposure compromises adult oocyte quality.« less

Expression of anti-Mullerian hormone in hens selected for different ovulation rates.

PubMed

Johnson, P A; Kent, T R; Urick, M E; Trevino, L S; Giles, J R

2009-05-01

In hens, the granulosa layer is the primary source of anti-Mullerian hormone (AMH), as it is in mammals. Small follicles express the greatest amount of Amh mRNA with less in the larger follicles. Laying hens have a distinct ovarian hierarchy of follicles while broiler breeder hens often have excessive follicle growth with a disrupted hierarchy. The objective of Experiment 1 was to examine Amh expression in two strains of hens differing in ovulatory efficiency. Amh expression was greater (P<0.01) in broiler breeder hens (n=6) as compared with laying hens (n=6). Experiment 2 was designed to examine whether alterations in follicular development due to diet, within the broiler breeder hens, were correlated with changes in the expression of Amh. Restricted feeding (RF) in broiler breeder hens promotes optimal follicular development. Egg production in broiler breeder hens on full feed (FF; n=8) was 78% that of hens on RF (n=9). The number of large follicles (P<0.05), total ovarian weight (P<0.01), and Amh mRNA expression were greater in FF hens as compared with RF hens (P<0.01). There was no difference in FSH receptor expression between the two groups. A direct nutritional effect was not supported because culture of granulosa cells with varying concentrations of glucose and insulin showed no effect on granulosa Amh expression. Finally, testis-conditioned medium resulted in a dose-related increase in granulosa cell proliferation, which could be inhibited by preincubation with AMH antibody. AMH may enhance granulosa cell proliferation through an autocrine or paracrine mechanism although excessive AMH may inhibit optimal follicle selection.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sobinoff, A.P.; Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308; Beckett, E.L.

Cigarette smoke is a reproductive hazard associated with pre-mature reproductive senescence and reduced clinical pregnancy rates in female smokers. Despite an increased awareness of the adverse effects of cigarette smoke exposure on systemic health, many women remain unaware of the adverse effects of cigarette smoke on female fertility. This issue is compounded by our limited understanding of the molecular mechanisms behind cigarette smoke induced infertility. In this study we used a direct nasal exposure mouse model of cigarette smoke-induced chronic obstructive pulmonary disease to characterise mechanisms of cigarette-smoke induced ovotoxicity. Cigarette smoke exposure caused increased levels of primordial follicle depletion,more » antral follicle oocyte apoptosis and oxidative stress in exposed ovaries, resulting in fewer follicles available for ovulation. Evidence of oxidative stress also persisted in ovulated oocytes which escaped destruction, with increased levels of mitochondrial ROS and lipid peroxidation resulting in reduced fertilisation potential. Microarray analysis of ovarian tissue correlated these insults with a complex mechanism of ovotoxicity involving genes associated with detoxification, inflammation, follicular activation, immune cell mediated apoptosis and membrane organisation. In particular, the phase I detoxifying enzyme cyp2e1 was found to be significantly up-regulated in developing oocytes; an enzyme known to cause molecular bioactivation resulting in oxidative stress. Our results provide a preliminary model of cigarette smoke induced sub-fertility through cyp2e1 bioactivation and oxidative stress, resulting in developing follicle depletion and oocyte dysfunction. - Highlights: • Cigarette smoke exposure targets developing follicle oocytes. • The antral follicle oocyte is a primary site of ovarian cigarette smoke metabolism. • Cyp2e1 is a major enzyme involved in ameliorating smoke-induced ovotoxicity. • Cigarette smoke causes oocyte mitochondrial ROS, impairing fertilisation.« less

A novel model for development, organization, and function of gonadotropes in fish pituitary.

PubMed

Golan, Matan; Biran, Jakob; Levavi-Sivan, Berta

2014-01-01

The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are key regulators of the reproductive axis in vertebrates. Despite the high popularity of zebrafish as a model organism for studying reproductive functions, to date no transgenic zebrafish with labeled gonadotropes have been introduced. Using gonadotropin regulatory elements from tilapia, we generated two transgenic zebrafish lines with labeled gonadotropes. The tilapia and zebrafish regulatory sequences were highly divergent but several conserved elements allowed the tilapia promoters to correctly drive the transgenes in zebrafish pituitaries. FSH cells reacted to stimulation with gonadotropin releasing hormone by proliferating and showing increased transgene fluorescence, whereas estrogen exposure caused a decrease in cell number and transgene fluorescence. Transgene fluorescence reflected the expression pattern of the endogenous fshb gene. Ontogenetic expression of the transgenes followed typical patterns, with FSH cells appearing early in development, and LH cells appearing later and increasing dramatically in number with the onset of puberty. Our transgenic lines provide a powerful tool for investigating the development, anatomy, and function of the reproductive axis in lower vertebrates.

Effects of vitrification cryopreservation on follicular morphology and stress relaxation behaviors of human ovarian tissues: sucrose versus trehalose as the non-permeable protective agent.

PubMed

Tian, Ting; Zhao, Gang; Han, Dan; Zhu, Kaixuan; Chen, Dawei; Zhang, Zhiguo; Wei, Zhaolian; Cao, Yunxia; Zhou, Ping

2015-04-01

Is sucrose more effective than trehalose in human ovarian tissue cryopreservation? The effect of sucrose as a cryoprotective agent (CPA) was not significantly different from that of trehalose in human ovarian tissue cryopreservation. Sugars have the ability to keep the cell membrane intact and can decrease the toxicity of CPAs. Sucrose is the most commonly used non-permeable CPA, while trehalose is rarely used in human ovarian tissue cryopreservation. Although various methods are utilized to evaluate the efficiency of human ovarian tissue cryopreservation, few studies have evaluated the effect of cryopreservation from the viewpoint of biomechanics. A total of 15 ovarian tissue samples were collected from 15 patients (20-41 years old) with benign ovarian tumors or malignancies, and each was dissected into six slices. Two slices were taken as the fresh control group. The remaining four slices were vitrified using different vitrification protocols. After warming, samples in each group were either fixed for histological evaluation or destined for stress relaxation test. The CPA solutions for the control and vitrified groups were composed of EDS and EDT (E, ethylene glycol; D, dimethylsulphoxide; S, sucrose; T, trehalose). The stress relaxation experiments were carried out at room temperature using a dynamic mechanical analyzer. Ovarian tissue samples were assessed for both their morphology and viscoelasticity. Stress relaxation data (SRD) were calculated as a percentage, representing the ability to maintain the initial stress after stretching. The percentage of morphologically normal follicles was compared between groups, which was represented by morphologic preservation ratio. The morphologic preservation ratio of the primordial follicles in the fresh control group (87.58%) was higher than that in group S (72.33%) (P = 0.000) and group T (79.56%) (P = 0.002). Although not statistically significant, compared with the S group, vitrification with T suggested a trend toward a higher morphologic preservation ratio of the primordial follicles. The SRD in the fresh control group (0.6433 ± 0.7233) was significantly different from that in group S (0.5200 ± 0.8331, P = 0.000) or in group T (0.5667 ± 0.6415, P = 0.000). However, no significant difference was found between groups S and T. Experimental samples were directly exposed to the air, which will result in a discrepancy in the viscoelastic properties between experimental tissues and in vivo tissues. Our study suggested a trend toward a higher morphologic preservation ratio of the primordial follicles after vitrification in trehalose compared with sucrose, which may provide a basis for further optimizing human ovarian tissue vitrification. In addition, it was possible to evaluate the effect of ovarian tissue cryopreservation from a biomechanics perspective. This study was supported by the grants from the Medical Scientific Research Subject, Health Ministry of Anhui Province (2010B014) and National Basic Research Program of China (973 Program) (2012CB944704), and the National Natural Science Foundation of China (Nos. 51276179 and 51476160). The authors declare that there is no conflict of interests regarding the publication of this original paper. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Ovarian cortex from high A4 cows secretes excess A4, and exhibits increased oxidative stress, macrophage markers and arrested follicle development which can be partially rescued by angiogenic VEGFA Is

USDA-ARS?s Scientific Manuscript database

We have identified a population of cows within the UNL physiology herd with excess androstenedione (A4) in follicular fluid and 17% reduction in calving rate. Because excess androgens can cause follicle arrest we hypothesized that 1) folliculogenesis would be disrupted in High A4 cows; and 2) vascul...

New insights into molecular pathways associated with flatfish ovarian development and atresia revealed by transcriptional analysis

PubMed Central

Tingaud-Sequeira, Angèle; Chauvigné, François; Lozano, Juanjo; Agulleiro, María J; Asensio, Esther; Cerdà, Joan

2009-01-01

Background The Senegalese sole (Solea senegalensis) is a marine flatfish of increasing commercial interest. However, the reproduction of this species in captivity is not yet controlled mainly because of the poor knowledge on its reproductive physiology, as it occurs for other non-salmonid marine teleosts that exhibit group-synchronous ovarian follicle development. In order to investigate intra-ovarian molecular mechanisms in Senegalese sole, the aim of the present study was to identify differentially expressed genes in the ovary during oocyte growth (vitellogenesis), maturation and ovarian follicle atresia using a recently developed oligonucleotide microarray. Results Microarray analysis led to the identification of 118 differentially expressed transcripts, of which 20 and 8 were monitored by real-time PCR and in situ hybridization, respectively. During vitellogenesis, many up-regulated ovarian transcripts had putative mitochondrial function/location suggesting high energy production (NADH dehydrogenase subunits, cytochromes) and increased antioxidant protection (selenoprotein W2a), whereas other regulated transcripts were related to cytoskeleton and zona radiata organization (zona glycoprotein 3, alpha and beta actin, keratin 8), intracellular signalling pathways (heat shock protein 90, Ras homolog member G), cell-to-cell and cell-to-matrix interactions (beta 1 integrin, thrombospondin 4b), and the maternal RNA pool (transducer of ERBB2 1a, neurexin 1a). Transcripts up-regulated in the ovary during oocyte maturation included ion transporters (Na+-K+-ATPase subunits), probably required for oocyte hydration, as well as a proteinase inhibitor (alpha-2-macroglobulin) and a vesicle calcium sensor protein (extended synaptotagmin-2-A). During follicular atresia, few transcripts were found to be up-regulated, but remarkably most of them were localized in follicular cells of atretic follicles, and they had inferred roles in lipid transport (apolipoprotein C-I), chemotaxis (leukocyte cell-derived chemotaxin 2,), angiogenesis (thrombospondin), and prevention of apoptosis (S100a10 calcium binding protein). Conclusion This study has identified a number of differentially expressed genes in the ovary that were not previously found to be regulated during ovarian development in marine fish. Specifically, we found evidence, for the first time in teleosts, of the activation of chemoattractant, angiogenic and antiapoptotic pathways in hypertrophied follicular cells at the onset of ovarian atresia. PMID:19754951

Oxygen consumption by bovine granulosa cells with prediction of oxygen transport in preantral follicles.

PubMed

Li, Dongxing; Redding, Gabe P; Bronlund, John E

2013-01-01

The rate of oxygen consumption by granulosa cells is a key parameter in mathematical models that describe oxygen transport across ovarian follicles. This work measured the oxygen consumption rate of bovine granulosa cells in vitro to be in the range 2.1-3.3×10⁻¹⁶ mol cell⁻¹ s⁻¹ (0.16-0.25 mol m⁻³ s⁻¹). The implications of the rates for oxygen transport in large bovine preantral follicles were examined using a mathematical model. The results indicate that oocyte oxygenation becomes increasingly constrained as preantral follicles grow, reaching hypoxic levels near the point of antrum formation. Beyond a preantral follicle radius of 134 µm, oxygen cannot reach the oocyte surface at typical values of model parameters. Since reported sizes of large bovine preantral follicles range from 58 to 145 µm in radius, this suggests that oocyte oxygenation is possible in all but the largest preantral follicles, which are on the verge of antrum formation. In preantral bovine follicles, the oxygen consumption rate of granulosa cells and fluid voidage will be the key determinants of oxygen levels across the follicle.

Dual protective role for Glutathione S-transferase class pi against VCD-induced ovotoxicity in the rat ovary

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keating, Aileen F.; Sen, Nivedita; Sipes, I. Glenn

2010-09-01

The occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively destroys ovarian small pre-antral follicles in rats and mice via apoptosis. Detoxification of VCD can occur through glutathione conjugation, catalyzed by glutathione S-transferase (GST) enzymes. Further, GST class pi (GSTp) can negatively regulate JNK activity through protein:protein interactions in extra-ovarian tissues. Dissociation of this protein complex in the face of chemical exposure releases the inhibition of pro-apoptotic JNK. Increased JNK activity during VCD-induced ovotoxicity has been shown in isolated ovarian small pre-antral follicles following in vivo dosing of rats (80 mg/kg/day; 15 days, i.p.). The present study investigated the pattern of ovarian GSTpmore » expression during VCD exposure. Additionally, the effect of VCD on an ovarian GSTp:JNK protein complex was investigated. PND4 F344 rat ovaries were incubated in control medium {+-} VCD (30 {mu}M) for 2-8 days. VCD increased ovarian GSTp mRNA (P < 0.05) relative to control on d4-d8; whereas GSTp protein was increased (P < 0.05) on d6-d8. A GSTp:JNK protein complex was detected by immunoprecipitation and Western blotting in ovarian tissues. Relative to control, the amount of GSTp-bound JNK was increased (P = 0.09), while unbound JNK was decreased (P < 0.05) on d6 of VCD exposure. The VCD-induced decrease in unbound JNK was preceded by a decrease in phosphorylated c-Jun which occurred on d4. These findings are in support of a possible dual protective role for GSTp in the rat ovary, consisting of metabolism of VCD and inhibition of JNK-initiated apoptosis.« less

Prophylactic salpingectomy in premenopausal low-risk women for ovarian cancer: primum non nocere.

PubMed

Morelli, Michele; Venturella, Roberta; Mocciaro, Rita; Di Cello, Annalisa; Rania, Erika; Lico, Daniela; D'Alessandro, Pietro; Zullo, Fulvio

2013-06-01

The objective of this study is to compare ovarian function and surgical outcomes between patients affected by benign uterine pathologies submitted to total laparoscopic hysterectomy (TLH) plus salpingectomy and women in which standard TLH with adnexal preservation was performed. We retrospectively compared data of 79 patients who underwent TLH plus bilateral salpingectomy (group A), with those of 79 women treated by standard TLH without adnexectomy (sTLH) (group B). Ovarian reserve modification, expressed as the difference between 3 months post-operative and pre-operative values of Anti-Müllerian Hormone (AMH), Follicle Stimulating Hormone (FSH), Antral Follicle Count (AFC), mean ovarian diameters and Peak Systolic Velocity (PSV), was recorded for each patient. For each surgical procedure, operative time, variation of hemoglobin level (ΔHb), postoperative hospital stay, postoperative return to normal activity, and complication rate were recorded as secondary outcomes. According to our post-hoc analysis, this equivalence study resulted to have a statistical power of 96.8%. Significant difference was not observed between groups with respect to ΔAMH (p=0.35), ΔFSH (p=0.15), ΔAFC (p=0.09), Δ mean ovarian diameters (p=0.57) and ΔPSV (p=0.61). In addition, secondary outcomes such as operative time (p=0.79), ΔHb (p=0.41), postoperative hospital stay (p=0.16), postoperative return to normal activity (p=0.11) and complication rate also did not show any significant difference. The addition of bilateral salpingectomy to TLH for prevention of ovarian cancer in women who do not carry a BRCA1/2 mutations do not show negative effects on the ovarian function. In addition, no perioperative complications are related to the salpingectomy step in TLH. Copyright © 2013 Elsevier Inc. All rights reserved.

Mechanistic Study on Triptorelin Action in Protecting From 5-FU-Induced Ovarian Damage in Rats.

PubMed

Wang, Ying; Tian, Xiaoyu; Liang, Lingxia; Wang, Yan; Wang, Ruifang; Cheng, Xiaolin; Yan, Zhen; Chen, Yawei; Qi, Pengwei

2014-01-01

Triptorelin, a kind of GnRH agonist, is widely used in the treatment of hormone-responsive cancers in the clinic. This study aimed to discover the underlying mechanism of triptorelin in protection from 5-fluorouracil (5-FU)-induced ovarian damage in Sprague-Dawley rats. In the present study, after using 5-FU to induce ovarian damage in rats, body weight and wet ovaries were weighed, the levels of estradiol (E2), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH) in blood were detected, and the expression of Bcl-2, Bax, and NF-κB was determined. It suggested that, compared to the control, body weight gain, the ratio of ovarian wet weight to body weight, primary follicle numbers, and the levels of AMH were significantly decreased, while the concentration of E2 and FSH was heavily increased following 5-FU administration. In contrast, after coadministration of triptorelin with 5-FU, the ratio of ovarian wet weight to body weight and the levels of AMH were significantly increased, whereas the level of E2 and FSH was decreased significantly when compared with the 5-FU group. Furthermore, at indicated times, 5-FU led to the reduced Bcl-2 and NF-κB expression and increased Bax expression while triptorelin plus 5-FU increased Bcl-2 and NF-κB expression and decreased Bax expression. It was indicated that triptorelin could protect rats from 5-FU-induced ovarian damage by modulation of hormones, Bcl-2, Bax, and NF-κB. These results might highlight the mechanism of triptorelin as a protective agent in clinical chemotherapy for ovarian damage.

Primary Ovarian Insufficiency

MedlinePlus

... POI. For example, women Fragile X syndrome or Turner syndrome are at higher risk. Certain diseases, such as ... Genetic disorders such as Fragile X syndrome and Turner syndrome A low number of follicles Autoimmune diseases, including ...

Novel biomarkers of perchlorate exposure in zebrafish

USGS Publications Warehouse

Mukhi, S.; Carr, J.A.; Anderson, T.A.; Patino, R.

2005-01-01

Perchlorate inhibits iodide uptake by thyroid follicles and lowers thyroid hormone production. Although several effects of perchlorate on the thyroid system have been reported, the utility of these pathologies as markers of environmental perchlorate exposures has not been adequately assessed. The present study examined time-course and concentration-dependent effects of perchlorate on thyroid follicle hypertrophy, colloid depletion, and angiogenesis; alterations in whole-body thyroxine (T4) levels; and somatic growth and condition factor of subadult and adult zebrafish. Changes in the intensity of the colloidal T4 ring previously observed in zebrafish also were examined immunohistochemically. Three-month-old zebrafish were exposed to ammonium perchlorate at measured perchlorate concentrations of 0, 11, 90, 1,131, and 11,480 ppb for 12 weeks and allowed to recover in clean water for 12 weeks. At two weeks of exposure, the lowest-observed-effective concentrations (LOECs) of perchlorate that induced angiogenesis and depressed the intensity of colloidal T4 ring were 90 and 1,131 ppb, respectively; other parameters were not affected (whole-body T4 was not determined at this time). At 12 weeks of exposure, LOECs for colloid depletion, hypertrophy, angiogenesis, and colloidal T4 ring were 11,480, 1,131, 90, and 11 ppb, respectively. All changes were reversible, but residual effects on angiogenesis and colloidal T4 ring intensity were still present after 12 weeks of recovery (LOEC, 11,480 ppb). Whole-body T 4 concentration, body growth (length and weight), and condition factor were not affected by perchlorate. The sensitivity and longevity of changes in colloidal T4 ring intensity and angiogenesis suggest their usefulness as novel markers of perchlorate exposure. The 12-week LOEC for colloidal T4 ring is the lowest reported for any perchlorate biomarker in aquatic vertebrates. ?? 2005 SETAC.

Functional Analysis of Nuclear Estrogen Receptors in Zebrafish Reproduction by Genome Editing Approach.

PubMed

Lu, Huijie; Cui, Yong; Jiang, Liwen; Ge, Wei

2017-07-01

Estrogens signal through both nuclear and membrane receptors with most reported effects being mediated via the nuclear estrogen receptors (nERs). Although much work has been reported on nERs in the zebrafish, there is a lack of direct genetic evidence for their functional roles and importance in reproduction. To address this issue, we undertook this study to disrupt all three nERs in the zebrafish, namely esr1 (ERα), esr2a (ERβII), and esr2b (ERβI), by the genome-editing technology clustered regularly interspaced short palindromic repeats and its associated nuclease (CRISPR/Cas9). Using this loss-of-function genetic approach, we successfully created three mutant zebrafish lines with each nER knocked out. In addition, we also generated all possible double and triple knockouts of the three nERs. The phenotypes of these mutants in reproduction were analyzed in all single, double, and triple nER knockouts in both females and males. Surprisingly, all three single nER mutant fish lines display normal reproductive development and function in both females and males, suggesting functional redundancy among these nERs. Further analysis of double and triple knockouts showed that nERs, especially Esr2a and Esr2b, were essential for female reproduction, and loss of these two nERs led to an arrest of folliculogenesis at previtellogenic stage II followed by sex reversal from female to male. In addition, the current study also revealed a unique role for Esr2a in follicle cell proliferation and transdifferentiation, follicle growth, and chorion formation. Taken together, this study provides the most comprehensive genetic analysis for differential functions of esr1, esr2a, and esr2b in fish reproduction. Copyright © 2017 Endocrine Society.

The use of serum anti-Mullerian hormone (AMH) levels and antral follicle count (AFC) to predict the number of oocytes collected and availability of embryos for cryopreservation in IVF.

PubMed

Kotanidis, L; Nikolettos, K; Petousis, S; Asimakopoulos, B; Chatzimitrou, E; Kolios, G; Nikolettos, N

2016-12-01

To investigate the predictive value of anti-Mullerian hormone (AMH) and antral follicle count (AFC) on the final number of oocytes retrieved and the availability of embryos for cryopreservation in in vitro fertilization (IVF) cycles. In this prospective study, one hundred and twenty women in their first IVF treatment were enrolled. The short stimulation agonist protocol was used for controlled ovarian hyperstimulation in all cases. Serum AMH levels were measured during the menstrual cycle preceding treatment. AFC was measured in cycle day 2, just before starting ovarian stimulation. A strong, positive correlation between AMH, AFC and the number of collected oocytes was found. The patients with available and suitable supplementary embryos for cryopreservation had higher levels of AMH and larger numbers of AFC. AMH and AFC appear to be valuable markers mainly for ovarian reserve and response to IVF treatment. Serum AMH levels and AFC are significantly associated with the number of retrieved oocytes. Also, a positive correlation with the availability of supernumerary embryos suitable for cryopreservation was observed.

LH prevents cisplatin-induced apoptosis in oocytes and preserves female fertility in mouse.

PubMed

Rossi, Valerio; Lispi, Monica; Longobardi, Salvatore; Mattei, Maurizio; Rella, Francesca Di; Salustri, Antonietta; De Felici, Massimo; Klinger, Francesca G

2017-01-01

Premature ovarian failure and female infertility are frequent side effects of anticancer therapies, owing to the extreme sensitivity of the ovarian reserve oocytes to the damaging effects of irradiation and chemotherapy on DNA. We report here a robust protective effect of luteinizing hormone (LH) on the primordial follicle pool of prepubertal ovaries against the cisplatin (Cs)-induced apoptosis. In vitro LH treatment of prepubertal ovarian fragments generated anti-apoptotic signals by a subset of ovarian somatic cells expressing LH receptor (LHR) through cAMP/PKA and Akt pathways. Such signals, reducing the oocyte level of pro-apoptotic TAp63 protein and favoring the repair of the Cs-damaged DNA in the oocytes, prevented their apoptosis. Noteworthy, in vivo administration to prepubertal female mice of a single dose of LH together with Cs inhibited the depletion of the primordial follicle reserve caused by the drug and preserved their fertility in reproductive age, preventing significant alteration in the number of pregnancy and of delivered pups. In conclusion, these findings establish a novel ovoprotective role for LH and further support the very attracting prospective to use physiological 'fertoprotective' approaches for preventing premature infertility and risks linked to precocious menopause in young patients who survived cancer after chemotherapy.

LH prevents cisplatin-induced apoptosis in oocytes and preserves female fertility in mouse

PubMed Central

Rossi, Valerio; Lispi, Monica; Longobardi, Salvatore; Mattei, Maurizio; Rella, Francesca Di; Salustri, Antonietta; De Felici, Massimo; Klinger, Francesca G

2017-01-01

Premature ovarian failure and female infertility are frequent side effects of anticancer therapies, owing to the extreme sensitivity of the ovarian reserve oocytes to the damaging effects of irradiation and chemotherapy on DNA. We report here a robust protective effect of luteinizing hormone (LH) on the primordial follicle pool of prepubertal ovaries against the cisplatin (Cs)-induced apoptosis. In vitro LH treatment of prepubertal ovarian fragments generated anti-apoptotic signals by a subset of ovarian somatic cells expressing LH receptor (LHR) through cAMP/PKA and Akt pathways. Such signals, reducing the oocyte level of pro-apoptotic TAp63 protein and favoring the repair of the Cs-damaged DNA in the oocytes, prevented their apoptosis. Noteworthy, in vivo administration to prepubertal female mice of a single dose of LH together with Cs inhibited the depletion of the primordial follicle reserve caused by the drug and preserved their fertility in reproductive age, preventing significant alteration in the number of pregnancy and of delivered pups. In conclusion, these findings establish a novel ovoprotective role for LH and further support the very attracting prospective to use physiological 'fertoprotective' approaches for preventing premature infertility and risks linked to precocious menopause in young patients who survived cancer after chemotherapy. PMID:27689876

Age-Related Normogram for Ovarian Antral Follicle Count in Women with Polycystic Ovary Syndrome and Comparison with Age Matched Controls Using Magnetic Resonance Imaging.

PubMed

Aiyappan, Senthil Kumar; Karpagam, Bulabai; Vadanika, V; Chidambaram, Prem Kumar; Vinayagam, S; Saravanan, K C

2016-01-01

Antral Follicle count (AFC) is a reliable marker for ovarian reserve. Previous studies have used transvaginal ultrasound for estimation of AFC, however we used magnetic resonance imaging (MRI) for estimation of AFC and for creating an age-related normogram in patients with polycystic ovary syndrome (PCOS) and compared it with normal patients. The aim of this study is to create an age related normogram for AFC in women with PCOS and to compare that with women without polycystic ovarian syndrome using MRI. A total of 1500 women were examined, out of which 400 fitted the criteria for PCOS. They all underwent MRI study and similar age matched women without PCOS also underwent MRI examination. Normogram for AFC were obtained using LMS software and a percentile chart was obtained. Normogram for AFC in PCOS women showed decline in number of AFC as the age progresses and the decline was linear. The normogram for AFC was compared with equal number of patients without PCOS and they also showed decline in AFC as the age progresses, however the decline was exponential and faster. Age related normogram for AFC is widely used and considered as best clinical predictor for ovarian response in assisted reproductive technology. Knowledge of ovarian reserve is important in PCOS and non-PCOS females as PCOS patients are at risk for ovarian hyperstimulation syndrome during gonadotrophin theraphy. MRI is an equally effective and in some times better alternative to transvaginal ultrasound as it has got its own advantages.

Regulation of Injury-Induced Ovarian Regeneration by Activation of Oogonial Stem Cells.

PubMed

Erler, Piril; Sweeney, Alexandra; Monaghan, James R

2017-01-01

Some animals have the ability to generate large numbers of oocytes throughout life. This raises the question whether persistent adult germline stem cell populations drive continuous oogenesis and whether they are capable of mounting a regenerative response after injury. Here we demonstrate the presence of adult oogonial stem cells (OSCs) in the adult axolotl salamander ovary and show that ovarian injury induces OSC activation and functional regeneration of the ovaries to reproductive capability. Cells that have morphological similarities to germ cells were identified in the developing and adult ovaries via histological analysis. Genes involved in germ cell maintenance including Vasa, Oct4, Sox2, Nanog, Bmp15, Piwil1, Piwil2, Dazl, and Lhx8 were expressed in the presumptive OSCs. Colocalization of Vasa protein with H3 mitotic marker showed that both oogonial and spermatogonial adult stem cells were mitotically active. Providing evidence of stemness and viability of adult OSCs, enhanced green fluorescent protein (EGFP) adult OSCs grafted into white juvenile host gonads gave rise to EGFP OSCs, and oocytes. Last, the axolotl ovaries completely regenerated after partial ovariectomy injury. During regeneration, OSC activation resulted in rapid differentiation into new oocytes, which was demonstrated by Vasa + /BrdU + coexpression. Furthermore, follicle cell proliferation promoted follicle maturation during ovarian regeneration. Overall, these results show that adult oogenesis occurs via proliferation of endogenous OSCs in a tetrapod and mediates ovarian regeneration. This study lays the foundations to elucidate mechanisms of ovarian regeneration that will assist regenerative medicine in treating premature ovarian failure and reduced fertility. Stem Cells 2017;35:236-247. © 2016 AlphaMed Press.

Use of multiple imaging modalities to detect ovarian cancer

NASA Astrophysics Data System (ADS)

Kanter, Elizabeth; Walker, Ross; Marion, Sam; Hoyer, Patricia; Barton, Jennifer K.

2005-04-01

Ovarian cancer is not a common cancer-approximately 25,000 new cases in 2004-but it is the fifth leading cause of death from cancer in women (over 16,000 in 2004). Little is known about the precursors and early stages of ovarian cancer partially due to the lack of human samples at the early stages. A cohesive model that incorporates ovarian cancer induction into a menopausal rodent would be well suited for comprehensive studies of ovarian cancer. Non-destructive imaging would allow carcinogenesis to be followed. Optical Coherence Tomography (OCT), Optical Coherence Microscopy (OCM) and Light-Induced Fluorescence (LIF) are minimally invasive optical modalities that allow both structural and biochemical changes to be noted. Rat ovaries were exposed to 4-vinylcyclohexene diepoxide (VCD) for 20 days in order to destroy the primordial follicles. Plain sutures and sutures coated with 7,12-dimethylbenz(a)anthracene (DMBA) were implanted in the right ovary, in order to produce epithelial based ovarian cancers (a plain suture was inserted in the control). Rats were sacrificed at 4 weeks and ovaries were harvested and imaged with a combined OCT/LIF system and with the OCM. Histology was preformed on the harvested ovaries and any pathology determined. Two of the ovaries were visually abnormal; the OCT/LIF imaging confirmed these abnormalities. The normal ovary OCM and OCT images show the organized structure of the ovary, the follicles, bursa and corpus lutea are visible. The OCM images show the disorganized structure of one of the abnormal ovaries. Overall this pilot study demonstrated the feasibility of both the animal model and optical imaging.

The evaluation of xenotransplantation of feline ovarian tissue vitrified by needle immersed vitrification technique into male immunodeficient mice.

PubMed

Demirel, Mürşide Ayşe; Acar, Duygu Baki; Ekim, Burcu; Çelikkan, Ferda Topal; Alkan, Kübra Karakaş; Salar, Seçkin; Erdemli, Esra Atabenli; Özkavukçu, Sinan; Yar, Seda Sağlam; Kanca, Halit; Baştan, Ayhan

2018-03-01

In this study, the efficiency of the "Needle Immersed Vitrification" technique was tested on cryopreserved feline ovarian tissue. For vitrification, ovarian fragments (0.5-1.5 mm 2 ) from each ovary were collected; the grafts were exposed to 7.5-15% ethylene glycol and 7.5-15% dimethyl sulfoxide at room temperature and stored in liquid nitrogen at least 1 week. Morphologic examinations, expression of genes such as B cell lymphoma 2, B-cell lymphoma-2-associated X protein, Bone morphogenetic protein 15, zone of polarizing activity, zona pellucida C protein and DNA (cytosine-5)-methyltransferase 1, ultrastructural analysis and viability tests were carried out from collected grafts. Light microscopy examinations revealed the percentage of morphologically normal primordial follicles in a fresh group which was significantly higher than the treatment groups (p < 0.001). Terminal deoxynucleotidyl transferase dUTP nick end labeling and anti-caspase-3 staining observed in oocytes, follicle cells, interstitial tissue showed higher rates of apoptosis for post-vitrification and -transplantation groups than freshly grafted ovarian tissues. Furthermore, we observed significant downregulation of zone of polarizing activity and zona pellucida C protein gene expression in vitrified ovarian tissue grafts than in the fresh grafts (p < 0.05). In conclusion, we suggest that the needle immersed vitrification method is a convenient, cheap, and feasible vitrification method for cat ovarian tissues. However, further studies need to be performed to determine more optimal vitrification solutions and equilibration times for the needle immersed vitrification method in order to adapt it for cat ovaries.

Modifiers of ovarian function in girls and women with classic galactosemia.

PubMed

Spencer, Jessica B; Badik, Jennifer R; Ryan, Emily L; Gleason, Tyler J; Broadaway, K Alaine; Epstein, Michael P; Fridovich-Keil, Judith L

2013-07-01

Classic galactosemia is a potentially lethal genetic disorder resulting from profound impairment of galactose-1P uridylyltransferase (GALT). More than 80% of girls and women with classic galactosemia experience primary or premature ovarian insufficiency despite neonatal diagnosis and rigorous lifelong dietary galactose restriction. The goal of this study was to test the relationship between markers of ovarian reserve, cryptic residual GALT activity, and spontaneous pubertal development in girls with classic galactosemia. This was a cross-sectional study with some longitudinal follow-up in a university research environment. Patients included girls and women with classic galactosemia and unaffected controls, <1 month to 30 years old. We evaluated plasma anti-Müllerian hormone (AMH) and FSH levels, antral follicle counts ascertained by ultrasound, and ovarian function as indicated by spontaneous vs assisted menarche. More than 73% of the pre- and postpubertal girls and women with classic galactosemia in this study, ages >3 months to 30 years, demonstrated AMH levels below the 95% confidence interval for AMH among controls of the same age, and both pre- and postpubertal girls and women with classic galactosemia also demonstrated abnormally low antral follicle counts relative to age-matched controls. Predicted residual GALT activity ≥ 0.4% significantly increased the likelihood that a girl with classic galactosemia would demonstrate an AMH level ≥ 0.1 ng/mL. A majority of girls with classic galactosemia demonstrate evidence of diminished ovarian reserve by 3 months of age, and predicted cryptic residual GALT activity is a modifier of ovarian function in galactosemic girls and women.

Changes in numbers of large ovarian follicles, plasma luteinizing hormone and estradiol-17beta concentrations and egg production figures in farmed ostriches throughout the year.

PubMed

Bronneberg, R G G; Stegeman, J A; Vernooij, J C M; Dieleman, S J; Decuypere, E; Bruggeman, V; Taverne, M A M

2007-06-01

In this study we described and analysed changes in the numbers of large ovarian follicles (diameter 6.1-9.0 cm) and in the plasma concentrations of luteinizing hormone (LH) and estradiol-17beta (E(2)beta) in relation to individual egg production figures of farmed ostriches (Struthio camelus spp.) throughout one year. Ultrasound scanning and blood sampling for plasma hormone analysis were performed in 9 hens on a monthly basis during the breeding season and in two periods of the non-breeding season. Our data demonstrated that: (1) large follicles were detected and LH concentrations were elevated already 1 month before first ovipositions of the egg production season took place; (2) E(2)beta concentrations increased as soon as the egg production season started; (3) numbers of large follicles, LH and E(2)beta concentrations were elevated during the entire egg production season; and that (4) numbers of large follicles, LH and E(2)beta concentrations decreased simultaneous with or following the last ovipositions of the egg production season. By comparing these parameters during the egg production season with their pre-and post-seasonal values, significant differences were found in the numbers of large follicles and E(2)beta concentrations between the pre-seasonal, seasonal and post-seasonal period; while LH concentrations were significantly different between the seasonal and post-seasonal period. In conclusion, our data demonstrate that changes in numbers of large follicles and in concentrations of LH and E(2)beta closely parallel individual egg production figures and provide some new cues that egg production in ostriches is confined to a marked reproductive season. Moreover, our data provide indications that mechanism, initiating, maintaining and terminating the egg production season in farmed breeding ostriches are quite similar to those already known for other seasonal breeding bird species.

Luteinizing hormone signaling phosphorylates and activates the cyclic GMP phosphodiesterase PDE5 in mouse ovarian follicles, contributing an additional component to the hormonally induced decrease in cyclic GMP that reinitiates meiosis.

PubMed

Egbert, Jeremy R; Yee, Siu-Pok; Jaffe, Laurinda A

2018-03-01

Prior to birth, oocytes within mammalian ovarian follicles initiate meiosis, but then arrest in prophase until puberty, when with each reproductive cycle, one or more follicles are stimulated by luteinizing hormone (LH) to resume meiosis in preparation for fertilization. Within preovulatory follicles, granulosa cells produce high levels of cGMP, which diffuses into the oocyte to maintain meiotic arrest. LH signaling restarts meiosis by rapidly lowering the levels of cGMP in the follicle and oocyte. Part of this decrease is mediated by the dephosphorylation and inactivation the NPR2 guanylyl cyclase in response to LH, but the mechanism for the remainder of the cGMP decrease is unknown. At least one cGMP phosphodiesterase, PDE5, is activated by LH signaling, which would contribute to lowering cGMP. PDE5 exhibits increased cGMP-hydrolytic activity when phosphorylated on serine 92, and we recently demonstrated that LH signaling phosphorylates PDE5 on this serine and increases its activity in rat follicles. To test the extent to which this mechanism contributes to the cGMP decrease that restarts meiosis, we generated a mouse line in which serine 92 was mutated to alanine (Pde5-S92A), such that it cannot be phosphorylated. Here we show that PDE5 phosphorylation is required for the LH-induced increase in cGMP-hydrolytic activity, but that this increase has only a modest effect on the LH-induced cGMP decrease in mouse follicles, and does not affect the timing of meiotic resumption. Though we show that the activation of PDE5 is among the mechanisms contributing to the cGMP decrease, these results suggest that another cGMP phosphodiesterase is also activated by LH signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

Effects of season and ovarian status on the outcome of long-term progesterone-based estrus synchronization protocols and ovulatory follicle development in Santa Inês ewes under subtropical conditions.

PubMed

Oliveira, M E F; Ayres, H; Oliveira, L G; Barros, F F P C; Oba, E; Bicudo, S D; Bartlewski, P M; Fonseca, J F; Vicente, W R R

2016-02-01

This study set out to investigate the efficiency of long-term estrus synchronization protocols and ovulatory follicle dynamics in ultrasonographically monitored Santa Inês ewes during lengthening (LD; September-October) and shortening photoperiods (SD; April-May), and the transitional period (TP; January). In addition, the influence of ovarian status (e.g., size of antral follicles and/or presence of corpora lutea) at the outset of the estrus synchronization protocols on the ensuing development of ovulatory follicles was examined. Seventy sexually mature Santa Inês ewes were subjected to one of the two estrus synchronization regimens; on Day 0 (random day of the estrous cycle or anovulatory period), the ewes were fitted with an intravaginal progesterone (P4)-releasing (controlled intrauterine drug release [CIDR]) device, which was left in place for 14 days (G-1CIDR, n = 35) or replaced on Day 7 (G-2CIDR, n = 35), and received an intramuscular injection of 10 mg of PGF2α. The ewes allocated to the G-1CIDR group had mean serum P4 concentrations less than 2 ng/mL during the last 4 days of the synchronization protocol. There were no differences (P > 0.05) in mean ovulation rates between the two protocols tested nor among the ewes varying in ovarian status or studied at different times of the year, but ovulations occurred ∼ 12 hours later in the TP compared with the SD period (P < 0.05). Ovulatory follicles emerged earlier (P < 0.05) in the G-1CIDR group than in the G-2CIDR group (Day 8.3 ± 0.5 vs. 9.2 ± 0.4) and during LD (Day 7.1 ± 0.6) compared with the TP (Day 9.1 ± 0.6) and SD (Day 9.9 ± 0.5 of the protocol). In conclusion, the replacement of CIDR devices prevented the occurrence of lower-than-normal luteal phase levels of P4 at the end of the 14-day estrus synchronization protocol. However, although this procedure and seasonal influences altered certain growth characteristics of ovulatory follicles, there were no effects of these factors on the mean ovulation rate. Copyright © 2016 Elsevier Inc. All rights reserved.

Changes in keratin 8/18 expression in human granulosa cell lineage are associated to cell death/survival events: potential implications for the maintenance of the ovarian reserve.

PubMed

Gaytan, F; Morales, C; Roa, J; Tena-Sempere, M

2018-04-01

Is keratin 8/18 (K8/K18) expression linked to cell death/survival events in the human granulosa cell lineage? A close association exists between changes in K8/K18 expression and cell death/survival events along the human granulosa cell lineage lifespan. In addition to their structural and mechanical functions, K8/K18 play essential roles regulating cell death, survival and differentiation in several non-gonadal epithelial tissues. Transfection of the granulosa-like tumor KGN cells with siRNA to interfere KRT8 and KRT18 expression increases FAS-mediated apoptosis, while an inverse association between K8/K18 expression and cell death has been found in the bovine antral follicles and corpus luteum. Yet, only fragmentary and inconclusive information exists regarding K8/K18 expression in the human ovary. Expression of K8/K18 was assessed by immunohistochemistry at different stages of the granulosa cell lineage, from flattened granulosa cells in primordial follicles to fully luteinized granulosa-lutein cells in the corpus luteum (including corpus luteum of pregnancy). Immunohistochemical detection of K8/K18 was conducted in 40 archival ovarian samples from women aged 17-39 years. K8/K18 expression was analyzed at the different stages of follicle development and corpus luteum lifespan. The proportions of primordial follicles showing all K8/K18-positive, all K8/K18 negative, or a mixture of K8/K18 negative and positive granulosa cells were quantified in 18 ovaries, divided into three age groups: ≤ 25 years (N = 6), 26-30 (N = 6) and 31-36 (N = 6) years. A total number of 1793 primordial, 750 transitional and 140 primary follicles were scored. A close association was found between changes in K8/K18 expression and cell death/cell survival events in the human granulosa cell lineage. Large secondary and early antral follicles (most of them undergoing atresia) and regressing corpora lutea displayed low/absent K8/K18 expression. Conversely, early growing and some large antral follicles, functional menstrual corpora lutea, as well as life-extended corpus luteum of pregnancy, in which cell death was scarce, showed high K8/K18 expression. Three sub-populations of primordial follicles were observed with respect to the presence of K8/K18 in their flattened granulosa cells, ranging from primordial follicles showing only positive granulosa cells [P0(+)], to others with a mixture of positive and negative cells [P0(+/-)] or follicles with only negative cells [P0(-)]. Significant age-related changes were found in the proportions of the different primordial follicle types. In relation to age, a positive correlation was found for P0(+) primordial follicles (R2= 0.7883, N = 18; P < 0.001), while negative correlations were found for P0(+/-) (R2 = 0.6853, N = 18; P < 0.001) and P0(-) (R2 = 0.6725, N = 18; P < 0.001) follicles. Furthermore, an age-related shift towards greater keratin expression was found in P0(+/-) follicles (χ2 = 19.07, P < 0.05). N/A. This is a descriptive study. Hence, a cause-and-effect relationship between K8/K18 expression and cell death/survival cannot be directly established. This study describes, for the first time, the existence of sub-populations of primordial follicles on the basis of K8/K18 expression in granulosa cells, and that their proportions change with age. While a progressive increase in K8/K18 expression cannot be ruled out, our data are consistent with the hypothesis that primordial follicles expressing low levels of K8/K18 are preferentially ablated by follicle attrition, while primordial follicles showing high K8/K18 levels are those predominantly recruited into the growing pool. This suggests that K8/K18 expression could constitute a novel factor regulating primordial follicle death/survival, and raises the possibility that alterations of K8/K18 expression could be involved in the accelerated depletion of the ovarian reserve leading to premature ovarian insufficiency. This work was supported by Grants BFU2011-025021 and BFU2014-57581-P (Ministerio de Economía y Competitividad, Spain; co-funded with EU funds from FEDER Program); project PIE14-00005 (Flexi-Met, Instituto de Salud Carlos III, Ministerio de Sanidad, Spain); Projects P08-CVI-03788 and P12-FQM-01943 (Junta de Andalucía, Spain); and EU research contract DEER FP7-ENV-2007-1. CIBER Fisiopatología de la Obesidad y Nutrición is an initiative of Instituto de Salud Carlos III. The authors have nothing to disclose in relation to the contents of this study.

21 CFR 522.533 - Deslorelin acetate.

Code of Federal Regulations, 2010 CFR

2010-04-01

...—(i) Amount. One implant per mare. (ii) Indications for use. For inducing ovulation within 48 hours in estrous mares with an ovarian follicle greater than 30 millimiters in diameter. Follicular size should be...

Quantitative Proteomic Analysis of Duck Ovarian Follicles Infected with Duck Tembusu Virus by Label-Free LC-MS

PubMed Central

Han, Kaikai; Zhao, Dongmin; Liu, Yuzhuo; Liu, Qingtao; Huang, Xinmei; Yang, Jing; An, Fengjiao; Li, Yin

2016-01-01

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. DTMUV infection mainly results in significant decreases in egg production in egg-laying ducks within 1–2 weeks post infection. However, information on the comparative protein expression of host tissues in response to DTMUV infection is limited. In the present study, the cellular protein response to DTMUV infection in duck ovarian follicles was analyzed using nano-flow high-performance liquid chromatography-electrospray tandem mass spectrometry. Quantitative proteomic analysis revealed 131 differentially expressed proteins, among which 53 were up regulated and 78 were down regulated. The identified proteins were involved in the regulation of essential processes such as cellular structure and integrity, RNA processing, protein biosynthesis and modification, vesicle transport, signal transduction, and mitochondrial pathway. Some selected proteins that were found to be regulated in DTMUV-infected tissues were screened by quantitative real-time PCR to examine their regulation at the transcriptional level, western blot analysis was used to validate the changes of some selected proteins on translational level. To our knowledge, this study is the first to analyze the proteomic changes in duck ovarian follicles following DTMUV infection. The protein-related information obtained in this study may be useful to understand the host response to DTMUV infection and the inherent mechanism of DTMUV replication and pathogenicity. PMID:27066001

Small glutamine-rich tetratricopeptide repeat-containing protein alpha is present in human ovaries but may not be differentially expressed in relation to polycystic ovary syndrome.

PubMed

Butler, Miriam S; Yang, Xing; Ricciardelli, Carmela; Liang, Xiaoyan; Norman, Robert J; Tilley, Wayne D; Hickey, Theresa E

2013-06-01

To evaluate the expression and function of small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), an androgen receptor (AR) molecular chaperone, in human ovarian tissues. Examine the effect of SGTA on AR subcellular localization in granulosa tumor cells (KGN) and SGTA expression in ovarian tissues. University-based research laboratory. Archived tissues from premenopausal women and granulosa cells from infertile women receiving assisted reproduction. None. AR subcellular localization and SGTA protein or mRNA levels. SGTA and AR proteins were expressed in the cytoplasm of KGN cells and exposure to androgen stimulated AR nuclear localization. SGTA protein knockdown increased AR nuclear localization at low (0-0.1 nmol/L) but not high (1-10 nmol/L) concentrations of androgen hormone. In ovarian tissues, SGTA was localized to the cytoplasm of granulosa cells at all stages of folliculogenesis and in thecal cells of antral follicles. SGTA protein levels were similar when comparing primordial and primary follicles within core biopsies (n = 40) from women with and without polycystic ovary syndrome (PCOS). Likewise, SGTA mRNA levels were not significantly different in granulosa cells from preovulatory follicles after hyperstimulation of women with and without PCOS. SGTA is present in human ovaries and has the potential to modulate AR signalling, but it may not be differentially expressed in PCOS. Copyright © 2013 American Society for Reproductive Medicine. All rights reserved.

Isolation and expression of the human gametocyte-specific factor 1 gene (GTSF1) in fetal ovary, oocytes, and preimplantation embryos.

PubMed

Huntriss, John; Lu, Jianping; Hemmings, Karen; Bayne, Rosemary; Anderson, Richard; Rutherford, Anthony; Balen, Adam; Elder, Kay; Picton, Helen M

2017-01-01

Gametocyte-specific factor 1 has been shown in other species to be required for the silencing of retrotransposons via the Piwi-interacting RNA (piRNA) pathway. In this study, we aimed to isolate and assess expression of transcripts of the gametocyte-specific factor 1 (GTSF1) gene in the human female germline and in preimplantation embryos. Complementary DNA (cDNA) libraries from human fetal ovaries and testes, human oocytes and preimplantation embryos and ovarian follicles isolated from an adult ovarian cortex biopsy were used to as templates for PCR, cloning and sequencing, and real time PCR experiments of GTSF1 expression. GTSF1 cDNA clones that covered the entire coding region were isolated from human oocytes and preimplantation embryos. GTSF1 mRNA expression was detected in archived cDNAs from staged human ovarian follicles, germinal vesicle (GV) stage oocytes, metaphase II oocytes, and morula and blastocyst stage preimplantation embryos. Within the adult female germline, expression was highest in GV oocytes. GTSF1 mRNA expression was also assessed in human fetal ovary and was observed to increase during gestation, from 8 to 21 weeks, during which time oogonia enter meiosis and primordial follicle formation first occurs. In human fetal testis, GTSF1 expression also increased from 8 to 19 weeks. To our knowledge, this report is the first to describe the expression of the human GTSF1 gene in human gametes and preimplantation embryos.

Lunar synchronization of in vitro steroidogenesis in ovaries of the golden rabbitfish, Siganus guttatus (Bloch).

PubMed

Rahman, Md Saydur; Takemura, Akihiro; Takano, Kazunori

2002-01-01

To assess the relationship between lunar cycle and steroidogenesis in the ovaries of the golden rabbitfish, Siganus guttatus, the intact follicles of oocytes were incubated in vitro with human chorionic gonadotropin (hCG) and seven steroid hormones, 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), 17alpha,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), 17alpha-hydroxyprogesterone (17alpha-OHP), progesterone (P), cortisol, estradiol-17beta (E2) and testosterone, during the two lunar phases, the new moon (1 week before spawning) and the first lunar quarter (just before spawning). Around the new moon, germinal vesicle breakdown (GVBD) could not be induced by addition of hCG or any steroid hormones. Around the first lunar quarter, GVBD was induced by addition of hCG, DHP, 20beta-S, 17alpha-OHP, P, and cortisol. DHP was the most potent steroid hormone. When the intact follicles of oocytes were incubated with hCG in both lunar phases, the production of E2 and DHP measured by enzyme immunoassay decreased and increased significantly from the new moon to the first lunar quarter, respectively. These results suggest that the ovarian follicles produce E2 around the new moon and DHP around the first lunar quarter and that the production/conversion of the steroid hormones is under the influence of gonadotropin(s). The synchronous increase in ovarian activity supports the hypothesis that lunar periodicity is a major factor for the ovarian development of S. guttatus.

Cryopreservation of in vitro matured oocytes in addition to ovarian tissue freezing for fertility preservation in paediatric female cancer patients before and after cancer therapy.

PubMed

Abir, R; Ben-Aharon, I; Garor, R; Yaniv, I; Ash, S; Stemmer, S M; Ben-Haroush, A; Freud, E; Kravarusic, D; Sapir, O; Fisch, B

2016-04-01

Is a protocol that combines in vitro maturation of germinal vesicle-stage oocytes and their vitrification with freezing of cortical ovarian tissue feasible for use in fertility preservation for both chemotherapy-naive paediatric patients as well as patients after initiation of cancer therapy? Follicle-containing ovarian tissue as well as oocytes that can undergo maturation in vitro can be obtained from paediatric patients (including prepubertal girls) both before and after cancer therapy. Anticancer therapy reduces the number of follicles/oocytes but this effect is less severe in young patients, particularly the paediatric age group. Autotransplantation of ovarian tissue has yielded to date 60 live births, including one from tissue that was cryostored in adolescence. However, it is assumed that autografting cryopreserved-thawed ovarian cortical tissue poses a risk of reseeding the malignancy. Immature oocytes can be collected from very young girls without hormonal stimulation and then matured in vitro and vitrified. We have previously shown that there is no difference in the number of ovarian cortical follicles between paediatric patients before and after chemotherapy. A prospective study was conducted in a cohort of 42 paediatric females with cancer (before and after therapy initiation) who underwent fertility preservation procedures in 2007-2014 at a single tertiary medical centre. The study group included girls and adolescent females with cancer: 22 before and 20 after chemotherapy. Following partial or complete oophorectomy, immature oocytes were either aspirated manually ex vivo from visible small antral follicles or filtered from spent media. Oocytes were incubated in oocyte maturation medium, and those that matured at 24 or 48 h were vitrified. Ovarian cortical tissue was cut and prepared for slow-gradual cryopreservation. Anti-Mullerian hormone (AMH) levels were measured in serum before and after oophorectomy. Ovarian tissue was successfully collected from 78.7% of the 42 patients. Oocytes were obtained from 20 patients before chemotherapy and 13 after chemotherapy. The youngest patients from whom oocytes were retrieved were aged 2 years (two atretic follicles) and 3 years. Of the 395 oocytes collected, ∼30% were atretic (29.6% in the pre-chemotherapy group, 37% in the post-chemotherapy group). One hundred twenty-one oocytes (31%) were matured in vitro and vitrified: 67.8% from patients before chemotherapy, the rest after chemotherapy. Mature oocytes suitable for vitrification were obtained from 16/20 patients before chemotherapy and from 12/13 patients after chemotherapy (maturation rate, 32 and 26.4%, respectively). There were significant correlations of the number of vitrified oocytes with patient age (more matured oocytes with older age) (P = 0.001) and with pre-oophorectomy AMH levels (P = 0.038 pre-chemotherapy group, P = 0.029 post-chemotherapy group). Oocytes suitable for vitrification were obtained both by manual aspiration of antral follicles (45%) and from rinse solutions after dissection. There were significantly more matured oocytes in the pre-chemotherapy group from aspiration than in the post-chemotherapy group after both aspiration (P < 0.033) and retrieval from rinsing fluids (P < 0.044). The number of pre-antral follicles per histological section did not differ in the pre- versus post-chemotherapy. AMH levels dropped by approximately 50% after ovarian removal in both groups, with a significant correlation between pre- and post-oophorectomy levels (P = 0.002 pre-chemotherapy group, P = 0.001 post-chemotherapy group). There were no patients between 5 years and 10 years old in the post-chemotherapy group, which might have affected some results and correlations. Oocytes from patients soon after chemotherapy might be damaged, and caution is advised when using them for fertility-restoration purposes. The viability, development capability and fertilization potential of oocytes from paediatric patients, especially prepubertal and after chemotherapy, are unknown, in particular oocytes recovered from the media after the tissue dissection step. Although more oocytes were collected and matured from chemotherapy-naïve paediatric patients, ovarian tissue and immature oocytes were also retrieved from young girls in whom cancer therapy has already been initiated. Our centre has established a protocol for potential maximal fertility preservation in paediatric female patients with cancer. Vitrified-in vitro-matured oocytes may serve as an important gamete source in paediatric female patients with cancer because the risk of reseeding the disease is avoided. Further studies are needed on the fertility-restoring potential of oocytes from paediatric and prepubertal patients, especially after exposure to chemotherapy. The study was conducted as part of the routine procedures for fertility preservation at our IVF unit. No funding outside of the IVF laboratory was received. Funding for the AMH measurements was obtained by a research grant from the Israel Science Foundation (to B.-A.I., ISF 13-1873). None of the authors have competing interests. N/A. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Relationship between serum anti-Mullerian hormone and intrafollicular AMH levels in PCOS women.

PubMed

Stracquadanio, M; Ciotta, L; Palumbo, M A

2018-03-01

Polycystic ovary syndrome is a complex disease characterized by various endocrine disorders that are the potential cause of anovulation and hyperandrogenism. Anti-Müllerian hormone expression is suspected to be overexpressed in PCOS granulosa cells. AMH acts as a regulator of folliculogenesis: it is produced by the granulosa cells of follicles from the stage of the primary follicle to the initial formation of the antrum. Serum and intrafollicular AMH levels are elevated in patients with PCOS due to increased number of small follicles and an increased secretion within each of these small follicles. This excess of AMH is strongly suspected to play a role in the characteristic follicular arrest of PCOS, through a negative action on aromatase expression and on FSH action. Value above 5 ng/ml or 35 pmol/l might be considered as a diagnostic criterion for PCOS. The aim of our study is to demonstrate the presence of higher AMH serum levels and higher AMH intrafollicular fluid level of PCOS patients, undergone to IVF cycles, compared to normovulatory patients. The results clearly indicate that blood and intrafollicular AMH levels are significantly higher in PCOS women comparing to the normovulatory population. Serum AMH level appears to be a good predictive marker for the risk ovarian hyperstimulation syndrome: thus, its evaluation should be recommended before starting a controlled ovarian stimulation for IVF.

Influence of puberty and antral follicle count on calving day in crossbred beef heifers.

PubMed

McNeel, Anthony K; Cushman, Robert A

2015-10-15

Lifetime productivity and longevity are greater in beef cows that give birth early in their first calving season. The ability of heifers to conceive early in the breeding season is traditionally thought to be a function of pubertal status; however, recent evidence suggests that antral follicle count is positively associated with calving day in pubertal beef heifers. Additionally, there is evidence to suggest that the total number of ovarian follicles may alter response to hormonal synchronization protocols. The objectives of this study were to confirm the beneficial influence of antral follicle count on calving day in beef heifers and to determine whether response to PGF2α is associated with differences in antral follicle counts. A 2 × 2 factorial experiment was designed to investigate the interaction between calving period (early vs. late) and PGF2α (control vs. PGF) on calving day and antral follicle count in yearling beef heifers (n = 95). As intended, calving day was less in the early calving period compared to the late calving period (P < 0.01). There were no differences in calving day in response to treatment with PGF2α (P > 0.05). There was a significant interaction between calving period and PGF2α on age at first calving (P < 0.01), such that heifers treated with PGF2α that gave birth early were younger than heifers treated with saline that gave birth early. Calf weaning weights were greater in the early calving group than in the late calving group (P < 0.01). Heifers that gave birth in the early calving group possessed more antral follicles at prebreeding ultrasonographic examination than heifers that gave birth in the late calving group (P = 0.05). These findings confirm that antral follicle counts are associated with calving day in pubertal beef heifers. The use of antral follicle counts as a prebreeding phenotype provides additional utility to reproductive tract scoring for commercial production because of its association with calving day. As a prebreeding ovarian phenotype, antral follicle counts may hold additional applicability for organic beef producers looking to reduce the length of their calving season without increasing the total number of replacement heifers retained. Published by Elsevier Inc.

Ovarian injury during cryopreservation and transplantation in mice: a comparative study between cryoinjury and ischemic injury.

PubMed

Lee, Jaewang; Kong, Hyun Sun; Kim, Eun Jung; Youm, Hye Won; Lee, Jung Ryeol; Suh, Chang Suk; Kim, Seok Hyun

2016-08-01

What is the main cause of ovarian injury during cryopreservation and transplantation in mice: cryoinjury or ischemic injury? Post-transplantation ischemia is the main cause of ovarian injury during cryopreservation and transplantation for restoring ovarian function. During cryopreservation and the transplantation of ovaries, cryoinjury and ischemic injury inevitably occur, which has a detrimental effect on ovarian quality and reserve. A total of 80 B6D2F1 female mice were randomly allocated to 2 control and 6 experimental groups according to the presence or the absence of transplantation (n = 10/group). The control groups consisted of fresh or vitrified-warmed controls that had the whole ovary fixed without transplantation (fresh and vitri-con, respectively). The experimental groups were further divided according to the presence of vitrification (fresh or vitrified-warmed) and the transplantation period (2 [D2], 7 [D7] or 21 [D21] days). In the control groups, fresh and vitrified-warmed ovaries were immediately fixed after the collection (fresh) and the vitrification-warming process (vitrification control, vitri-con), respectively. Of those experimental groups, three were auto-transplanted with fresh whole ovary (FrOT; FrOT-D2, FrOT-D7 and FrOT-D21). For the other three groups, the ovaries were harvested and stored in liquid nitrogen for 1 week after vitrification and then warmed to auto-transplant the vitrified whole ovaries (vitrified ovary [VtOT]; VtOT-D2, VtOT-D7 and VtOT-D21). After 2, 7 or 21 days of grafting, the grafts and blood sera were collected for analysis by hematoxylin-eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, CD31 immunohistochemistry and follicle-stimulating hormone enzyme-linked immunosorbent assay. The vitrification-warming procedure decreased the proportion of intact follicles (Grade 1, G1) (vitri-con 50.3% versus fresh 64.2%) but there was a larger decrease due to ischemic injury after transplantation (FrOT-D2: 42.5%). The percentage of apoptotic follicles was significantly increased in the vitrified-warmed ovary group compared with the fresh control, but it increased more after transplantation without vitrification (fresh: 0.9%, vitri-con: 6.0% and FrOT-D2: 26.8%). The mean number of follicles per section and percentage of CD31-positive area significantly decreased after vitrification but decreased to a larger extent after transplantation (number of follicles, fresh: 30.3 ± 3.6, vitri-con: 20.6 ± 2.9, FrOT-D2: 17.9 ± 2.1; CD31-positive area, fresh: 10.6 ± 1.3%, vitri-con: 5.7 ± 0.9% and FrOT-D2: 4.2 ± 0.4%). Regarding the G1 follicle ratio and CD31-positive area per graft, only the FrOT groups significantly recovered with time after transplantation (G1 follicle ratio, FrOT-D2: 42.5%, FrOT-D7: 56.1% and FrOT-D21: 70.7%; CD31-positive area, FrOT-D2: 4.2 ± 0.4%, FrOT-D7: 5.4 ± 0.6% and FrOT-D21: 7.5 ± 0.8%). Although there was no significant difference between the two transplantation groups at each evaluation, the serum follicle-stimulating hormone level of both groups significantly decreased over time. It is unclear how far these results can be extrapolated from mice to the human ovary. Minimizing ischemic injury should be the first priority rather than preventing cryoinjury alone, and decreasing the combination of cryoinjury and ischemic injury is necessary to improve ovarian quality after cryopreservation and transplantation. This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C0055). The authors have no conflict of interest to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effects of excess metabolizable protein on ovarian function and circulating amino acids of beef cows: 2. Excessive supply in varying concentrations from corn gluten meal.

PubMed

Geppert, T C; Meyer, A M; Perry, G A; Gunn, P J

2017-04-01

In the dairy industry, excess dietary CP is consistently correlated with decreased conception rates. However, amount of excess CP effects on reproductive function in beef cattle is largely undefined. The objective of this experiment was to determine the effects of excess metabolizable protein (MP) supplementation from a moderately abundant rumen undegradable protein (RUP) source (corn gluten meal: 62% RUP) on ovarian function and circulating amino acid (AA) concentrations in beef cows consuming low quality forage. Non-pregnant, non-lactating beef cows (n=16) were allocated by age, BW and body condition score (BCS) to 1 of 2 isocaloric supplements designed to maintain BW for 60 days. Cows had ad libitum access to corn stalks and were individually offered a corn gluten meal-based supplement daily at 125% (MP125) or 150% (MP150) of National Research Council (NRC) MP requirements. After a 20-day supplement adaptation period, cows were synchronized for ovulation. After 10 days of synchronization, follicular growth was reset with gonadotropin releasing hormone. Daily thereafter, transrectal ultrasonography was performed to diagram ovarian follicular waves, and blood samples were collected for hormone, metabolite and AA analyses. After 7 days of observation of estrus, corpus luteum (CL) size was determined via ultrasound. Data were analyzed using the MIXED procedures of SAS. No differences (P⩾0.21) in BW and BCS existed throughout the study; however, plasma urea N at ovulation was greater (P=0.04) in MP150. Preovulatory ovarian follicle size at dominance, duration of dominance, size at spontaneous luteolysis, length of proestrus and wavelength were not different (P⩾0.11) between treatments. However, ovulatory follicles were larger (P=0.04) and average antral follicle count was greater (P=0.01) in MP150 than MP125. Estradiol concentration and ratio of estradiol to ovulatory follicle volume were not different due to treatment (P⩾0.25). While CL volume 7 days post-estrus was greater (P<0.01) in MP150 than MP125, circulating progesterone 7 days post-estrus and ratio of progesterone to CL volume were not different (P⩾0.21). Total AA were not different (P⩾0.76) at study initiation or completion; however, as a percent of total AA, branched-chain AA at ovulation were greater (P=0.02) in MP150. In conclusion, supplementation of CP at 150% of NRC MP requirements from a moderately undegradable protein source may enhance growth of the ovulatory follicle and subsequent CL compared with MP supplementation at 125% of NRC MP requirements.

Anti-Müllerian hormone is produced heterogeneously in primate preantral follicles and is a potential biomarker for follicle growth and oocyte maturation in vitro.

PubMed

Xu, Jing; Xu, Fuhua; Letaw, John H; Park, Byung S; Searles, Robert P; Ferguson, Betsy M

2016-12-01

The main goals of this study were to investigate the expression of anti-Müllerian hormone (AMH) and its receptor (AMHR2) during follicular development in primates, and to evaluate the potential of AMH as a biomarker for follicle growth and oocyte maturation in vitro. The mRNA and protein expression of AMH and AMHR2 were determined using isolated follicles and ovarian sections from rhesus macaques (n = 4) by real-time PCR and immunohistochemistry, respectively. Isolated secondary follicles were cultured individually. Follicle growth and media AMH concentrations were assessed by ELISA. The mRNA expression profiles, obtained from RNA sequencing, of in vitro- and in vivo-developed antral follicles were compared. Secondary follicles from additional animals (n = 35) were cultured. Follicle growth, oocyte maturation, and media AMH concentrations were evaluated for forecasting follicular development in vitro by AMH levels. AMH immunostaining was heterogeneous in the population of preantral follicles that were also stained for AMHR2. The mRNA expression profiles were comparable between in vivo- and in vitro-developed follicles. AMH levels produced by growing follicles were higher than those of nongrowing follicles in culture. With a cutoff value of 1.40 ng/ml, 85 % of nongrowing follicles could be identified while eliminating only 5 % of growing follicles. Growing follicles that generated metaphase II-stage oocytes secreted greater amounts of AMH than did those yielding immature germinal vesicle-stage oocytes. AMH, co-expressed with AMHR2, was produced heterogeneously by preantral follicles in macaques with levels correlated positively with follicle growth and oocyte maturation. AMH may serve as a biomarker for primate follicular development in vitro.

Encapsulated Three-Dimensional Culture Supports Development of Nonhuman Primate Secondary Follicles1

PubMed Central

Xu, Min; West-Farrell, Erin R.; Stouffer, Richard L.; Shea, Lonnie D.; Woodruff, Teresa K.; Zelinski, Mary B.

2009-01-01

In vitro ovarian follicle cultures may provide fertility-preserving options to women facing premature infertility due to cancer therapies. An encapsulated three-dimensional (3-D) culture system utilizing biomaterials to maintain cell-cell communication and support follicle development to produce a mature oocyte has been developed for the mouse. We tested whether this encapsulated 3-D system would also support development of nonhuman primate preantral follicles, for which in vitro growth has not been reported. Three questions were investigated: Does the cycle stage at which the follicles are isolated affect follicle development? Does the rigidity of the hydrogel influence follicle survival and growth? Do follicles require luteinizing hormone (LH), in addition to follicle-stimulating hormone (FSH), for steroidogenesis? Secondary follicles were isolated from adult rhesus monkeys, encapsulated within alginate hydrogels, and cultured individually for ≤30 days. Follicles isolated from the follicular phase of the menstrual cycle had a higher survival rate (P < 0.05) than those isolated from the luteal phase; however, this difference may also be attributed to differing sizes of follicles isolated during the different stages. Follicles survived and grew in two hydrogel conditions (0.5% and 0.25% alginate). Follicle diameters increased to a greater extent (P < 0.05) in the presence of FSH alone than in FSH plus LH. Regardless of gonadotropin treatment, follicles produced estradiol, androstenedione, and progesterone by 14–30 days in vitro. Thus, an alginate hydrogel maintains the 3-D structure of individual secondary macaque follicles, permits follicle growth, and supports steroidogenesis for ≤30 days in vitro. This study documents the first use of the alginate system to maintain primate tissue architecture, and findings suggest that encapsulated 3-D culture will be successful in supporting the in vitro development of human follicles. PMID:19474063

Polycystic ovarian disease: animal models.

PubMed

Mahajan, D K

1988-12-01

The reproductive systems of human beings and other vertebrates are grossly similar. In the ovary particularly, the biochemical and physiologic processes are identical not only in the formation of germ cells, the development of primordial follicles and their subsequent growth to Graafian follicles, and eventual ovulation but also in anatomic structure. In a noncarcinogenic human ovary, hypersecretion of androgen causes PCOD. Such hypersecretion may result from a nonpulsatile, constant elevated level of circulating LH or a disturbance in the action of neurotransmitters in the hypothalamus. In studying the pathophysiology of PCOD in humans, one must be aware of the limitations for manipulating the hypothalamic-pituitary axis. Although the rat is a polytocous rodent, the female has a regular ovarian cyclicity of 4 or 5 days, with distinct proestrus, estrus, and diestrus phases. Inasmuch as PCOD can be experimentally produced in the rat, that species is a good model for studying the pathophysiology of human PCOD. These PCOD models and their validity have been described: (1) estradiol-valerate, (2) DHA, (3) constant-light (LL), and (4) neonatally androgenized. Among these, the LL model is noninvasive and seems superior to the others for study of the pathophysiology of PCOD. The production of the polycystic ovarian condition in the rat by the injection of estrogens or androgens in neonate animals, or estradiol or DHA in adult rats, or the administration of antigonadotropins to these animals all cause a sudden appearance of the persistent estrus state by disturbing the metabolic and physiologic processes, whereas exposure of the adult rat to LL causes polycystic ovaries gradually, similar to what is seen in human idiopathic PCOD. After about 50 days of LL, the rat becomes anovulatory and the ovaries contain thickened tunica albuginea and many atretic follicles, and the tertiary follicles are considerably distended and cystic. The granulosa and theca cells appear normal histologically, although some of the stromal cells appear hypertrophic. The anatomic features consequent to polycystic ovaries resulting from LL are similar to those in human PCOD, and both rat and human PCOD ovarian cells still retain the ability to respond to FSH/LH, LHRH, and unilateral ovariectomy. In the estradiol valerate rat model, although the anatomy and physiology of the ovary resemble those of PCOD patients, the progressive degeneration of the hypothalamus and the altered response of the pituitary to LHRH make this model inappropriate for studying the hypothalamic-pituitary-ovarian axis in the polycystic ovary condition.(ABSTRACT TRUNCATED AT 400 WORDS)

Human fallopian tube epithelium co-culture with murine ovarian follicles reveals crosstalk in the reproductive cycle

PubMed Central

Zhu, Jie; Xu, Yuanming; Rashedi, Alexandra S.; Pavone, Mary Ellen; Kim, J. Julie; Woodruff, Teresa K.; Burdette, Joanna E.

2016-01-01

Study question Do interactions between human fallopian tube epithelium and murine follicles occur during an artificial reproductive cycle in a co-culture system in vitro? Summary answer In a co-culture system, human fallopian tissues responded to the menstrual cycle mimetic by changes in morphology and levels of secreted factors, and increasing murine corpus luteum progesterone secretion. What is known already The entire fallopian tube epithelium, including ciliated and secretory cells, can be regulated in the reproductive cycle. Currently, there are no in vitro culture models that can monitor fallopian tissues in real time in response to factors produced by the ovary. In addition, there are no reports on the impact of fallopian tissue on ovarian function during the menstrual cycle. Study design, samples/materials, methods Human fallopian tissue (n = 24) was obtained by routine hysterectomies from women (aged 26–50 years, mean age = 43.6) who had not undergone exogenous hormonal treatment for at least 3 months prior to surgery. CD1 female mice were used for ovarian follicle isolation. The human fallopian epithelium layers were either co-cultured with five murine multilayer secondary follicles (150–180 μm follicles, encapsulated in one alginate gel bead) for 15 days or received stepwise steroid hormone additions for 13 days. The fallopian tissue morphology and cilia beating rate, as measured by an Andor Spinning Disk Confocal, were investigated. Oviduct-specific glycoprotein 1 (OVGP1), human insulin-like growth factor 1 (hIGF1), vascular endothelial growth factor A (VEGF-A) and interleukin 8 (IL8) as biological functional markers were measured either by ELISA or western blot to indicate dynamic changes in the fallopian epithelium during the reproductive cycle generated by mouse follicles or by stepwise steroid hormone induction. Three or four patients in each experiment were recruited for replicates. Data were presented as mean ± SD and further analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. Main results and the role of chance The cultured fallopian tube epithelium responded to exogenous steroid hormone stimulation, as demonstrated by enhanced cilia beating rate (~25% increase, P = 0.04) and an increase in OVGP1 secretion (P = 0.02) in response to 1 nM estradiol (E2) treatment when compared with 0.1 nM E2. Conversely, 10 nM progesterone plus 1 nM E2 suppressed cilia beating rate by ~30% (P = 0.008), while OVGP1 secretion was suppressed by 0.1 nM E2 plus 50 nM progesterone (P = 0.002 versus 1 nM E2 alone). Human fallopian tube epithelium was co-cultured with murine secondary follicles to mimic the human menstrual cycle. OVGP1 and VEGF-A secretion from fallopian tissue was similar with stepwise hormone treatment and when cultured with murine follicles. However, the secretion patterns of hIGF1 and IL8 differed in the luteal phase when comparing steroid treatment with follicle co-culture. In co-culture, hIGF1 secretion was suppressed in the luteal versus follicular phase (P = 0.005) but stepwise hormone treatment had no effect on hIGF1. In co-culture, IL8 secretion was also suppressed on luteal phase day 15 (P = 0.013) versus follicular phase day 7, but IL8 secretion increased continuously under high E2/progesterone treatment (P = 0.003 for D13 versus D3). In the co-culture system, the corpus luteum continuously produced progesterone in the presence of fallopian tube tissue until Day 18 while, without fallopian tissue, progesterone started to drop from Day 13. Limitations, reasons for caution One limitation of this study is that murine follicles were used to mimic the human menstrual cycle. However, although secretion patterns of peptide hormones such as inhibins and activins differ in mice and humans, the co-culture system used here did reveal interactions between the tissues that govern reproductive function. Wider implications of the findings In vitro co-culture models of fallopian reproductive tissues with ovarian follicles can provide an important tool for understanding fertility and for uncovering the mechanisms responsible for reduced fertility. In addition, the role of oviductal secretions and how they influence ovarian function, such as the production of progesterone during the menstrual cycle, can be uncovered using this model. Large-scale data None. Study funding and competing interest(s) This work was funded by grants from the NIH (UH3TR001207), the American Cancer Society (RSG-12-230-01-TBG) and NIH (R01EB014806). The authors declare no competing financial interest. PMID:27542947

Human fallopian tube epithelium co-culture with murine ovarian follicles reveals crosstalk in the reproductive cycle.

PubMed

Zhu, Jie; Xu, Yuanming; Rashedi, Alexandra S; Pavone, Mary Ellen; Kim, J Julie; Woodruff, Teresa K; Burdette, Joanna E

2016-11-01

Do interactions between human fallopian tube epithelium and murine follicles occur during an artificial reproductive cycle in a co-culture system in vitro? In a co-culture system, human fallopian tissues responded to the menstrual cycle mimetic by changes in morphology and levels of secreted factors, and increasing murine corpus luteum progesterone secretion. The entire fallopian tube epithelium, including ciliated and secretory cells, can be regulated in the reproductive cycle. Currently, there are no in vitro culture models that can monitor fallopian tissues in real time in response to factors produced by the ovary. In addition, there are no reports on the impact of fallopian tissue on ovarian function during the menstrual cycle. Human fallopian tissue (n = 24) was obtained by routine hysterectomies from women (aged 26-50 years, mean age = 43.6) who had not undergone exogenous hormonal treatment for at least 3 months prior to surgery. CD1 female mice were used for ovarian follicle isolation. The human fallopian epithelium layers were either co-cultured with five murine multilayer secondary follicles (150-180 μm follicles, encapsulated in one alginate gel bead) for 15 days or received stepwise steroid hormone additions for 13 days. The fallopian tissue morphology and cilia beating rate, as measured by an Andor Spinning Disk Confocal, were investigated. Oviduct-specific glycoprotein 1 (OVGP1), human insulin-like growth factor 1 (hIGF1), vascular endothelial growth factor A (VEGF-A) and interleukin 8 (IL8) as biological functional markers were measured either by ELISA or western blot to indicate dynamic changes in the fallopian epithelium during the reproductive cycle generated by mouse follicles or by stepwise steroid hormone induction. Three or four patients in each experiment were recruited for replicates. Data were presented as mean ± SD and further analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. The cultured fallopian tube epithelium responded to exogenous steroid hormone stimulation, as demonstrated by enhanced cilia beating rate (~25% increase, P = 0.04) and an increase in OVGP1 secretion (P = 0.02) in response to 1 nM estradiol (E 2 ) treatment when compared with 0.1 nM E 2 . Conversely, 10 nM progesterone plus 1 nM E 2 suppressed cilia beating rate by ~30% (P = 0.008), while OVGP1 secretion was suppressed by 0.1 nM E 2 plus 50 nM progesterone (P = 0.002 versus 1 nM E 2 alone). Human fallopian tube epithelium was co-cultured with murine secondary follicles to mimic the human menstrual cycle. OVGP1 and VEGF-A secretion from fallopian tissue was similar with stepwise hormone treatment and when cultured with murine follicles. However, the secretion patterns of hIGF1 and IL8 differed in the luteal phase when comparing steroid treatment with follicle co-culture. In co-culture, hIGF1 secretion was suppressed in the luteal versus follicular phase (P = 0.005) but stepwise hormone treatment had no effect on hIGF1. In co-culture, IL8 secretion was also suppressed on luteal phase day 15 (P = 0.013) versus follicular phase day 7, but IL8 secretion increased continuously under high E 2 /progesterone treatment (P = 0.003 for D13 versus D3). In the co-culture system, the corpus luteum continuously produced progesterone in the presence of fallopian tube tissue until Day 18 while, without fallopian tissue, progesterone started to drop from Day 13. One limitation of this study is that murine follicles were used to mimic the human menstrual cycle. However, although secretion patterns of peptide hormones such as inhibins and activins differ in mice and humans, the co-culture system used here did reveal interactions between the tissues that govern reproductive function. In vitro co-culture models of fallopian reproductive tissues with ovarian follicles can provide an important tool for understanding fertility and for uncovering the mechanisms responsible for reduced fertility. In addition, the role of oviductal secretions and how they influence ovarian function, such as the production of progesterone during the menstrual cycle, can be uncovered using this model. None. This work was funded by grants from the NIH (UH3TR001207), the American Cancer Society (RSG-12-230-01-TBG) and NIH (R01EB014806). The authors declare no competing financial interest. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Amniotic fluid stem cells prevent follicle atresia and rescue fertility of mice with premature ovarian failure induced by chemotherapy.

PubMed

Xiao, Guan-Yu; Liu, I-Hsuan; Cheng, Chun-Chun; Chang, Chia-Chun; Lee, Yen-Hua; Cheng, Winston Teng-Kuei; Wu, Shinn-Chih

2014-01-01

Chemotherapy used to treat cancer may cause irreversible premature ovarian failure (POF). Of late, amniotic fluid stem cells (AFSCs) provide a novel source for regenerative medicine because of their primitive stage, low immunogenicity, and easy accessibility. In this study, we isolated AFSCs from transgenic mice that ubiquitously express enhanced green fluorescence protein (EGFP). These AFSCs exhibited morphologies, immunophenotypes, and mesoderm trilineage differentiation potentials similar to mesenchymal stem cells (MSCs). Further, AFSCs proliferated faster than MSCs and expressed OCT4, a marker for pluripotency. To investigate their potential in recovering fertility in POF model, AFSCs were transplanted into the ovaries of mice with POF six weeks post induction using chemotherapeutic drugs, busulfan and cyclophosphamide. AFSCs could rescue the reproductive ability of mice with POF by preventing follicle atresia and sustaining the healthy follicles. Notably, the transplanted AFSCs did not differentiate into granulosa and germline cells in vivo. After one month, the decreased numbers of transplanted AFSCs accompanied with the reduced beneficial effects indicated that the therapeutic efficacy were directly from AFSCs. These findings demonstrated the therapeutic effects of AFSCs and suggested the promise of AFSCs for treating infertility and POF caused by chemotherapy.

Reversible ovulatory failure associated with the development of luteinized unruptured follicles in women with inflammatory arthritis taking non-steroidal anti-inflammatory drugs.

PubMed

Smith, G; Roberts, R; Hall, C; Nuki, G

1996-05-01

The case histories of three young women with ankylosing spondylitis, rheumatoid arthritis and a seronegative inflammatory polyarthritis undergoing investigations for infertility are presented. In each, non-steroidal anti-inflammatory drug (NSAID) therapy was associated with the recurrent development of luteinized unruptured ovarian follicles and normal ovulation following drug withdrawal. It is suggested that NSAID therapy may be an important and frequently overlooked cause of anovulation and infertility.

Characterization of freshly retrieved preantral follicles using a low-invasive, mechanical isolation method extended to different ruminant species.

PubMed

Langbeen, A; Jorssen, E P A; Fransen, E; Rodriguez, A P A; García, M Chong; Leroy, J L M R; Bols, P E J

2015-10-01

Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 μm for primordial follicles, 47 ± 6.3 μm for primary follicles and 67.1 ± 13.1 μm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin-eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.

Quantifying growing versus non-growing ovarian follicles in the mouse.

PubMed

Uslu, Bahar; Dioguardi, Carola Conca; Haynes, Monique; Miao, De-Qiang; Kurus, Meltem; Hoffman, Gloria; Johnson, Joshua

2017-01-13

A standard histomorphometric approach has been used for nearly 40 years that identifies atretic (e.g., dying) follicles by counting the number of pyknotic granulosa cells (GC) in the largest follicle cross-section. This method holds that if one pyknotic granulosa nucleus is seen in the largest cross section of a primary follicle, or three pyknotic cells are found in a larger follicle, it should be categorized as atretic. Many studies have used these criteria to estimate the fraction of atretic follicles that result from genetic manipulation or environmental insult. During an analysis of follicle development in a mouse model of Fragile X premutation, we asked whether these 'historical' criteria could correctly identify follicles that were not growing (and could thus confirmed to be dying). Reasoning that the fraction of mitotic GC reveals whether the GC population was increasing at the time of sample fixation, we compared the number of pyknotic nuclei to the number of mitotic figures in follicles within a set of age-matched ovaries. We found that, by itself, pyknotic nuclei quantification resulted in high numbers of false positives (improperly categorized as atretic) and false negatives (improperly categorized intact). For preantral follicles, scoring mitotic and pyknotic GC nuclei allowed rapid, accurate identification of non-growing follicles with 98% accuracy. This method most often required the evaluation of one follicle section, and at most two serial follicle sections to correctly categorize follicle status. For antral follicles, we show that a rapid evaluation of follicle shape reveals which are intact and likely to survive to ovulation. Combined, these improved, non-arbitrary methods will greatly improve our ability to estimate the fractions of growing/intact and non-growing/atretic follicles in mouse ovaries.

Metabolic and reproductive status are not improved from 11 to 25 day post-partum in non-weaned primiparous rabbit does.

PubMed

Garcia-Garcia, R M; Sakr, O G; Arias-Alvarez, M; Velasco, B; Lorenzo, P L; Rebollar, P G

2012-03-01

The aim of present work was to analyze the body reserves and ovarian features of lactating primiparous rabbit does under extensive reproductive management (artificial insemination (AI) at 25 days post-partum (dpp)) compared with the common insemination rhythm at 11 dpp. A total of 48 primiparous Californian×New Zealand White rabbit does suckling 8 kits were used to assess liveweight, estimated body composition, serum metabolic and endocrine parameters (oestradiol and progesterone concentrations) and ovarian features like follicle population and atresia rate, and oocyte maturation. Rabbit does were randomly allocated in two experimental groups: (a) lactating does euthanized at early post-partum period (11 dpp) according to a semi-intensive rhythm (n=24), and (b) lactating does euthanized at later post-partum period (25 dpp) according to a more extensive rhythm (n=24). Liveweight, body energy content, lipid depots and serum non-esterified fatty acids (NEFA) concentrations decreased from parturition to post-partum period (P<0.05). In addition, serum protein and glucose concentrations increased in the post-partum period (P<0.05). Similar oestradiol and progesterone levels were found in rhythms as well as similar follicle population and nuclear and cytoplasmic maturation rates measured as metaphase II and cortical granule migration, respectively in both post-partum times. However, the number of preovulatory follicles on the ovarian surface was lower (P<0.05) and the atresia rate tended to be higher with a lower percentage of healthy follicles (P<0.1) in ovaries from females of extensive group. In conclusion, the body reserves, serum metabolic parameters and oocyte quality of primiparous non-weaned rabbits does at the late post-partum time (25 days) were not improved. Thus this reproductive management did not present any advantages compared to earlier post-partum (11 days) reproductive rhythm. Copyright © 2012 Elsevier B.V. All rights reserved.

Antral follicle count predicts natural menopause in a population-based sample: the Coronary Artery Risk Development in Young Adults Women's Study.

PubMed

Wellons, Melissa F; Bates, Gordon Wright; Schreiner, Pamela J; Siscovick, David S; Sternfeld, Barbara; Lewis, Cora E

2013-08-01

The timing of menopause is associated with multiple chronic diseases. Tools that predict this milestone have relevance for clinical and research purposes. Among infertile women, a positive relationship exists between antral follicle count (AFC) and response to controlled ovarian hyperstimulation, a marker of ovarian reserve. However, an age-independent relationship between AFC and menopause has not been demonstrated. Thus, our objective was to evaluate the relationship between AFC measured in women aged 34 to 49 years and incident natural menopause during 7 years of follow-up. The Coronary Artery Risk Development in Young Adults Study is a longitudinal community-based study (Chicago, IL; Birmingham, AL; Minneapolis, MN; and Oakland, CA) begun in 1985-1986. In 2002-2003, the Coronary Artery Risk Development in Young Adults Women's Study measured follicle-stimulating hormone (FSH) levels and performed a transvaginal ultrasound protocol that included AFC (2-10 mm follicles on both ovaries). Incident natural menopause was assessed by surveys in 2005-2006 and 2009-2010. In our sample (n = 456), the median (interquartile range) AFC and FSH level were 5 (2-9) and 7.8 (5.6-11.0) mIU/mL, respectively, at a mean (range) age of 42 (34-49) years in 2002-2003. One hundred one women reported natural menopause by 2009-2010. In Cox models, current smoking, stable menses, FSH level higher than 13 mIU/mL, and AFC of 4 or less were independently associated with incident natural menopause. Compared with AFC higher than 4, those with an AFC of 4 or less were nearly twice as likely to have undergone menopause during 7 years of follow-up (hazard ratio, 1.89; 95% CI, 1.19-3.02) after adjustment for covariates. AFC has been found to be independently associated with natural menopause during 7 years of follow-up after controlling for other markers of ovarian aging.

Polycystic ovarian disease: US features in 104 patients.

PubMed

Yeh, H C; Futterweit, W; Thornton, J C

1987-04-01

Ultrasonographic (US) study was performed in 25 healthy women and 104 patients with polycystic ovarian disease (PCOD). Although the average size of ovaries in the PCOD patients was much larger than that of the healthy women, 29.7% of ovaries in the PCOD patients were normal in size. The shapes of the ovaries (roundness index) in PCOD patients were not different from those of the healthy women. There was no significant correlation between the size and shape of the ovaries. Bilaterally enlarged, globular-shaped ovaries were rare and usually asymmetric in size. The most important feature of PCOD on US scans is the bilaterally increased numbers of developing follicles (0.5-0.8 cm in size), usually more than five in each ovary. Although maturing follicles (1.5-2.9 cm) are much rarer in PCOD patients (13.5%) than in healthy women (36%), the incidences of follicular cysts (greater than 3 cm) was about the same in both.

The reproductive endocrinology of the menopausal transition.

PubMed

Butler, Laura; Santoro, Nanette

2011-06-01

The menopause transition is a dynamic process that begins with the first appearance of menstrual irregularity and ends with a woman's final menstrual period. As ovarian follicle numbers dwindle, the hypothalamic-pituitary-ovarian axis enters a state of compensated failure. In this state, elevated FSH is capable of maintaining relatively regular folliculogenesis and ovulation, but fertility is reduced. Eventually, this state of compensated failure cannot be sustained, and the ovary becomes unable to produce functioning follicles. Recent multicenter studies from several countries have addressed the pattern of change in hormones and a model form reproductive aging has been developed that helps explain the changes in hormone patterns and fertility that accompany menopause. Perhaps more important, the hormonal changes of the menopausal transition may be predictive of future disease risk. This review will undertake an explanation of the current literature on this topic. Copyright © 2011 Elsevier Inc. All rights reserved.

Melatonin in human preovulatory follicular fluid

NASA Technical Reports Server (NTRS)

Brzezinski, Amnon; Seibel, Machelle M.; Lynch, Harry J.; Deng, Mei-Hua; Wurtman, Richard J.

1987-01-01

Melatonin, the major hormone of the pineal gland, has antigonadotrophic activity in many mammals and may also be involved in human reproduction. Melatonin suppresses steroidogenesis by ovarian granulosa and luteal cells in vitro. To determine if melatonin is present in the human ovary, preovulatory follicular fluids (n = 32) from 15 women were assayed for melatonin by RIA after solvent extraction. The fluids were obtained by laparoscopy or sonographically controlled follicular puncture from infertile women undergoing in vitro fertilization and embryo transfer. All patients had received clomiphene citrate, human menopausal gonadotropin, and hCG to stimulate follicle formation. Blood samples were obtained by venipuncture 30 rain or less after follicular aspiration. All of the follicular fluids contained melatonim, in concentrations substantially higher than those in the corresponding serum. A positive correlation was found between follicular fluid and serum melatonin levels in each woman; these observations indicate that preovulatory follicles contain substantial amounts of melatonin that may affect ovarian steroidogenesis.

[Total oxidative status of peritoneal fluid in women with endometriosis].

PubMed

Polak, Grzegorz; Kotarski, Jan

2010-12-01

Pathophysiology of endometriosis remains enigmatic despite extensive investigations. Accumulating data suggest that oxidative stress in the peritoneal cavity may be implicated in the pathogenesis of endometriosis. The aim of our study was to evaluate the oxidative status of peritoneal fluid (PF) in women with and without endometriosis. Sixty-five women participated in the study 40 women with endometriosis constituted the study group and 25 patients with functional follicle ovarian cysts comprised the reference group. Total oxidative status of PF was determined using a commercially available colorimetric assay kit (Immundiagnostic AG, Cat. nr. KC5100). Women with endometriosis had significantly higher PF oxidative status compared to women with follicle ovarian cysts. No significant difference in the peritoneal oxidative status was found between patients with stage I/II endometriosis, and women with stage III/IV endometriotic disease. Disrupted oxidative status in the peritoneal cavity of women with endometriosis plays a role in the pathogenesis of the disease.

Behavioral indicators to detect ovarian phase in the dromedary she-camel.

PubMed

Padalino, B; Rateb, S A; Ibrahim, N B; Monaco, D; Lacalandra, G M; El-Bahrawy, K A

2016-06-01

This pilot study was conducted to test the hypothesis that female camels behave differently in various ovarian phases in the presence of a restrained male camel. The aim was to identify behavioral patterns which could be used as indicators to detect ovulatory phase by visual observation in the presence of a restrained virile bull. Twenty-four healthy, nonpregnant, and nonlactating adult females were used. Transrectal ultrasonography was performed for each animal once a week over a 3-week period to determine the phase of the ovarian cycle. Females were considered to be in the ovulatory phase (O) when there was at least one preovulatory follicle (12<Ø<19 mm) protruding from the ovarian surface, and in the nonovulatory phase (NO), when growing follicles, regressing follicles, or corpora lutea were detected. Immediately after examination, each female was freely exposed to a restrained bull for 15 minutes, and her behaviors were filmed. The videos were analyzed through a focal animal-sampling ethogram (states: looking at the male; looking outside; standing close to the male; searching; and lying down; events: interaction with the male; urination; defecation; sound emission; and steps). A score for tail position (tail score: 1 = close to the vulva, 2 = horizontal, 3 = vertical) and for interest in the bull (male time score: from 1 to 5; 1 = <20% of observation period spent near the bull; 5 = more than 80%) were recorded. Ovulatory phase camels showed higher interest in the male than nonovulatory phases: they stood close to the male for longer periods (P = 0.0159), interacted with the male more frequently (P = 0.0004), and tended to lie down in front of him (P = 0.1202). Moreover, ovulatory phase had a significant effect on male time score (P < 0.01), mature follicular ovarian phase being associated with higher scores. Seeking the male has already been proposed as a behavioral indicator of estrus in camels, this has now been confirmed using a standardized ethogram. The present results clarify that camels behave differently in different ovarian phases and that monitoring their behavior in the presence of a restrained bull could help detect their ovulatory phase. This would have profound implications for enhancing fertility in dromedary camels by improving timing of mating or artificial insemination. Copyright © 2016 Elsevier Inc. All rights reserved.

The Hippo Signaling Pathway Regulates Ovarian Function via the Proliferation of Ovarian Germline Stem Cells.

PubMed

Ye, Haifeng; Li, Xiaoyan; Zheng, Tuochen; Hu, Chuan; Pan, Zezheng; Huang, Jian; Li, Jia; Li, Wei; Zheng, Yuehui

2017-01-01

To improve the separation, identification and cultivation of ovarian germline stem cells (OGSCs), to clarify the relationship between the Hippo signaling pathway effector YAP1 and the proliferation and differentiation of OGSCs in vitro and to identify the major contribution of Hippo signaling to ovarian function. Two-step enzymatic separation processes and magnetic separation were used to isolate and identify OGSCs by determining the expression of Mvh, Oct4, Nanog, Fragilis and Stella markers. Then, YAP1, as the main effector molecule in the Hippo signaling pathway, was chosen as the target gene of the study. Lentivirus containing overexpressed YAP1 or a YAP1-targeted shRNA was transduced into OGSCs. The effects of modulating the Hippo signaling pathway on the proliferation, differentiation, reproduction and endocrine function of ovaries were observed by microinjecting the lentiviral vectors with overexpressed YAP1 or YAP1 shRNA into infertile mouse models or natural mice of reproductive age. (1) The specific expression of Mvh, Oct4, Nanog, Fragilis and Stella markers was observed in isolated stem cells. Thus, the isolated cells were preliminarily identified as OGSCs. (2) The co-expression of LATS2, MST1, YAP1 and MVH was observed in isolated OGSCs. Mvh and Oct4 expression levels were significantly increased in OGSCs overexpressing YAP1 compared to GFP controls. Consistently, Mvh and Oct4 levels were significantly decreased in cells expressing YAP1-targeted shRNA. (3) After 14-75 days of YAP1 overexpression in infertile mouse models, we detected follicle regeneration in ovaries, the activation of primordial follicles and increased birth rate, accompanied by increasing levels of E2 and FSH. (4) However, we detected decreasing follicles in ovaries, lower birth rate, and decreasing E2 and FSH in serum from healthy mice of reproductive age following YAP1 shRNA expression. Methods for the isolation, identification and culture of OGSCs were successfully established. Further results indicate that isolated OGSCs can specifically recognize Hippo signaling molecules and that manipulation of YAP1 expression can be used to regulate the proliferation and differentiation of OGSCs, as well as ovarian function in mice. This study suggests that the Hippo signaling pathway may represent a new molecular target for the regulation of mouse ovarian functional remodeling. © 2017 The Author(s)Published by S. Karger AG, Basel.

The effects of whole ovarian perfusion and cryopreservation on endothelial cell-related gene expression in the ovarian medulla and pedicle.

PubMed

Onions, V J; Webb, R; Pincott-Allen, C; Picton, H M; Campbell, B K

2013-04-01

Fertility preservation by whole ovarian cryopreservation requires successful cryopreservation of both the ovary and its vascular supply. Previous work has indicated detrimental effects of both perfusion and cryopreservation on the ovarian vasculature. This study assessed the effects of blood perfusion, alone or in combination with cryopreservation, on functional effects in the follicle population and ovarian function in vivo following short-term autotransplantation of the tissue after vascular reanastomosis and measured acute changes in endothelial cell-related gene expression within the ovarian medulla and pedicle. Following autotransplantation for 7 days, primordial, transitional and primary follicle densities were significantly reduced (P < 0.05) and stromal Ki67 and caspase-3 expression significantly increased (P < 0.05) in cryopreserved but not fresh or perfused whole ovaries. There was evidence of clot formation and fluorescent microsphere (FMS) extravasation in the medulla of all cryopreserved ovaries, indicating vascular damage. Utilizing a customized RT-PCR array or conventional RT-PCR, we found that perfusion alone resulted in down-regulation in the expression of caspase 6 and thrombospondin 1 (THBS1) genes in the medulla. Following additional cryopreservation, endothelial nitric oxide synthase (eNOS), endothelin 1, endothelin receptor A and Bcl-2 expression were significantly (P < 0.05) down-regulated. In the pedicle, both perfusion and cryopreservation caused a (P < 0.05) down-regulation of eNOS and THBS1, and an up-regulation in Bax expression. Perfusion also caused a down-regulation of TNF and up-regulation of endothelin-2 expression (P < 0.05). In conclusion, this study has identified a number of endothelial cell-related genes expressed in the medulla which are acutely affected by both cryopreservation and perfusion, supporting the hypothesis that both interventions have deleterious effects on endothelial cell function.

Modifiers of Ovarian Function in Girls and Women With Classic Galactosemia

PubMed Central

Spencer, Jessica B.; Badik, Jennifer R.; Ryan, Emily L.; Gleason, Tyler J.; Broadaway, K. Alaine; Epstein, Michael P.

2013-01-01

Context: Classic galactosemia is a potentially lethal genetic disorder resulting from profound impairment of galactose-1P uridylyltransferase (GALT). More than 80% of girls and women with classic galactosemia experience primary or premature ovarian insufficiency despite neonatal diagnosis and rigorous lifelong dietary galactose restriction. Objective: The goal of this study was to test the relationship between markers of ovarian reserve, cryptic residual GALT activity, and spontaneous pubertal development in girls with classic galactosemia. Design and Setting: This was a cross-sectional study with some longitudinal follow-up in a university research environment. Patients: Patients included girls and women with classic galactosemia and unaffected controls, <1 month to 30 years old. Main Outcome Measures: We evaluated plasma anti-Müllerian hormone (AMH) and FSH levels, antral follicle counts ascertained by ultrasound, and ovarian function as indicated by spontaneous vs assisted menarche. Results: More than 73% of the pre- and postpubertal girls and women with classic galactosemia in this study, ages >3 months to 30 years, demonstrated AMH levels below the 95% confidence interval for AMH among controls of the same age, and both pre- and postpubertal girls and women with classic galactosemia also demonstrated abnormally low antral follicle counts relative to age-matched controls. Predicted residual GALT activity ≥0.4% significantly increased the likelihood that a girl with classic galactosemia would demonstrate an AMH level ≥0.1 ng/mL. Conclusions: A majority of girls with classic galactosemia demonstrate evidence of diminished ovarian reserve by 3 months of age, and predicted cryptic residual GALT activity is a modifier of ovarian function in galactosemic girls and women. PMID:23690308

The mare: a 1000-pound guinea pig for study of the ovulatory follicular wave in women.

PubMed

Ginther, O J

2012-03-15

The mare is a good comparative model for study of ovarian follicles in women, owing to striking similarities in follicular waves and the mechanism for selection of a dominant follicle. Commonality in follicle dynamics between mares and women include: (1) a ratio of 2.2:1 (mare:woman) in diameter of the largest follicle at wave emergence when the wave-stimulating FSH surge reaches maximum, in diameter increase of the two largest follicles between emergence and the beginning of deviation between the future dominant and subordinate follicles, in diameter of each of the two largest follicles at the beginning of deviation, and in maximum diameter of the preovulatory follicle; (2) emergence of the future ovulatory follicle before the largest subordinate follicle; (3) a mean interval of 1 day between emergence of individual follicles of the wave; (4) percentage increase in diameter of follicles for the 3 days before deviation; (5) deviation 3 or 4 days after emergence; (6) 25% incidence of a major anovulatory follicular wave emerging before the ovulatory wave; (7) 40% incidence of a predeviation follicle preceding the ovulatory wave; (8) small but significant increase in estradiol and LH before deviation; (9) cooperative roles of FSH and insulin-like growth factor 1 and its proteases in the deviation process; (10) age-related effects on the follicles and oocytes; (11) approximate 37-hour interval between administration of hCG and ovulation; and (12) similar gray-scale and color-Doppler ultrasound changes in the preovulatory follicle. In conclusion, the mare may be the premier nonprimate model for study of follicle dynamics in women. Copyright © 2012 Elsevier Inc. All rights reserved.

Regulation of cell death and cell survival gene expression during ovarian follicular development and atresia.

PubMed

Jiang, Jin-Yi; Cheung, Carmen K M; Wang, Yifang; Tsang, Benjamin K

2003-01-01

Mammalian ovarian follicular development and atresia is closely regulated by the cross talk of cell death and cell survival signals, which include endocrine hormones (gonadotropins) and intra-ovarian regulators (gonadal steroids, cytokines and growth factors). The fate of the follicle is dependent on a delicate balance in the expression and actions of factors promoting follicular cell proliferation, growth and differentiation and of those inducing programmed cell death (apoptosis). As an important endocrine hormone, FSH binds to its granulosa cell receptors and promotes ovarian follicle survival and growth not only by stimulating proliferation and estradiol secretion of these cells, but also inhibiting the apoptosis by up-regulating the expression of intracellular anti-apoptotic proteins, such as XIAP and FLIP. In addition, intra-ovarian regulators, such as TGF-alpha and TNF-alpha, also play an important role in the control of follicular development and atresia. In response to FSH, Estradiol-17 beta synthesized from the granulosa cells stimulates thecal expression of TGF-alpha, which in turn increases granulosa cell XIAP expression and proliferation. The death receptor and ligand, Fas and Fas ligand, are expressed in granulosa cells following gonadotropin withdrawal, culminating in caspase-mediated apoptosis and follicular atresia. In contrast, TNF-alpha has both survival and pro-apoptotic function in the follicle, depending on the receptor subtype activated, but has been shown to promote granulosa cell survival by increasing XIAP and FLIP expression via the IkappaB-NFkappaB pathway. The pro-apoptotic action of TNF-alpha is mediated through the activation of caspases, via its receptor- (i.e. Caspases-8 and -3) and mitochrondria- (i.e. Caspase-9 and -3) death pathways. In the present manuscript, we have reviewed the actions and interactions of gonadotropins and intra-ovarian regulators in the control of granulosa cell fate and ultimately follicular destiny. We have highlighted the role and regulation of granulosa cell XIAP and FLIP expression, as well as their interactions with the death signaling pathways in the maintenance of granulosa cell survival during follicular development. We have provided strong evidence for these intracellular survival factors as key determinants for ovarian follicular destiny (growth versus atresia), the expression of which is regulated by a highly integrated endocrine, paracrine and autocrine mechanism. Further studies in these aspects will lead to a better understanding of the molecular and cellular regulation of follicular development and atresia, and provide invaluable insight into novel strategies in assisted reproduction in human infertility as well as in increasing reproductive efficiency in livestock industries.

Clinically applied procedures for human ovarian tissue cryopreservation result in different levels of efficacy and efficiency.

PubMed

Bastings, Lobke; Westphal, Johan R; Beerendonk, Catharina C M; Bekkers, Ruud L M; Zusterzeel, Petra L M; Hendriks, Jan C M; Braat, Didi D M; Peek, Ronald

2016-12-01

Different protocols are being used worldwide for the cryopreservation of human ovarian tissue for fertility preservation purposes. The efficiency and efficacy of the majority of these protocols has not been extensively evaluated, possibly resulting in sub-optimally cryopreserved ovarian tissue. To address the impact of this issue, we assessed the effects of two clinically successful human ovarian tissue slow-freezing cryopreservation procedures on the quality of the cryopreserved tissue. To differentiate between cryopreservation ( C ) versus thawing ( T ) related effects, four combinations of these two (A and B) very different cryopreservation/thawing protocols (A C A T , A C B T , B C A T , B C B T ) were studied. Before and after cryopreservation and thawing, the percentage of living and morphologically normal follicles, as well as the overall tissue viability, was assessed. Our experiments revealed that the choice of the cryopreservation protocol noticeably affected the overall tissue viability and percentage of living follicles, with a higher viability after protocol B C when compared to A C . No statistically significant differences in tissue viability were observed between the two thawing protocols, but thawing protocol B T required considerably more human effort and materials than thawing protocol A T . Tissue morphology was best retained using the B C A T combination. Our results indicate that extensive and systematical evaluation of clinically used protocols is warranted.