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Sample records for rat ovarian follicles

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

    PubMed Central

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

    2016-01-01

    Introduction 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. Methods 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. Results 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). Conclusion 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

  2. Follicle Development of Xenotransplanted Sheep Ovarian Tissue into Male and Female Immunodeficient Rats

    PubMed Central

    Tahaei, Leila Sadat; Eimani, Hussein; Hajmusa, Ghazaleh; Fathi, Rouhollah; Rezazadeh Valojerdi, Mojtaba; Shahverdi, Abdolhossein; Eftekhari-Yazdi, Poopak

    2015-01-01

    Background This study aimed to assess follicle survival after xenotransplantation of sheep ovarian tissue into male and female immunodeficient rats. We evaluated the effects of gonadotropin treatment on follicular development in the transplanted tissue. Materials and Methods In this experimental study, sheep ovarian cortical strips were transplanted into the neck back muscles of 8 male and 8 female immunodeficient, castrated rats. Fourteen days after surgery, each rat was treated with human menopausal gonadotropin (hMG) for 9 weeks. One day after the last injection, ovarian tissues were removed and fixed for histology assessment. Histology analyses were performed before and after grafting. Estradiol (E2) levels were measured before and after gonadectomy, and at the end of the experiment. The control group consisted of 7 male and 7 female noncastrated/non-grafted rats and the sham group comprised 7 male and 7 female castrated/ non-grafted rats for comparison of serum E2 concentrations. Results The percentage of primordial follicles decreased after transplantation in male (25.97%) and female (24.14%) rats compared to the control group (ovarian tissue nongrafted; 37.51%). Preantral follicles increased in the male (19.5%) and female (19.49%) transplanted rats compared to the control group (11.4%). Differences in antral follicles between male (0.06 ± 0.0%) and female (0.06 ± 0.0%) rats were not noticeable compared to control (1.25 ± 0.0%) rats. We observed a significantly higher percent of mean E2 secretion in grafted males compared to grafted females (P˂0.05). Conclusion Despite significant differences in E2 secretion between xenografted male and female rats, we observed no statistical differences in terms of follicular development. PMID:26644859

  3. Follicle Development of Xenotransplanted Sheep Ovarian Tissue into Male and Female Immunodeficient Rats.

    PubMed

    Tahaei, Leila Sadat; Eimani, Hussein; Hajmusa, Ghazaleh; Fathi, Rouhollah; Rezazadeh Valojerdi, Mojtaba; Shahverdi, Abdolhossein; Eftekhari-Yazdi, Poopak

    2015-01-01

    This study aimed to assess follicle survival after xenotransplantation of sheep ovarian tissue into male and female immunodeficient rats. We evaluated the effects of gonadotropin treatment on follicular development in the transplanted tissue. In this experimental study, sheep ovarian cortical strips were transplanted into the neck back muscles of 8 male and 8 female immunodeficient, castrated rats. Fourteen days after surgery, each rat was treated with human menopausal gonadotropin (hMG) for 9 weeks. One day after the last injection, ovarian tissues were removed and fixed for histology assessment. Histology analyses were performed before and after grafting. Estradiol (E2) levels were measured before and after gonadectomy, and at the end of the experiment. The control group consisted of 7 male and 7 female noncastrated/non-grafted rats and the sham group comprised 7 male and 7 female castrated/ non-grafted rats for comparison of serum E2 concentrations. The percentage of primordial follicles decreased after transplantation in male (25.97%) and female (24.14%) rats compared to the control group (ovarian tissue nongrafted; 37.51%). Preantral follicles increased in the male (19.5%) and female (19.49%) transplanted rats compared to the control group (11.4%). Differences in antral follicles between male (0.06 ± 0.0%) and female (0.06 ± 0.0%) rats were not noticeable compared to control (1.25 ± 0.0%) rats. We observed a significantly higher percent of mean E2 secretion in grafted males compared to grafted females (P˂0.05). Despite significant differences in E2 secretion between xenografted male and female rats, we observed no statistical differences in terms of follicular development.

  4. Xenobiotic Effects on Ovarian Preantral Follicles1

    PubMed Central

    Mark-Kappeler, Connie J.; Hoyer, Patricia B.; Devine, Patrick J.

    2011-01-01

    Women are born with a finite population of ovarian follicles, which are slowly depleted during their reproductive years until reproductive failure (menopause) occurs. The rate of loss of primordial follicles is determined by genetic and environmental influences, but certain toxic exposures can accelerate this process. Ionizing radiation reduces preantral follicle numbers in rodents and humans in a dose-dependent manner. Cigarette smoking is linked to menopause occurring 1–4 yr earlier than with nonsmokers, and components of smoke, polycyclic aromatic hydrocarbons, can cause follicle depletion in rodents or in ovaries in vitro. Chemotherapeutic agents, such as alkylating drugs and cisplatin, also cause loss of preantral ovarian follicles. Effects depend on dose, type, and reactivity of the drug, and the age of the individual. Evidence suggests DNA damage may underlie follicle loss induced by one common alkylating drug, cyclophosphamide. Occupational exposures have also been linked to ovarian damage. In an industrial setting, 2-bromopropane caused infertility in men and women, and it can induce ovarian follicle depletion in rats. Solvents, such as butadiene, 4-vinylcyclohexene, and their diepoxides, can also cause specific preantral follicle depletion. The mechanism(s) underlying effects of the latter compound may involve alterations in apoptosis, survival factors such as KIT/Kit Ligand, and/or the cellular signaling that maintains primordial follicle dormancy. Estrogenic endocrine disruptors may alter follicle formation/development and impair fertility or normal development of offspring. Thus, specific exposures are known or suspected of detrimentally impacting preantral ovarian follicles, leading to early ovarian failure. PMID:21697514

  5. Soy isoflavones administered to rats from weaning until sexual maturity affect ovarian follicle development by inducing apoptosis.

    PubMed

    Wang, Wenxiang; Sun, Yan; Liu, Jin; Li, Yuchen; Li, Hong; Xiao, Shihua; Weng, Shaozheng; Zhang, Wenchang

    2014-10-01

    Twenty-one-day-old female Wistar rats were treated daily with orally administered soy isoflavones (SIFs) at concentrations of 50, 100, or 200 mg/kg body weight from weaning until sexual maturity (3 mo.), and ovarian follicle development was evaluated. At the end of the treatment period, the ultrastructure of the ovarian granulosa cells was examined by transmission electron microscopy. The apoptotic cell death of ovarian granulosa cells was detected using TUNEL staining. The mRNA expression levels of caspase-3, caspase-8, caspase-9, Bcl2, Bax, and Fas were determined by real-time quantitative PCR. The protein expression levels of caspase-3, Bcl2, Bax, and Fas were determined by western blotting. Our data showed that exposure to SIFs resulted in morphological changes consistent with ovarian granulosa cell apoptosis. The percentage of TUNEL-positive granulosa cells was increased. The mRNA expression levels of the apoptosis-related genes caspase-3, caspase-8, caspase-9, Bax, and Fas increased significantly. The protein levels of Bax, Fas, and cleaved caspase-3 were also increased. These results indicate that the exposure of rats to modest doses of SIFs from weaning until sexual maturity can affect ovarian follicle development by inducing apoptosis. The mechanism of SIF-induced alterations in ovarian follicle development may involve the activation of Fas-mediated and Bcl2/Bax-mediated apoptotic signaling pathways. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Chocolate cysts from ovarian follicles.

    PubMed

    Jain, S; Dalton, M E

    1999-11-01

    To study the development of chocolate cysts by serial transvaginal ultrasonographic tracking of ovarian follicles. Retrospective study. Department of Obstetrics and Gynaecology, Sunderland Royal Hospital, Sunderland, United Kingdom. We reviewed case notes of all patients who underwent laparoscopy for diathermy to endometriosis/ovarian diathermy/aspiration of ovarian cysts from 1989 to 1998. Twelve women with histories of infertility and proven chocolate cysts with documented ultrasonographic findings were included in the study. Serial ultrasonographic tracking of ovarian follicles in the cycle leading to the development of the chocolate cysts, followed by ultrasonographic tracking of cysts for 3 months and laparoscopy. Development and formation of chocolate cysts. The diagnosis of chocolate cysts was confirmed laparoscopically in all patients and histopathologically in four. Ultrasound confirmed that they had all developed from follicles. Chocolate cysts can develop from ovarian follicles.

  7. 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

  8. The effects of microwave emitted by cellular phones on ovarian follicles in rats.

    PubMed

    Gul, Abdulaziz; Celebi, Hacer; Uğraş, Serdar

    2009-11-01

    The aim of this study was to investigate whether there were any toxic effects of microwaves of cellular phones on ovaries in rats. In this study, 82 female pups of rats, aged 21 days (43 in the study group and 39 in the control group) were used. Pregnant rats in the study group were exposed to mobile phones that were placed beneath the polypropylene cages during the whole period of pregnancy. The cage was free from all kinds of materials, which could affect electromagnetic fields. A mobile phone in a standby position for 11 h and 45 min was turned on to speech position for 15 min every 12 h and the battery was charged continuously. On the 21st day after the delivery, the female rat pups were killed and the right ovaries were removed. The volumes of the ovaries were measured and the number of follicles in every tenth section was counted. The analysis revealed that in the study group, the number of follicles was lower than that in the control group. The decreased number of follicles in pups exposed to mobile phone microwaves suggest that intrauterine exposure has toxic effects on ovaries. We suggest that the microwaves of mobile phones might decrease the number of follicles in rats by several known and, no doubt, countless unknown mechanisms.

  9. Effects of ionizing radiation and pretreatment with (D-Leu6,des-Gly10) luteinizing hormone-releasing hormone ethylamide on developing rat ovarian follicles

    SciTech Connect

    Jarrell, J.; YoungLai, E.V.; McMahon, A.; Barr, R.; O'Connell, G.; Belbeck, L.

    1987-10-01

    To assess the effects of a gonadotropin-releasing hormone agonist, (D-Leu6,des-Gly10) luteinizing hormone-releasing hormone ethylamide, in ameliorating the damage caused by ionizing radiation, gonadotropin-releasing hormone agonist was administered to rats from day 22 to 37 of age in doses of 0.1, 0.4, and 1.0 microgram/day or vehicle and the rats were sacrificed on day 44 of age. There were no effects on estradiol, progesterone, luteinizing, or follicle-stimulating hormone, nor an effect on ovarian follicle numbers or development. In separate experiments, rats treated with gonadotropin-releasing hormone agonist in doses of 0.04, 0.1, 0.4, or 1.0 microgram/day were either irradiated or sham irradiated on day 30 and all groups sacrificed on day 44 of age. Irradiation produced a reduction in ovarian weight and an increase in ovarian follicular atresia. Pretreatment with the agonist prevented the reduction in ovarian weight and numbers of primordial and preantral follicles but not healthy or atretic antral follicles. Such putative radioprotection should be tested on actual reproductive performance.

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

    SciTech Connect

    Myllymaeki, Sari . E-mail: saanmy@utu.fi; Haavisto, Tapio; Vainio, Minna; Toppari, Jorma; Paranko, Jorma

    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 have 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.

  11. Gene bionetworks that regulate ovarian primordial follicle assembly.

    PubMed

    Nilsson, Eric; Zhang, Bin; Skinner, Michael K

    2013-07-23

    Primordial follicle assembly is the process by which ovarian primordial follicles are formed. During follicle assembly oocyte nests break down and a layer of pre-granulosa cells surrounds individual oocytes to form primordial follicles. The pool of primordial follicles formed is the source of oocytes for ovulation during a female's reproductive life. The current study utilized a systems approach to detect all genes that are differentially expressed in response to seven different growth factor and hormone treatments known to influence (increase or decrease) primordial follicle assembly in a neonatal rat ovary culture system. One novel factor, basic fibroblast growth factor (FGF2), was experimentally determined to inhibit follicle assembly. The different growth factor and hormone treatments were all found to affect similar physiological pathways, but each treatment affected a unique set of differentially expressed genes (signature gene set). A gene bionetwork analysis identified gene modules of coordinately expressed interconnected genes and it was found that different gene modules appear to accomplish distinct tasks during primordial follicle assembly. Predictions of physiological pathways important to follicle assembly were validated using ovary culture experiments in which ERK1/2 (MAPK1) activity was increased. A number of the highly interconnected genes in these gene networks have previously been linked to primary ovarian insufficiency (POI) and polycystic ovarian disease syndrome (PCOS). Observations have identified novel factors and gene networks that regulate primordial follicle assembly. This systems biology approach has helped elucidate the molecular control of primordial follicle assembly and provided potential therapeutic targets for the treatment of ovarian disease.

  12. Gene bionetworks that regulate ovarian primordial follicle assembly

    PubMed Central

    2013-01-01

    Background Primordial follicle assembly is the process by which ovarian primordial follicles are formed. During follicle assembly oocyte nests break down and a layer of pre-granulosa cells surrounds individual oocytes to form primordial follicles. The pool of primordial follicles formed is the source of oocytes for ovulation during a female’s reproductive life. Results The current study utilized a systems approach to detect all genes that are differentially expressed in response to seven different growth factor and hormone treatments known to influence (increase or decrease) primordial follicle assembly in a neonatal rat ovary culture system. One novel factor, basic fibroblast growth factor (FGF2), was experimentally determined to inhibit follicle assembly. The different growth factor and hormone treatments were all found to affect similar physiological pathways, but each treatment affected a unique set of differentially expressed genes (signature gene set). A gene bionetwork analysis identified gene modules of coordinately expressed interconnected genes and it was found that different gene modules appear to accomplish distinct tasks during primordial follicle assembly. Predictions of physiological pathways important to follicle assembly were validated using ovary culture experiments in which ERK1/2 (MAPK1) activity was increased. Conclusions A number of the highly interconnected genes in these gene networks have previously been linked to primary ovarian insufficiency (POI) and polycystic ovarian disease syndrome (PCOS). Observations have identified novel factors and gene networks that regulate primordial follicle assembly. This systems biology approach has helped elucidate the molecular control of primordial follicle assembly and provided potential therapeutic targets for the treatment of ovarian disease. PMID:23875758

  13. Cigarette smoke impaired maturation of ovarian follicles and normal growth of uterus inner wall of female wild-type and hypertensive rats.

    PubMed

    Lee, Hae-Miru; Kim, Cho-Won; Hwang, Kyung-A; Sung, Jae-Hyuck; Lee, Jin-Kyu; Choi, Kyung-Chul

    2017-07-06

    Cigarette smoke (CS) is well known to be very harmful to human body functions such as fertility, reproduction, and development. CS is considered to more affect patients with hypertension (HT). To estimate the effect of CS associated with female rat's fertility, we examined the histopathological characteristics of the uterus and ovary which were obtained from the female rats exposed to smoke of the standard cigarette (3R4F) for 4 weeks (10h a week) according to the OECD guidelines. The female wild-type Wistar Kyoto (WK) rats (WTR) and spontaneously hypertensive WK rats (SHR) were used to compare the effect of CS on healthy and hypertensive rats. After CS exposure, we manufactured tissue slides from uterine and ovarian samples and evaluated the maturation of follicles of ovary and cell proliferation in the uterus by H&E staining and immunohistochemistry (IHC). In IHC analysis on ovarian tissues, the expression of proliferating cell nuclear antigen (PCNA) and the number of follicles were decreased by CS exposure. On the contrary, PCNA expression and cell proliferation in the uterine inner layers were increased by CS exposure. The protein expression of C/EBP homologous protein (CHOP), an endoplasmic reticulum (ER)-stress marker, and BAX, a pro-apoptotic protein, was decreased by CS exposure. This phenomenon was more exacerbated in SHR rats than in WTR rats. Taken together, acute exposure to CS induced the decreased maturation of ovarian follicles and abnormal over-growth of uterine inner wall, leading to a harmful effect on female rat's normal function. In addition, this harmful effect of CS may be displayed more seriously in rats with HT. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Differences between rats and mice in the involvement of the aryl hydrocarbon receptor in 4-vinylcyclohexene diepoxide-induced ovarian follicle loss

    SciTech Connect

    Thompson, Kary E.; Bourguet, Shannon M.; Christian, Patricia J.; Benedict, Jamie C.; Sipes, I. Glenn; Flaws, Jodi A.; Hoyer, Patricia B. . E-mail: hoyer@u.arizona.edu

    2005-03-01

    Repeated dosing with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively depletes small pre-antral follicles in the ovaries of rats and mice via apoptosis. The aryl hydrocarbon receptor (AhR) plays a role in mediating the effects of several xenobiotics. Therefore, this study was designed to investigate a potential role of the AhR in VCD-induced ovotoxicity. Female F344 rats, C57BL/6 mice, or AhR-deficient (-/-, AhRKO) mice were dosed daily (15 days) with vehicle, VCD (80 mg/kg, i.p.) and/or the AhR antagonist, alpha-naphthoflavone (ANF; 80 mg/kg, i.p.). Compared with controls, VCD caused a 60% reduction (P < 0.05) in primordial and primary follicles in mice and rats. Concurrent dosing with ANF protected against the VCD-induced follicle loss in rats, but not in mice. As with AhR-intact mice and rats, VCD induced a 70% loss (P < 0.05) of small pre-antral follicles in AhRKO mice. AhR mRNA expression was increased (P < 0.05) by VCD dosing in small pre-antral follicles isolated from ovaries of rats but not mice. AhR protein in rats was increased by VCD dosing in oocyte nuclei in primordial and primary follicles when measured by immunofluorescence and confocal microscopy. In rat small pre-antral follicles, apoptosis-associated caspase-3-like activity was increased (P < 0.05) by VCD treatment, decreased (P < 0.05) by ANF treatment, and unaffected by VCD plus ANF treatment. VCD had no effect on expression of GST Ya1 or GST Ya2 mRNA or CYP 1A1 protein in rats. Taken together, these findings demonstrate a difference between rats and mice in the potential involvement of AhR as related to VCD-induced ovotoxicity. Whereas, AhR appears to be involved in rats, no evidence for a similar role in mice was obtained. Overall, these findings point out that there can be mechanistic species differences in ovarian responses to xenobiotic chemicals.

  15. Control of ovarian primordial follicle activation

    PubMed Central

    2012-01-01

    The ovarian follicles develop initially from primordial follicles. The majority of ovarian primordial follicles are maintained quiescently as a reserve for the reproductive life span. Only a few of them are activated and develop to an advanced follicular stage. The maintenance of dormancy and activation of primordial follicles are controlled by coordinated actions of a suppressor/activator with close communications with somatic cells and intra-oocyte signaling pathways. Many growth factors and signaling pathways have been identified and the transforming growth factor-beta superfamily plays important roles in early folliculogenesis. However, the mechanism of maintaining the dormancy and survival of primordial follicles has remained unknown for decades. Recently, since the first finding that all primordial follicles are activated prematurely in mice deficient forkhead box O3a, phosphatidylinositol 3 kinase/phosphatase and tensin homolog (PTEN) signaling pathway was reported to be important in the regulation of dormancy and initial follicular activation. With these informations on early folliculogenesis, clinical application can be expected such as in vitro maturation of immature oocytes or in vitro activation of follicles by PTEN inhibitor in cryopreserved ovarian cortical tissues for fertility preservation. PMID:22563545

  16. Gene Bionetwork Analysis of Ovarian Primordial Follicle Development

    PubMed Central

    Nilsson, Eric E.; Savenkova, Marina I.; Schindler, Ryan; Zhang, Bin; Schadt, Eric E.; Skinner, Michael K.

    2010-01-01

    Ovarian primordial follicles are critical for female reproduction and comprise a finite pool of gametes arrested in development. A systems biology approach was used to identify regulatory gene networks essential for primordial follicle development. Transcriptional responses to eight different growth factors known to influence primordial follicles were used to construct a bionetwork of regulatory genes involved in rat primordial follicle development. Over 1,500 genes were found to be regulated by the various growth factors and a network analysis identified critical gene modules involved in a number of signaling pathways and cellular processes. A set of 55 genes was identified as potential critical regulators of these gene modules, and a sub-network associated with development was determined. Within the network two previously identified regulatory genes were confirmed (i.e., Pdgfa and Fgfr2) and a new factor was identified, connective tissue growth factor (CTGF). CTGF was tested in ovarian organ cultures and found to stimulate primordial follicle development. Therefore, the relevant gene network associated with primordial follicle development was validated and the critical genes and pathways involved in this process were identified. This is one of the first applications of network analysis to a normal developmental process. These observations provide insights into potential therapeutic targets for preventing ovarian disease and promoting female reproduction. PMID:20661288

  17. Ovarian follicle vascularization in fasted pig.

    PubMed

    Barboni, Barbara; Barbara, Barboni; Martelli, Alessandra; Alessandra, Martelli; Berardinelli, Paolo; Paolo, Berardinelli; Russo, Valentina; Valentina, Russo; Turriani, Maura; Maura, Turriani; Bernabò, Nicola; Nicola, Bernabò; Lucidi, Pia; Pia, Lucidi; Mattioli, Mauro; Mauro, Mattioli

    2004-09-01

    The authors have investigated in the different classes of ovarian follicles the vascular area, the blood vessel distribution, the vascular endothelial growth factor (VEGF) mRNA expression and the VEGF secretion during equine chorionic gonadotropin (eCG) induced follicle growth in prepubertal gilts fed ad libitum or fasted. Immunohistochemistry staining of Von Willebrand factor showed that fasting caused a dramatic increase in the vascular area of medium-large tertiary follicles. The increase involved the two concentric vessel networks and the area between them that, becoming crossed by several anastomosis, modified the whole vessel architecture. Both in situ hybridization and in vitro culture experiments demonstrate that granulosa cells from medium-large follicles are engaged in a copious VEGF production upon eCG stimulation both in gilts fed ad libitum or fasted. More surprisingly, the production of VEGF becomes diffuse amongst theca cells of fasted animals thus recruiting a compartment that in condition of normal feeding regimen appears nearly quiescent. In conclusion, the data presented describe a local angiogenic process that develops in the follicle wall of growing antral follicle in case of acute severe food restriction. The mechanism, essentially confined to follicles that potentially approach ovulation, appears to assume the meaning of a local compensatory mechanism that may help maintaining adequate nutrient delivery to follicles that undergo ovulation.

  18. The effects of electromagnetic fields on the number of ovarian primordial follicles: An experimental study.

    PubMed

    Bakacak, Murat; Bostancı, Mehmet Sühha; Attar, Rukset; Yıldırım, Özge Kizilkale; Yıldırım, Gazi; Bakacak, Zeyneb; Sayar, Hamide; Han, Agahan

    2015-06-01

    The aim of this study was to evaluate the effect of an electromagnetic field (EMF), generated close to the ovaries, on primordial follicles. A total of 16 rats were used in this study. The study group consisted of rats exposed to an EMF in the abdominal region for 15 min/d for 15 days. Both the study and control group were composed of eight rats. After the treatment period of 15 days, the ovaries of the rats were extracted, and sections of ovarian tissue were taken for histological evaluation. The independent samples t test was used to compare the two groups. In the study group, the means of the right and left ovarian follicle numbers were 34.00 ± 10.20 and 36.00 ± 10.53, respectively. The average total ovarian follicle number was 70.00 ± 19.03. In the control group, the means of the right and left ovarian follicle numbers were 78.50 ± 25.98 and 71.75 ± 29.66, respectively, and the average total ovarian follicle number was 150.25 ± 49.53. The comparisons of the means of the right and left ovarian follicle numbers and the means of the total ovarian follicle numbers between the study and control groups indicated that the study group had significantly fewer follicles (p < 0.001, p = 0.011, and p = 0.002, respectively). This study found a significant decrease in the number of ovarian follicles in rats exposed to an EMF. Further clinical studies are needed to reveal the effects of EMFs on ovarian reserve and infertility. Copyright © 2015. Published by Elsevier Taiwan.

  19. Automated follicle analysis in ovarian ultrasound

    NASA Astrophysics Data System (ADS)

    Krivanek, Anthony; Liang, Weidong; Sarty, Gordon E.; Pierson, Roger A.; Sonka, Milan

    1998-06-01

    For women undergoing assisted reproductive therapy, ovarian ultrasound has become an invaluable tool for monitoring the growth and assessing the physiological status of individual follicles. Measurements of the size and shape of follicles are the primary means of evaluation by physicians. Currently, follicle wall segmentation is achieved by manual tracing which is time consuming and susceptible to inter- operator variation. We are introducing a completely automated method of follicle wall isolation which provides faster, more consistent analysis. Our automated method is a 4-step process which employs watershed segmentation and a knowledge-based graph search algorithm which utilizes a priori information about follicle structure for inner and outer wall detection. The automated technique was tested on 36 ultrasonographic images of woman's ovaries. Five images from this set were omitted due to poor image quality. Validation of the remaining 31 ultrasound images against manually traced borders has shown an average rms error of 0.61 +/- 0.40 mm for inner border and 0.61 +/- 0.31 mm for outer border detection. Quantitative comparison of the computer-defined borders and the user-defined borders advocates the accuracy of our automated method of follicle analysis.

  20. On the granulosa cells of ovarian follicles. II. Identification of different morphological patterns of granulosa cells in evolutive follicles.

    PubMed

    Zecchi, S; Repice, F; Balboni, G C

    1981-03-15

    An attempt has been made for identifying different types of granulosa cells in the wall of cavitary ovarian follicles. Human, porcine and rat ovaries have been examined at the light and electron microscopes. Some smears of granulosa cells as well as human foetal ovaries have been also studied. These preliminary results seem to confirm that in the granulosa layer of evolutive follicles the cells may present some different morphological and histochemical features.

  1. Effects of aqueous extract from Asparagus officinalis L. roots on hypothalamic-pituitary-gonadal axis hormone levels and the number of ovarian follicles in adult rats

    PubMed Central

    Karimi Jashni, Hojatollah; Kargar Jahromi, Hossein; Ghorbani Ranjbary, Ali; Kargar Jahromi, Zahra; Khabbaz Kherameh, Zahra

    2016-01-01

    Background: Asparagus is a plant with high nutritional, pharmaceutical, and industrial values. Objective: The present study aimed to evaluate the effect of aqueous extract of asparagus roots on the hypothalamic-pituitary-gonadal axis hormones and oogenesis in female rats. Materials and Methods: In this experimental study, 40 adult female Wistar rats were divided into five groups, which consist 8 rats. Groups included control, sham and three experimental groups receiving different doses (100, 200, 400 mg/kg/bw) of aqueous extract of asparagus roots. All dosages were administered orally for 28 days. Blood samples were taken from rats to evaluate serum levels of Gonadotropin releasing hormone (GnRH), follicular stimulating hormone (FSH), Luteinal hormone (LH), estrogen, and progesterone hormones. The ovaries were removed, weighted, sectioned, and studied by light microscope. Results: Dose-dependent aqueous extract of asparagus roots significantly increased serum levels of GnRH, FSH, LH, estrogen, and progestin hormones compared to control and sham groups. Increase in number of ovarian follicles and corpus luteum in groups treated with asparagus root extract was also observed (p<0.05). Conclusion: Asparagus roots extract stimulates secretion of hypothalamic- pituitary- gonadal axis hormones. This also positively affects oogenesis in female rats. PMID:27200420

  2. Inhibitory Actions of Anti-Müllerian Hormone (AMH) on Ovarian Primordial Follicle Assembly

    PubMed Central

    Nilsson, Eric E.; Schindler, Ryan; Savenkova, Marina I.; Skinner, Michael K.

    2011-01-01

    The current study was designed to investigate the actions of Anti-Müllerian Hormone (AMH) on primordial follicle assembly. Ovarian primordial follicles develop from the breakdown of oocyte nests during fetal development for the human and immediately after birth in rodents. AMH was found to inhibit primordial follicle assembly and decrease the initial primordial follicle pool size in a rat ovarian organ culture. The AMH expression was found to be primarily in the stromal tissue of the ovaries at this period of development, suggesting a stromal-epithelial cell interaction for primordial follicle assembly. AMH was found to promote alterations in the ovarian transcriptome during primordial follicle assembly with over 200 genes with altered expression. A gene network was identified suggesting a potential central role for the Fgf2/Nudt6 antisense transcript in the follicle assembly process. A number of signal transduction pathways are regulated by AMH actions on the ovarian transcriptome, in particular the transforming growth factor – beta (TGFß) signaling process. AMH is the first hormone/protein shown to have an inhibitory action on primordial follicle assembly. Due to the critical role of the primordial follicle pool size for female reproduction, elucidation of factors, such as AMH, that regulate the assembly process will provide insights into potential therapeutics to manipulate the pool size and female reproduction. PMID:21637711

  3. Inhibitory actions of Anti-Müllerian Hormone (AMH) on ovarian primordial follicle assembly.

    PubMed

    Nilsson, Eric E; Schindler, Ryan; Savenkova, Marina I; Skinner, Michael K

    2011-01-01

    The current study was designed to investigate the actions of Anti-Müllerian Hormone (AMH) on primordial follicle assembly. Ovarian primordial follicles develop from the breakdown of oocyte nests during fetal development for the human and immediately after birth in rodents. AMH was found to inhibit primordial follicle assembly and decrease the initial primordial follicle pool size in a rat ovarian organ culture. The AMH expression was found to be primarily in the stromal tissue of the ovaries at this period of development, suggesting a stromal-epithelial cell interaction for primordial follicle assembly. AMH was found to promote alterations in the ovarian transcriptome during primordial follicle assembly with over 200 genes with altered expression. A gene network was identified suggesting a potential central role for the Fgf2/Nudt6 antisense transcript in the follicle assembly process. A number of signal transduction pathways are regulated by AMH actions on the ovarian transcriptome, in particular the transforming growth factor-beta (TGFß) signaling process. AMH is the first hormone/protein shown to have an inhibitory action on primordial follicle assembly. Due to the critical role of the primordial follicle pool size for female reproduction, elucidation of factors, such as AMH, that regulate the assembly process will provide insights into potential therapeutics to manipulate the pool size and female reproduction.

  4. Alginate: A Versatile Biomaterial to Encapsulate Isolated Ovarian Follicles.

    PubMed

    Vanacker, Julie; Amorim, Christiani A

    2017-02-28

    In vitro culture of ovarian follicles isolated or enclosed in ovarian tissue fragments and grafting of isolated ovarian follicles represent a potential alternative to restore fertility in cancer patients who cannot undergo cryopreservation of embryos or oocytes or transplantation of frozen-thawed ovarian tissue. In this regard, respecting the three-dimensional (3D) architecture of isolated follicles is crucial to maintaining their proper follicular physiology. To this end, alginate hydrogel has been widely investigated using follicles from numerous animal species, yielding promising results. The goal of this review is therefore to provide an overview of alginate applications utilizing the biomaterial as a scaffold for 3D encapsulation of isolated ovarian follicles. Different methods of isolated follicle encapsulation in alginate are discussed in this review, as its use of 3D alginate culture systems as a tool for in vitro follicle analysis. Possible improvements of this matrix, namely modification with arginine-glycine-aspartic acid peptide or combination with fibrin, are also summarized. Encouraging results have been obtained in different animal models, and particularly with isolated follicles encapsulated in alginate matrices and grafted to mice. This summary is designed to guide the reader towards development of next-generation alginate scaffolds, with enhanced properties for follicle encapsulation.

  5. C-type natriuretic peptide stimulates ovarian follicle development.

    PubMed

    Sato, Yorino; Cheng, Yuan; Kawamura, Kazuhiro; Takae, Seido; Hsueh, Aaron J W

    2012-07-01

    C-type natriuretic peptide (CNP) encoded by the NPPC (Natriuretic Peptide Precursor C) gene expressed in ovarian granulosa cells inhibits oocyte maturation by activating the natriuretic peptide receptor (NPR)B (NPRB) in cumulus cells. RT-PCR analyses indicated increased NPPC and NPRB expression during ovarian development and follicle growth, associated with increases in ovarian CNP peptides in mice. In cultured somatic cells from infantile ovaries and granulosa cells from prepubertal animals, treatment with CNP stimulated cGMP production. Also, treatment of cultured preantral follicles with CNP stimulated follicle growth whereas treatment of cultured ovarian explants from infantile mice with CNP, similar to FSH, increased ovarian weight gain that was associated with the development of primary and early secondary follicles to the late secondary stage. Of interest, treatment with FSH increased levels of NPPC, but not NPRB, transcripts in ovarian explants. In vivo studies further indicated that daily injections of infantile mice with CNP for 4 d promoted ovarian growth, allowing successful ovulation induction by gonadotropins. In prepubertal mice, CNP treatment alone also promoted early antral follicle growth to the preovulatory stage, leading to efficient ovulation induction by LH/human chorionic gonadotropin. Mature oocytes retrieved after CNP treatment could be fertilized in vitro and developed into blastocysts, allowing the delivery of viable offspring. Thus, CNP secreted by growing follicles is capable of stimulating preantral and antral follicle growth. In place of FSH, CNP treatment could provide an alternative therapy for female infertility.

  6. Induction of Ovarian Primordial Follicle Assembly by Connective Tissue Growth Factor CTGF

    PubMed Central

    Schindler, Ryan; Nilsson, Eric; Skinner, Michael K.

    2010-01-01

    Primordial follicle assembly is a process that occurs when oocyte nests break down to form individual primordial follicles. The size of this initial pool of primordial follicles in part determines the reproductive lifespan of the female. Connective tissue growth factor (CTGF) was identified as a potential regulatory candidate for this process in a previous microarray analysis of follicle development. The current study examines the effects of CTGF and associated transforming growth factor beta 1 (TGFβ-1) on follicle assembly. Ovaries were removed from newborn rat pups and placed in an organ culture system. The ovaries treated with CTGF for two days were found to have an increased proportion of assembled follicles. CTGF was found to regulate the ovarian transcriptome during primordial follicle assembly and an integrative network of genes was identified. TGFβ-1 had no effect on primordial follicle assembly and in combination with CTGF decreased oocyte number in the ovary after two days of culture. Over ten days of treatment only the combined treatment of CTGF and TGFβ-1 was found to cause an increase in the proportion of assembled follicles. Interestingly, treatment with TGFβ-1 alone resulted in fewer total oocytes in the ovary and decreased the primordial follicle pool size after ten days of culture. Observations indicate that CTGF alone or in combination with TGFβ-1 stimulates primordial follicle assembly and TGFβ-1 can decrease the primordial follicle pool size. These observations suggest the possibility of manipulating primordial follicle pool size and influencing female reproductive lifespan. PMID:20886044

  7. Induction of ovarian primordial follicle assembly by connective tissue growth factor CTGF.

    PubMed

    Schindler, Ryan; Nilsson, Eric; Skinner, Michael K

    2010-09-24

    Primordial follicle assembly is a process that occurs when oocyte nests break down to form individual primordial follicles. The size of this initial pool of primordial follicles in part determines the reproductive lifespan of the female. Connective tissue growth factor (CTGF) was identified as a potential regulatory candidate for this process in a previous microarray analysis of follicle development. The current study examines the effects of CTGF and associated transforming growth factor beta 1 (TGFβ-1) on follicle assembly. Ovaries were removed from newborn rat pups and placed in an organ culture system. The ovaries treated with CTGF for two days were found to have an increased proportion of assembled follicles. CTGF was found to regulate the ovarian transcriptome during primordial follicle assembly and an integrative network of genes was identified. TGFβ-1 had no effect on primordial follicle assembly and in combination with CTGF decreased oocyte number in the ovary after two days of culture. Over ten days of treatment only the combined treatment of CTGF and TGFβ-1 was found to cause an increase in the proportion of assembled follicles. Interestingly, treatment with TGFβ-1 alone resulted in fewer total oocytes in the ovary and decreased the primordial follicle pool size after ten days of culture. Observations indicate that CTGF alone or in combination with TGFβ-1 stimulates primordial follicle assembly and TGFβ-1 can decrease the primordial follicle pool size. These observations suggest the possibility of manipulating primordial follicle pool size and influencing female reproductive lifespan.

  8. Interrelationships between ovarian follicles grown in culture and possible mediators.

    PubMed

    Sirotkin, Alexander V; Florkovičová Koničková, Iveta; Schaeffer, Hans-Jorg; Laurincik, Jozef; Harrath, Abdel Halim

    2017-03-01

    Dominance or cooperation between ovarian follicles can determine the number of ovulations and fecundity, but interrelationships between follicles in mono- and poly-ovulatory species and their mechanisms are poorly understood. The goals of this work were to determine the existence and compare the character of mutual influence of cultured ovarian follicles from a mono-ovulatory species (cow) with established follicular dominance with those from a poly-ovulatory species (pig), in which interrelationship between follicles remain unknown, and to examine the role of ovarian cell proliferation, the insulin-like growth factor I (IGF-I)- oxytocin (OT) system, and steroid hormones in mediating interrelationships among ovarian follicles. Bovine and porcine ovarian follicles were isolated and cultured alone and in pairs, and the percentage of growing follicles was calculated. Porcine follicles were cultured alone and in pairs after addition of exogenous OT and IGF-I (100ngmL(-1)) or inactivation of endogenous OT and IGF-I by antisera against these hormones (1%). Proliferation of porcine follicular cells was assessed by SDS PAGE-Western immunoblotting, the release of IGF-I, progesterone, androstenedione and estradiol by cultured porcine ovarian follicles was analyzed by RIA/EIA. Overall, our observations suggest (1) competition/dominance (mutual suppression of growth) in bovine ovarian follicles, (2) cooperation (mutual support of growth) in porcine ovarian follicles, (3) that this mutual growth of porcine ovarian follicles was caused by the promotion of cell proliferation, (4) that this mechanism was probably not involved in bovine follicular dominance, (5) that communication between both porcine and bovine follicles affects their secretory activity, and (6) that both follicular dominance in cows and cooperation of follicles in pigs can be mediated by either down- or up-regulation of the IGF-I-OT system, which in turn affects follicular steroidogenesis and promotes follicular

  9. Ovarian follicle dynamics of female Greater Scaup during egg production

    USGS Publications Warehouse

    Gorman, K.B.; Flint, P.L.; Esler, Daniel; 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.

  10. Inhibin in individual buffalo ovarian follicles in relation to size.

    PubMed

    Palta, P; Prakash, B S; Manik, R S; Madan, M L

    1996-06-01

    A sensitive and specific radioimmunoassay was validated and applied for measurement of inhibin in follicular fluid (bFF) obtained from individual buffalo ovarian follicles. Follicular size was measured with an ultrasound machine and follicles were categorized as small, medium and large. Presence of inhibin was detected in all the antral follicles above 3 mm diameter. Inhibin concentration showed a positive relationship (R = 0.27, P < 0.01) with follicular diameter and was significantly higher (P < 0.05) in bFF from medium and large follicles (8.44 +/- 0.54 and 7.70 +/- 0.45 micrograms/ml, respectively) in comparison to that from small follicles (5.74 +/- 0.80 micrograms/ml). Total inhibin content was highly correlated (R = 0.92, P < 0.001) with follicular diameter and the inhibin content was higher (P < 0.001) in large > medium > small follicles.

  11. Abnormal growth of ovarian antral follicles in breast cancer patients.

    PubMed

    Byskov, A G; McNatty, K P; Westergaard, L; Larsen, J K; Grinsted, J; Peters, H

    1983-07-01

    Ovarian antral follicles from patients with breast cancer were compared with follicles from healthy women. Steroid levels in the follicular fluid and the health status of the follicles were evaluated. Follicles were judged to be healthy or atretic by flow cytometric determinations of the deoxyribonucleic acid content of aspirated granulosa cell nuclei. Fifteen of the 25 follicles (60%) from the cancer patients contained unmeasurable or abnormally low steriod levels (i.e., less than 100 ng/ml) which were significantly (P less than 0.001) lower than in follicles of the same health status from healthy women (500 to 1000 ng/ml). It is speculated whether substances other than the usual follicular steriods are produced by the cancer patients, which stimulate mitotic activity of the granulosa cells.

  12. 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

  13. Toxicity of Gossypol from Cottonseed Cake to Sheep Ovarian Follicles

    PubMed Central

    Câmara, Antônio Carlos Lopes; Gadelha, Ivana Cristina Nunes; Borges, Pedro Augusto Cordeiro; de Paiva, Silvano Alves; Melo, Marília Martins; Soto-Blanco, Benito

    2015-01-01

    Gossypol, a polyphenol compound produced by cotton plant, has proven reproductive toxicity, but the effects of gossypol on sheep ovaries are unknown. This study was aimed to determine the in vitro and in vivo effects of gossypol on the ovarian follicles of sheep. This trial was divided into two experiments. In the first one, we used twelve non-pregnant, nulliparous, Santa Inês crossbred ewes, which were randomly distributed into two equal groups and fed diets with and without cottonseed cake. Feed was offered at 1.5% of the animal’s body weight for 63 days. The concentrations of total and free gossypol in the cottonseed cake were 3.28 mg/g and 0.11 mg/g, respectively. Throughout the trial period, no animal showed clinical signs of toxicity and no effects on body weight were observed. However, there was a significantly lower number of viable ovarian follicles (20.6%) and higher number of atretic follicles (79.4%) in the gossypol-fed sheep compared to the control (85.1 and 34.9%, respectively). These findings were observed at all stages of follicular development. In the second experiment, eight ovaries from slaughterhouse were cultured with different concentrations of gossypol acetic acid (0, 5, 10 and 20 μg/mL) for 24 hours or seven days. The in vitro action of gossypol resulted in a significant decrease in viable ovarian follicles, especially the primary and transition follicles, and a significant increase in the number of atretic follicles after 24 hours of culture. These follicles were greatly affected when cultured with gossypol for seven days. It is concluded that gossypol present in cotton seeds directly acts on ovarian follicles in sheep to increase atresia. PMID:26600470

  14. Follicle Viability after Vitrification of Bovine Ovarian Tissue.

    PubMed

    Guedes, Janaína de Souza; Rodrigues, Jhenifer Kliemchen; Campos, Ana Luisa Menezes; Moraes, Camila Cruz de; Caetano, João Pedro Junqueira; Marinho, Ricardo Mello

    2017-08-31

    Purpose The present study aimed to evaluate the impact of vitrification on the viability of follicles using a three-dimensional (3D) in vitro culture. Methods Bovine ovarian tissue samples (n = 5) obtained from slaughterhouses were utilized. The cortex was cut into small fragments of 2 × 3 × 0.5 mm using a tissue slicer. From these fragments, secondary follicles were first isolated by mechanical and enzymatic methods, then encapsulated in alginate gel and individually cultured for 20 days. Additional fragments of the same ovarian tissue were vitrified in a solution containing 25% glycerol and 25% ethylene glycol. After warming, the follicles underwent the same follicular isolation process that was performed for the fresh follicles. Results A total of 61 follicles were isolated, 51 from fresh ovarian tissue, and 10 from vitrified tissue. After the culture, the vitrified and fresh follicles showed 20% and 43.1% survival rates respectively (p = 0.290), with no significant differences. At the end of the culture, there were no significant differences in follicular diameter between the vitrified (422.93 ± 85.05 µm) and fresh (412.99 ± 102.55 µm) groups (p = 0.725). Fresh follicles showed higher mean rate of antrum formation when compared with vitrified follicles (47.1% and 20.0% respectively), but without significant difference (p = 0.167). Conclusions The follicles were able to develop, grow and form antrum in the 3D system after vitrification, despite the lower results obtained with the fresh tissue. Thieme Revinter Publicações Ltda Rio de Janeiro, Brazil.

  15. Regulators of ovarian preantral follicle development.

    PubMed

    McGee, Elizabeth A; Raj, Renju S

    2015-05-01

    Preantral follicle development has become an increasingly recognized area of study in the last two decades. Factors that regulate the growth survival and differentiation of these small, yet complex structures have been identified. The field of fertility preservation and a need for increased numbers of mature oocytes for stem cell research revealed how little we knew of how follicles got from the primordial stage to the antral stage with a healthy and competent oocyte inside. This work discusses the role of gonadotropins in regulating preantral follicles and also the role of the TGF-β family members and their associated Smad signaling molecules in preantral follicle development. Preantral follicle development is a necessary step to fertility in females and further understanding of this process is essential for progress in both infertility care and the enlarging field of in vitro folliculogenesis.

  16. Mouse ovarian follicles secrete factors affecting the growth and development of like-sized ovarian follicles in vitro.

    PubMed

    Spears, Norah; Baker, Stuart; Srsen, Vlastimil; Lapping, Rebecca; Mullan, Julie; Nelson, Robert; Allison, Vivian

    2002-12-01

    A series of experiments have been carried out to determine whether follicles secrete factors able to affect the growth and development of other, like-sized follicles. Late preantral mouse ovarian follicles were either cocultured or cultured in media conditioned by previously cultured follicles. In particular, the experiments examined whether follicles do secrete such factors, whether the level of FSH in the culture media can affect that process, and what the nature of such secretory factor(s) might be. First, pairs of follicles were cocultured across a polycarbonate membrane containing pores. This showed that communication between the follicles resulted in the stimulation of growth and that the stimulation was due, at least in part, to the production of secretory factor(s). In subsequent experiments, follicles were cultured in media that had been preconditioned by previously cultured follicles. The concentration of FSH in the cultures determined the effect of the conditioned media: conditioned media was stimulatory to follicle growth when levels of FSH remained high throughout the culture, but inhibitory when FSH levels were dropped midway through the cultures. Heat inactivation removed this inhibitory effect, showing that the factor was likely to be a protein; addition of follistatin to the conditioned media did not alter its effect, indicating that the factor was unlikely to be activin. We have shown through a series of culture experiments that mouse follicles secrete factor(s) that can affect the development of other like-sized follicles when cultured from the late preantral to Graafian stages. Furthermore, we have shown that the effect (or production) of such factors is dependent on the FSH environment of the follicles.

  17. Charged iron particles, components of space radiation, destroy ovarian follicles.

    PubMed

    Mishra, Birendra; Ortiz, Laura; Luderer, Ulrike

    2016-08-01

    Do charged iron particles, components of space radiation, cause premature ovarian failure? Exposure to charged iron particles causes ovarian DNA damage, oxidative damage and apoptosis, resulting in premature ovarian failure. The ovary is very sensitive to follicle destruction by low linear energy transfer (LET) radiation, such as X-rays and γ-rays. However, it is completely unknown whether high-LET radiation, such as charged iron particles, also destroys ovarian follicles. Twelve week old C57BL/6J female mice were exposed to single doses of 0, 5, 30 or 50 cGy (n = 8/group) charged iron particles (LET = 179 keV/µm) at energy of 600 MeV/u. Two groups were irradiated at the highest dose, one fed AIN-93M chow and the other fed AIN-93M chow supplemented with 150 mg/kg diet alpha lipoic acid (ALA). We quantified the numbers of ovarian follicles, measured serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations, and analyzed histone H2AX phosphorylation, oxidative damage and apoptosis markers in the ovarian follicles. H2AX phosphorylation, lipid peroxidation, protein nitration and apoptosis were highly induced in ovarian follicles at 6 h and remained increased 1 week after irradiation. As a result, numbers of healthy ovarian follicles were significantly and dose-dependently depleted at 1 and 8 weeks post-irradiation, with 57, 84 and 99% decreases in primordial follicles at 8 weeks at the 5, 30 and 50 cGy doses, respectively (P < 0.05 versus 0 cGy). Consistent with near-total depletion of ovarian follicles in the 50 cGy group, serum concentrations of FSH and LH were significantly elevated at 8 weeks. Dietary supplementation with ALA partially prevented the adverse ovarian effects of 50 cGy iron particles. About 21% of the estimated radiation dose from exposure to galactic cosmic rays during a multi-year Mars mission will be due to high-LET particles, of which iron is only one. The effects of galactic cosmic rays, which contain a mixture of

  18. Roles of Gremlin1 and Gremlin2 in Regulating Ovarian Primordial to Primary Follicle Transition

    PubMed Central

    Nilsson, Eric E.; Larsen, Ginger; Skinner, Michael K.

    2014-01-01

    A network of extracellular signaling factors has previously been shown to act in concert to control the ovarian primordial to primary follicle transition. The current study was designed to investigate the roles of the endogenous bone morphogenetic protein (BMP) inhibitors Gremlin1 (GREM1) and Gremlin2 (GREM2) in primordial follicle transition in the rat ovary. GREM1 and GREM2 treatment were found to reverse the effects of Anti-Müllerian Hormone (AMH) to inhibit follicle transition in a whole ovary culture system. GREM1 reversed the effect of bone morphogenetic protein 4 (BMP4) to stimulate primordial follicle transition. Immunohistochemical studies showed that GREM2, but not GREM1, was present in primordial follicles suggesting GREM2 may regulate primordial follicle transition in vivo. Co-immunoprecipitation studies indicated that GREM2 directly binds to AMH, as well as to BMP4. Transcriptome analyses of ovaries treated with GREM2 or GREM1 yielded negligible numbers of differentially expressed genes, suggesting the immediate effects of GREM2 or GREM1 appear to be at the level of protein-protein interactions, rather than direct actions on the cells. A number of other ovarian growth factors were found to influence the expression of Grem2. Observations suggest that Grem2 is a part of the signaling network of growth factors that regulate the primordial to primary follicle transition. Insights into the regulatory networks affecting the pool of primordial follicles is important to understand the molecular basis for reproductive diseases such as primary ovarian insufficiency (POI). PMID:24614542

  19. Roles of Gremlin 1 and Gremlin 2 in regulating ovarian primordial to primary follicle transition.

    PubMed

    Nilsson, Eric E; Larsen, Ginger; Skinner, Michael K

    2014-06-01

    A network of extracellular signaling factors has previously been shown to act in concert to control the ovarian primordial to primary follicle transition. The current study was designed to investigate the roles of the endogenous bone morphogenetic protein (BMP) inhibitors Gremlin 1 (GREM1) and GREM2 in primordial follicle transition in the rat ovary. GREM1 and GREM2 treatments were found to reverse the effects of anti-Müllerian hormone (AMH) to inhibit follicle transition in a whole-ovary culture system. GREM1 reversed the effect of BMP4 to stimulate primordial follicle transition. Immunohistochemical studies showed that GREM2, but not GREM1, was present in primordial follicles suggesting that GREM2 may regulate primordial follicle transition in vivo. Co-immunoprecipitation studies indicated that GREM2 directly binds to AMH, as well as to BMP4. Transcriptome analyses of ovaries treated with GREM2 or GREM1 yielded negligible numbers of differentially expressed genes, suggesting that the immediate effects of GREM2 or GREM1 appear to be at the level of protein-protein interactions, rather than direct actions on the cells. A number of other ovarian growth factors were found to influence the expression of Grem2. Observations suggest that Grem2 is a part of the signaling network of growth factors that regulate the primordial to primary follicle transition. Insights into the regulatory networks affecting the pool of primordial follicles are important to understand the molecular basis for reproductive diseases such as primary ovarian insufficiency. © 2014 Society for Reproduction and Fertility.

  20. Ovarian follicle selection and granulosa cell differentiation.

    PubMed

    Johnson, A L

    2015-04-01

    The reproductive strategy for avian species that produce a sequence (or clutch) of eggs is dependent upon the maintenance of a small cohort of viable, undifferentiated (prehierarchal) follicles. It is from this cohort that a single follicle is selected on an approximate daily basis to initiate rapid growth and final differentiation before ovulation. This review describes a working model in which follicles within this prehierarchal cohort are maintained in an undifferentiated state by inhibitory cell signaling until the time of selection. Ultimately, follicle selection represents a process in which a single undifferentiated follicle per day is predicted to escape such inhibitory mechanisms to begin rapid growth and final maturation before ovulation. Several processes initiated within the granulosa cell layer at selection are dependent upon G protein-coupled receptors signaling via cyclic adenosine monophosphate (cAMP), and several critical processes are described herein. Finally, reference is made to several practical outcomes that can result from understanding the process of selection, including applications within the poultry industry. Proximal factors and processes that mediate follicle selection can either extend or decrease the length of the laying sequence, and thus directly influence overall egg production. In particular, any aberration that results in the selection of more than one follicle per day will result in decreased egg production. More generally, in wild birds these processes are modified by prevailing environmental conditions and by social interactions to influence clutch size. The elucidation of cellular processes that regulate follicle selection can assist in the development of assisted reproductive technologies for application in threatened and endangered avian species. © 2014 Poultry Science Association Inc.

  1. Involvement of Notch signaling in early chick ovarian follicle development.

    PubMed

    Li, Jun; Zhao, Dan; Guo, Changquan; Li, Jian; Mi, Yuling; Zhang, Caiqiao

    2016-01-01

    The formation of primordial follicles is a crucial process in the establishment of follicle pools required for the female's reproductive life span. For laying hens, ample follicles are a prerequisite for high laying performance. Notch signaling plays critical roles in germ cell cysts breakdown and in the formation of primordial follicles. Here, we investigated the role of Notch signaling in the ovarian development of post-hatch chicks. Results showed that around post-hatch day 4 (H4), the germ cell cysts broke apart, oocytes became surrounded by squamous pregranulosa cells, and the primordial follicles were then formed. Subsequently, we detected the expression of Notch signaling-related genes including Notch receptors (Notch1, 2), ligands (Jag1, 2 and Dll1, 4), and target genes (Hes1, Hey1). These genes all showed expression at H4 and some of these genes were up-regulated during primordial follicle formation. To evaluate the Notch signaling requirement for early follicular development, we adopted an in vitro ovary culture system. Suppression of Notch signaling by γ-secretase inhibitor induced a decrease of primordial follicles and an increase of germ cells in cysts. Attenuating Notch signaling also inhibited the phosphatidylinositol 3-kinase/protein kinase B pathways and suppressed cadherin expression. These results suggest that Notch signaling is endowed with an indispensable role in primordial follicle formation in post-hatch chicks.

  2. The Mare Model to Study the Effects of Ovarian Dynamics on Preantral Follicle Features

    PubMed Central

    Alves, Kele A.; Alves, Benner G.; Gastal, Gustavo D. A.; de Tarso, Saulo G. S.; Gastal, Melba O.; Figueiredo, José R.; Gambarini, Maria L.; Gastal, Eduardo L.

    2016-01-01

    Ovarian tissue collected by biopsy procedures allows the performance of many studies with clinical applications in the field of female fertility preservation. The aim of the present study was to investigate the influence of reproductive phase (anestrous vs. diestrous) and ovarian structures (antral follicles and corpus luteum) on the quality, class distribution, number, and density of preantral follicles, and stromal cell density. Ovarian fragments were harvested by biopsy pick-up procedures from mares and submitted to histological analysis. The mean preantral follicle and ovarian stromal cell densities were greater in the diestrous phase and a positive correlation of stromal cell density with the number and density of preantral follicles was observed. The mean area (mm2) of ovarian structures increased in the diestrous phase and had positive correlations with number of preantral follicles, follicle density, and stromal cell density. Biopsy fragments collected from ovaries containing an active corpus luteum had a higher follicle density, stromal cell density, and proportion of normal preantral follicles. In conclusion, our results showed: (1) the diestrous phase influenced positively the preantral follicle quality, class distribution, and follicle and stromal cell densities; (2) the area of ovarian structures was positively correlated with the follicle and stromal cell densities; and (3) the presence of an active corpus luteum had a positive effect on the quality of preantral follicles, and follicle and stromal densities. Therefore, herein we demonstrate that the presence of key ovarian structures favors the harvest of ovarian fragments containing an appropriate number of healthy preantral follicles. PMID:26900687

  3. High salt intake negatively impacts ovarian follicle development.

    PubMed

    Wang, Guang; Yeung, Cheung-Kwan; Zhang, Jing-Li; Hu, Xi-Wen; Ye, Yu-Xiang; Yang, Yong-Xia; Li, Jiang-Chao; Lee, Kenneth Ka Ho; Yang, Xuesong; Wang, Li-Jing

    2015-07-01

    Many human disorders induce high salinity in tissues and organs, interfering with their normal physiological functions. Using a mouse model, we demonstrated that high salt intake caused infertility. Specifically, we established that high salinity dramatically affects ovarian follicle development and the extent of follicular atresia. However, it did not significantly influence the primordial follicles. TUNEL assays revealed that high salt intake inhibited follicle development by inducing the granulosa and theca cells that surround the oocytes to undergo apoptosis. Furthermore, immunohistological staining for the proliferation markers Ki67 and PH3 showed that high salt intake also repressed granulosa cell proliferation. In vitro testing of granulosa cells also confirmed that high salt significantly repressed cell proliferation and promoted cell apoptosis. In summary, high salt consumption negatively impacts reproductive functions in female mice by interfering with ovarian folliculogenesis.

  4. 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

  5. Remote ischemic preconditioning on neovascularization and follicle viability on ovary autotransplantation in rats.

    PubMed

    Damous, L L; Silva, S M; Simões, R S; Morello, R J; Carbonel, A P F; Simões, M J; Montero, E F S

    2008-04-01

    Verify the optimum remote vascular occlusion time to reduce ovarian injury in autologous transplants in rats. Twenty-four adult female rats were assigned to four groups: GC (control group): bilateral oophorectomy followed by ovary transplant; GIPC (ischemic preconditioning group): remote ischemic preconditioning at the iliac artery for 5, 10, and 15 minutes (GIPC-5, GIPC-20, and GIPC-15) previous to bilateral oophorectomy and ovarian transplantation. The right ovary was fixed in the retroperitoneum. Euthanasia was performed 4 days after the surgical procedure. The follicles were counted and classified as developing versus atretic. The immunohistochemical assay identified vascular factor of endothelial growth (VEGF) in the ovarian stroma and assessed the proliferation capacity by means of the Ki-67 in the ovarian follicles. Every group showed an inflammatory infiltrate, luteous body, and ovarian follicles in several phases of development. The ischemic preconditioning groups displayed greater amounts of viable ovarian follicles and increased vascularization and vasodilatation than the control group. GIPC-15 showed the highest amount of viable follicles compared to the others (P < .05 GIPC-15 vs GC; GIPC-15 vs GIPC-5). More VEGF-labeled cells were observed in GIPC-10 than the control group (P < .05, GIPC-10 vs GC). The proliferation index assessed by Ki-67 marking showed GC: 80%; GIPC-5: 76%; GIPC-10: 67%; and GIPC-15: 64% (P > .05). The PCI-15 cohort seem to be the most adequate timing to achieve functional support and preservation of a greater number of viable ovarian follicles.

  6. Preantral follicle density in ovarian biopsy fragments and effects of mare age.

    PubMed

    Alves, K A; Alves, B G; Gastal, G D A; Haag, K T; Gastal, M O; Figueiredo, J R; Gambarini, M L; Gastal, E L

    2017-04-01

    The aims of the present study were to: (1) evaluate preantral follicle density in ovarian biopsy fragments within and among mares; (2) assess the effects of mare age on the density and quality of preantral follicles; and (3) determine the minimum number of ovarian fragments and histological sections needed to estimate equine follicle density using a mathematical model. The ovarian biopsy pick-up method was used in three groups of mares separated according to age (5-6, 7-10 and 11-16 years). Overall, 336 preantral follicles were recorded with a mean follicle density of 3.7 follicles per cm(2). Follicle density differed (P<0.05) among animals, ovarian fragments from the same animal, histological sections and age groups. More (P<0.05) normal follicles were observed in the 5-6 years (97%) than the 11-16 years (84%) age group. Monte Carlo simulations showed a higher probability (90%; P<0.05) of detecting follicle density using two experimental designs with 65 histological sections and three to four ovarian fragments. In summary, equine follicle density differed among animals and within ovarian fragments from the same animal, and follicle density and morphology were negatively affected by aging. Moreover, three to four ovarian fragments with 65 histological sections were required to accurately estimate follicle density in equine ovarian biopsy fragments.

  7. Gonadal soma controls ovarian follicle proliferation through Gsdf in zebrafish.

    PubMed

    Yan, Yi-Lin; Desvignes, Thomas; Bremiller, Ruth; Wilson, Catherine; Dillon, Danielle; High, Samantha; Draper, Bruce; Buck, Charles Loren; Postlethwait, John

    2017-08-30

    Aberrant signaling between germ cells and somatic cells can lead to reproductive disease and depends on diffusible signals, including transforming growth factor-beta (TGFB) -family proteins. The TGFB-family protein Gsdf (gonadal soma derived factor) controls sex determination in some fish and is a candidate for mediating germ cell/soma signaling. Zebrafish expressed gsdf in somatic cells of bipotential gonads and expression continued in ovarian granulosa cells and testicular Sertoli cells. Homozygous gsdf knockout mutants delayed leaving the bipotential gonad state, but then became a male or a female. Mutant females ovulated a few oocytes, then became sterile, accumulating immature follicles. Female mutants stored excess lipid and down-regulated aromatase, gata4, insulin receptor, estrogen receptor, and genes for lipid metabolism, vitellogenin, and steroid biosynthesis. Mutant females contained less estrogen and more androgen than wild-types. Mutant males were fertile. Genomic analysis suggests that Gsdf, Bmp15, and Gdf9, originated as paralogs in vertebrate genome duplication events. In zebrafish, gsdf regulates ovarian follicle maturation and expression of genes for steroid biosynthesis, obesity, diabetes, and female fertility, leading to ovarian and extra-ovarian phenotypes that mimic human polycystic ovarian syndrome (PCOS), suggesting a role for a related TGFB signaling molecule in the etiology of PCOS. Developmental Dynamics, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  8. Transforming growth factor-beta: its role in ovarian follicle development.

    PubMed

    Rosairo, Davina; Kuyznierewicz, Ileana; Findlay, Jock; Drummond, Ann

    2008-12-01

    Ovarian follicular growth and differentiation in response to transforming growth factor-beta (TGFB) was investigated using postnatal and immature ovarian models. TGFB ligand and receptor mRNAs were present in the rat ovary 4-12 days after birth and at day 25. In order to assess the impact of TGFB1 on follicle growth and transition from the primordial through to the primary and preantral stages of development, we established organ cultures with 4-day-old rat ovaries. After 10 days in culture with FSH, TGFB1, or a combination of the two, ovarian follicle numbers were counted and an assessment of atresia was undertaken using TUNEL. Preantral follicle numbers declined significantly when treated with the combination of FSH and TGFB1, consistent with our morphological appraisal suggesting an increase in atretic primary and preantral follicles. To investigate the mechanisms behind the actions of TGFB1, we isolated granulosa cells and treated them with FSH and TGFB1. Markers of proliferative, steroidogenic, and apoptotic capacity were measured by real-time PCR. Cyclin D2 mRNA expression by granulosa cells was significantly increased in response to the combination of FSH and TGFB. The expression of forkhead homolog in rhabdomyosarcoma (Foxo1) mRNA by granulosa cells was significantly reduced in the presence of both FSH and TGFB1, individually and in combination regimes. By contrast, the expression of steroidogenic enzymes/proteins was largely unaffected by TGFB1. These data suggest an inhibitory role for TGFB1 (in the presence of FSH) in follicle development and progression.

  9. A non-neuronal cholinergic system of the ovarian follicle.

    PubMed

    Mayerhofer, Artur; Kunz, Lars

    2005-11-01

    We have recently provided evidence that acetylcholine (ACh) is a non-neuronal intraovarian signalling molecule, produced by granulosa cells (GCs) and which appears to act as signalling factor in the growing follicle. The ACh biosynthesis enzyme, choline-acetyltransferase (ChAT), is expressed only in growing, antral follicles in rodent and primate species. This restriction to follicle stages, which depend on the activity of follicle-stimulating hormone (FSH), may suggest that ACh could be an as yet unknown local mediator of FSH actions. In respect of ACh actions, our ongoing studies indicate that they may be exerted via different muscarinic ACh-receptors (MR) in GCs, but also in oocytes in an overlapping fashion. To elucidate functional details we have studied cultured human GCs isolated from preovulatory follicles. Activation of MRs increases intracellular calcium and, e.g., induces the master transcription factor egr-1, implying involvement in cell differentiation events. ACh agonists also activate a calcium-activated potassium channel (BK(Ca)) resulting in membrane hyperpolarization, which allows activation of other voltage-dependent ion channels. Experimental modulation of the chain of these events causes altered steroidogenesis, implying a crucial role of ACh in endocrine functions. Further ACh actions include phosphorylation of the gap junction molecule connexin 43 and disruption of intercellular communication between GCs. This may allow strongly coupled GCs to escape from the functional syncytium of the follicle in order to initiate proliferation. Proliferation is indeed strongly increased in cultured human GCs when treated with cholinergic agents. The repertoire of ACh/MR actions is far from being fully appreciated and may include epigenetic regulation in healthy growing follicles. Although many aspects of the ovarian cholinergic system, including, for instance, influence of follicular ACh on the MR-bearing oocyte, remain to be examined. The present data

  10. Glycomic analyses of ovarian follicles during development and atresia

    PubMed Central

    Hatzirodos, Nicholas; Nigro, Julie; Irving-Rodgers, Helen F.; Vashi, Aditya V.; Hummitzsch, Katja; Caterson, Bruce; Sullivan, Thomas R.; Rodgers, Raymond J.

    2012-01-01

    To examine the detailed composition of glycosaminoglycans during bovine ovarian follicular development and atresia, the specialized stromal theca layers were separated from the stratified epithelial granulosa cells of healthy (n = 6) and atretic (n = 6) follicles in each of three size ranges: small (3–5 mm), medium (6-9 mm) and large (10 mm or more) (n = 29 animals). Fluorophore-assisted carbohydrate electrophoresis analyses (on a per cell basis) and immunohistochemistry (n = 14) were undertaken. We identified the major disaccharides in thecal layers and the membrana granulosa as chondroitin sulfate-derived ∆uronic acid with 4-sulfated N-acetylgalactosamine and ∆uronic acid with 6-sulfated N-acetylgalactosamine and the heparan sulfate-derived Δuronic acid with N-acetlyglucosamine, with elevated levels in the thecal layers. Increasing follicle size and atresia was associated with increased levels of some disaccharides. We concluded that versican contains 4-sulfated N-acetylgalactosamine and it is the predominant 4-sulfated N-acetylgalactosamine proteoglycan in antral follicles. At least one other non- or 6-sulfated N-acetylgalactosamine proteoglycan(s), which is not decorin or an inter-α-trypsin inhibitor family member, is present in bovine antral follicles and associated with hitherto unknown groups of cells around some larger blood vessels. These areas stained positively for chondroitin/dermatan sulfate epitopes [antibodies 7D4, 3C5, and 4C3], similar to stem cell niches observed in other tissues. The sulfation pattern of heparan sulfate glycosaminoglycans appears uniform across follicles of different sizes and in healthy and atretic follicles. The heparan sulfate products detected in the follicles are likely to be associated with perlecan, collagen XVIII or betaglycan. PMID:22057033

  11. Alginate encapsulation supports the growth and differentiation of human primordial follicles within ovarian cortical tissue.

    PubMed

    Laronda, Monica M; Duncan, Francesca E; Hornick, Jessica E; Xu, Min; Pahnke, Jennifer E; Whelan, Kelly A; Shea, Lonnie D; Woodruff, Teresa K

    2014-08-01

    In vitro follicle growth (IVFG) is an investigational fertility preservation technique in which immature follicles are grown in culture to produce mature eggs that can ultimately be fertilized. Although progress has been made in growing primate primary and secondary follicles in vitro, it has been a relatively greater challenge to isolate and culture primordial follicles. The purpose of this study was to develop methods to grow human primordial follicles in vitro using alginate hydrogels. We obtained human ovarian tissue for research purposes through the National Physicians Cooperative from nationwide sites and used it to test two methods for culturing primordial follicles. First, primordial follicles were isolated from the ovarian cortex and encapsulated in alginate hydrogels. Second, 1 mm × 1 mm pieces of 500 μm-thick human ovarian cortex containing primordial follicles were encapsulated in alginate hydrogels, and survival and follicle development within the tissue was assessed for up to 6 weeks. We found that human ovarian tissue could be kept at 4 °C for up to 24 h while still maintaining follicle viability. Primordial follicles isolated from ovarian tissue did not survive culture. However, encapsulation and culture of ovarian cortical pieces supported the survival, differentiation, and growth of primordial and primary follicles. Within several weeks of culture, many of the ovarian tissue pieces had formed a defined surface epithelium and contained growing preantral and antral follicles. The early stages of in vitro human follicle development require the support of the native ovarian cortex.

  12. Piscine follicle-stimulating hormone triggers progestin production in gilthead seabream primary ovarian follicles.

    PubMed

    Zapater, Cinta; Chauvigné, François; Scott, Alexander P; Gómez, Ana; Katsiadaki, Ioanna; Cerdà, Joan

    2012-11-01

    Ovarian growth (vitellogenesis) in most lower vertebrates is mediated by estradiol-17beta (E2) secreted by the follicles in response to follicle-stimulating hormone (Fsh), whereas oocyte maturation and ovulation are mediated by progestins, such as 17alpha,20beta-dihydroxypregn-4-en-3-one (17,20beta-P), produced in response to luteinizing hormone (Lh). In teleosts, follicular synthesis of 17,20beta-P at the time of maturation is due primarily to up-regulation of the enzymes P450c17-II (Cyp17a2) and 20beta-hydroxysteroid dehydrogenase (Cbr1). Here, we show that follicular cells associated with primary growth (previtellogenic) oocytes of the gilthead seabream also express cyp17a2 and cbr1, in addition to P450c17-I (cyp17a1) and aromatase (cyp19a1), enzymes required for E2 synthesis. Ovaries containing only oogonia and early primary ovarian follicles had a 60-fold higher concentration of 17,20beta-P than ovaries in the succeeding stages and had a higher expression of cbr1 and Fsh receptor (fshra). Stimulation of explants of primary follicles in vitro with recombinant piscine Fsh (rFsh), which specifically activates the seabream Fshra, promoted a rapid accumulation of 17,20beta-P, and synthesis was sustained by an external supply of 17alpha-hydroxyprogesterone. In the presence of Cbr1 inhibitors, rFsh-mediated 17,20beta-P production was reduced, with a concomitant increase in testosterone and E2 synthesis. In primary explants, rFsh up-regulated cyp17a2 and cbr1 transcription and simultaneously down-regulated cyp17a1 and cyp19a1 steady-state mRNA levels within 24 h. In contrast, in explants containing vitellogenic follicles, rFsh had no effect on cyp17a2 and cbr1 expression, but increased that of cyp17a1 and cyp19a1. These data suggest a functional Fshra-activated Cyp17a2/Cbr1 steroidogenic pathway in gilthead seabream primary ovarian follicles triggering the production of 17,20beta-P.

  13. Vasoactive intestinal peptide can promote the development of neonatal rat primordial follicles during in vitro culture.

    PubMed

    Chen, Niannian; Li, Yu; Wang, Wenjun; Ma, Yun; Yang, Dongzi; Zhang, Qingxue

    2013-01-01

    Recruitment of primordial follicles is essential for female fertility. Some of the intraovarian growth factors involved in the initiation of primordial follicle growth have been identified, but the exact mechanisms regulating follicle activation are poorly understood. Strong evidence indicates that vasoactive intestinal peptide (VIP), a neuropeptide found in ovarian nerves, plays a role in the physiology of follicle development and function. The aim of the present study was to determine whether VIP might regulate the activation and growth of neonatal rat primordial follicles in an in vitro culture system. Ovaries from 4-day-old rats were cultured for 14 days in medium containing 10(-7) M VIP. At the end of the culture, the developmental stages and viability of the follicles were evaluated using histological sections. Immunohistochemistry studies for proliferating cell nuclear antigen (PCNA) were performed to assess the mitotic activity of granulosa cells. In addition, the expression level of kit ligand (KL) mRNA was examined after culture. Histology showed that primordial follicles could survive and start to grow in vitro. The proportion of primordial follicles was decreased and the proportion of early primary follicles increased after in vitro culture with VIP. Immunolocalization of PCNA showed that follicle growth was initiated after VIP treatment. The expression level of KL mRNA was increased in the VIP treatment group. Thus, VIP can promote primordial follicle development, possibly mediated in part through upregulating the expression of KL.

  14. Cox-1 Suppression and Follicle Depletion in the Etiology of Menopause- Associated Ovarian Cancer

    DTIC Science & Technology

    2008-04-01

    during the release of the ovum and the cell proliferation that occurs postovulation to repair the ovarian surface epithelium have been proposed to...for the release of the ovum .14–16 After rupture of the follicle and the release of the ovum at the ovarian surface, the oestrogen-producing follicle

  15. Two classes of ovarian primordial follicles exhibit distinct developmental dynamics and physiological functions.

    PubMed

    Zheng, Wenjing; Zhang, Hua; Gorre, Nagaraju; Risal, Sanjiv; Shen, Yan; Liu, Kui

    2014-02-15

    In the mammalian ovary, progressive activation of primordial follicles serves as the source of fertilizable ova, and disorders in the development of primordial follicles lead to various ovarian diseases. However, very little is known about the developmental dynamics of primordial follicles under physiological conditions, and the fates of distinct populations of primordial follicles also remain unclear. In this study, by generating the Foxl2-CreER(T2) and Sohlh1-CreER(T2) inducible mouse models, we have specifically labeled and traced the in vivo development of two classes of primordial follicles, the first wave of simultaneously activated follicles after birth and the primordial follicles that are gradually activated in adulthood. Our results show that the first wave of follicles exists in the ovaries for ∼3 months and contributes to the onset of puberty and to early fertility. The primordial follicles at the ovarian cortex gradually replace the first wave of follicles and dominate the ovary after 3 months of age, providing fertility until the end of reproductive life. Moreover, by tracing the time periods needed for primordial follicles to reach various advanced stages in vivo, we were able to determine the exact developmental dynamics of the two classes of primordial follicles. We have now revealed the lifelong developmental dynamics of ovarian primordial follicles under physiological conditions and have clearly shown that two classes of primordial follicles follow distinct, age-dependent developmental paths and play different roles in the mammalian reproductive lifespan.

  16. Two classes of ovarian primordial follicles exhibit distinct developmental dynamics and physiological functions

    PubMed Central

    Zheng, Wenjing; Zhang, Hua; Gorre, Nagaraju; Risal, Sanjiv; Shen, Yan; Liu, Kui

    2014-01-01

    In the mammalian ovary, progressive activation of primordial follicles serves as the source of fertilizable ova, and disorders in the development of primordial follicles lead to various ovarian diseases. However, very little is known about the developmental dynamics of primordial follicles under physiological conditions, and the fates of distinct populations of primordial follicles also remain unclear. In this study, by generating the Foxl2-CreERT2 and Sohlh1-CreERT2 inducible mouse models, we have specifically labeled and traced the in vivo development of two classes of primordial follicles, the first wave of simultaneously activated follicles after birth and the primordial follicles that are gradually activated in adulthood. Our results show that the first wave of follicles exists in the ovaries for ∼3 months and contributes to the onset of puberty and to early fertility. The primordial follicles at the ovarian cortex gradually replace the first wave of follicles and dominate the ovary after 3 months of age, providing fertility until the end of reproductive life. Moreover, by tracing the time periods needed for primordial follicles to reach various advanced stages in vivo, we were able to determine the exact developmental dynamics of the two classes of primordial follicles. We have now revealed the lifelong developmental dynamics of ovarian primordial follicles under physiological conditions and have clearly shown that two classes of primordial follicles follow distinct, age-dependent developmental paths and play different roles in the mammalian reproductive lifespan. PMID:24087793

  17. 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

  18. 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.

  19. Cystic ovarian follicles and thyroid activity in the dairy cow.

    PubMed

    Mutinati, M; Rizzo, A; Sciorsci, R L

    2013-05-01

    Thyroid activity affects the functionality of the reproductive axis and thyroid dysfunction has been associated with ovarian hyperstimulation syndrome and polycystic ovarian syndrome, in human medicine. This study investigates serum17- estradiol, progesterone, thyrotropic and thyroid hormone levels, in cyclic dairy cows on heat (Group H) and in dairy cows with ovarian follicular cysts (Group FC). Both 17- estradiol and progesterone serum concentrations were statistically higher in cystic than in cyclic cows (estradiol: 8.51±1.91 vs 6.32±1pg/mL) (progesterone: 0.49±0.17 vs 0.13±0.03ng/mL), whereas TSH and fT4 serum concentrations were statistically lower in cows with cystic ovarian follicles (COF), compared to cyclic ones (TSH: 2.48±1.31 vs 3.56±1.03ng/mL) (fT4: 5.86±1.69 vs 8.63±1.08). fT3 serum levels were similar, in both cystic and cyclic subjects (2.94±0.65 vs 3.02±0.9, respectively). Based on these results it was decided to examine the function of the thyrothropic axis of dairy cows in a similar manner to that conducted on humans. If severe hypothyroidism should be found, a hormone replacement therapy could be attempted in cystic cows refractory to "ordinary" therapies.

  20. Mechanisms of fibroblast growth factor signaling in the ovarian follicle.

    PubMed

    Price, Christopher A

    2016-02-01

    Fibroblast growth factors (FGFs) have been shown to alter growth and differentiation of reproductive tissues in a variety of species. Within the female reproductive tract, the effects of FGFs have been focused on the ovary, and the most studied one is FGF2, which stimulates granulosa cell proliferation and decreases differentiation (decreased steroidogenesis). Other FGFs have also been implicated in ovarian function, and this review summarizes the effects of members of two subfamilies on ovarian function; the FGF7 subfamily that also contains FGF10, and the FGF8 subfamily that also contains FGF18. There are data to suggest that FGF8 and FGF18 have distinct actions on granulosa cells, despite their apparent similar receptor binding properties. Studies of non-reproductive developmental biology also indicate that FGF8 is distinct from FGF18, and that FGF7 is also distinct from FGF10 despite similar receptor binding properties. In this review, the potential mechanisms of differential action of FGF7/FGF10 and FGF8/FGF18 during organogenesis will be reviewed and placed in the context of follicle development. A model is proposed in which FGF8 and FGF18 differentially activate receptors depending on the properties of the extracellular matrix in the follicle. © 2016 Society for Endocrinology.

  1. 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

  2. COX-1 Suppression and Follicle Depletion in the Etiology of Menopause-Associated Ovarian Cancer

    DTIC Science & Technology

    2009-10-01

    repetitive wounding during the release of the ovum and the cell proliferation that occurs postovulation to repair the ovarian surface epithelium have been...and proteolytic enzymes, which leads to rupture of the ovarian surface-epithelial layer for the release of the ovum .14–16 After rupture of the...follicle and the release of the ovum at the ovarian surface, the oestrogen-producing follicle is converted to a progesterone-producing corpus luteum

  3. Evidence for selective expression of angiotensin II receptors on atretic follicles in the rat ovary: an autoradiographic study

    SciTech Connect

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

    1988-06-01

    Ovarian angiotensin II (Ang II) receptors display a cyclical pattern of variation during the rat estrous cycle. Ang II receptors, estimated by the specific binding of the Ang II receptor antagonist (/sup 125/I)iodo-(Sar1,Ile8) Ang II to ovarian membranes, were lowest at estrus (binding site density (Bmax) = 35 +/- 2 fmol/mg; binding site affinity (KD) = 2.0 +/- 0.2 nM) and highest at diestrus I (Bmax = 59 +/- 3 fmol/mg; KD = 1.6 +/- 0.1 nM). We have previously shown that Ang II receptors in the rat ovary predominantly exist on the granulosa cell layer of a subpopulation of follicles. Our present studies show that the Ang II receptor-containing follicles in the rat ovary are mainly atretic (approximately 80%) or show signs of early atresia (approximately 15%) during all stages of the estrous cycle. A small number of Ang II receptor-containing follicles were healthy (approximately 5%). In contrast to the Ang II receptor-containing follicles, the FSH receptor-containing follicles were predominantly healthy (greater than 90%). Follicles which contained both Ang II receptors and FSH receptors were mainly early atretic. Since Ang II receptor-containing follicles in the rat ovary were mainly atretic these studies suggest that in the rat Ang II may be a major factor in regulating the function of atretic ovarian follicles.

  4. COX-1 Suppression and Follicle Depletion in the Etiology of Menopause-Associated Ovarian Cancer

    DTIC Science & Technology

    2008-10-01

    The repetitive wounding during the release of the ovum and the cell proliferation that occurs postovulation to repair the ovarian surface epithelium...of the ovarian surface- epithelial layer for the release of the ovum .14–16 After rupture of the follicle and the release of the ovum at the ovarian

  5. Ultrasound image attributes of human ovarian dominant follicles during natural and oral contraceptive cycles

    PubMed Central

    Birtch, Rebecca L; Baerwald, Angela R; Olatunbosun, Olufemi A; Pierson, Roger A

    2005-01-01

    Background Computer-assisted analyses were used to examine ultrasound image attributes of human dominant ovarian follicles that developed during natural and oral contraceptive (OC) cycles. We hypothesized that image attributes of natural cycle follicles would quantitatively differ from those in OC cycles and that OC cycle follicles would possess image attributes indicative of atresia. Methods Dominant ovarian follicles of 18 clinically normal women were compared using transvaginal ultrasonography for the 7 days before ovulation during a natural cycle (n = 9) or the 7 days before peak estradiol in women using OC (n = 11). Follicles were analyzed using region and line techniques designed to compare the image attributes numerical pixel value (NPV), pixel heterogeneity (PH) and area under the curve (AUC). Results NPV was higher in OC cycle follicles with region analysis and tended to be higher with line analysis (p = 0.005 and p = 0.06, respectively). No differences were observed in two other image attributes (AUC and PH), measured with either technique, between natural and OC cycle follicles. Conclusion The increased NPV value of OC cycle follicles and lack of differences in PH and AUC values between natural cycle and OC cycle follicles did not support the hypothesis that OC cycle follicles would show ultrasonographically detectable signs of atresia. Image attributes observed in OC cycle follicles were not clearly indicative of atresia nor were they large enough to preclude preovulatory physiologic status in OC cycle follicles. PMID:15829004

  6. Development of cryopreservation protocols for early stage zebrafish (Danio rerio) ovarian follicles using controlled slow cooling.

    PubMed

    Tsai, S; Rawson, D M; Zhang, T

    2009-05-01

    Cryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, cryopreservation of early stage zebrafish ovarian follicles was studied for the first time using controlled slow freezing. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and ovarian follicle developmental stage were investigated. Stages I and II ovarian follicles were frozen in 4M methanol and 3M DMSO in either L-15 medium or KCl buffer. Ovarian follicle viability was assessed using trypan blue, FDA+PI staining and ADP/ATP assay. The results showed that KCl buffer was more beneficial than L-15 medium, methanol was more effective than DMSO, optimum cooling rates were 2-4 degrees C/min, stepwise removal of cryoprotectant improved ovarian follicle viability significantly and stage I ovarian follicles were more sensitive to freezing. The results also showed that FDA+PI staining and ADP/ATP assay were more sensitive than TB staining. The highest follicle viabilities after post-thaw incubation for 2h obtained with FDA+PI staining were 50.7+/-4.0% although ADP/ATP ratios of the cryopreserved follicles were significantly increased indicating increased cell damage. Studies are currently being carried out on in vitro maturation of these cryopreserved ovarian follicles.

  7. Lysophosphatidic acid expression in theca cells depends on the type of bovine ovarian follicle.

    PubMed

    Sinderewicz, E; Grycmacher, K; Boruszewska, D; Kowalczyk-Zięba, I; Woclawek-Potocka, I

    2017-02-01

    Lysophosphatidic acid (LPA) exerts various actions on the mammalian reproductive system. In cows, LPA stimulates the synthesis and secretion of luteotropic factors in the ovary, which affects the growth and development of ovarian follicles. The role of LPA in granulosa cells, oocyte and oocyte-cumulus complex (COC) has previously been investigated; but its role in the theca layer, which is an important structural and functional component of the ovarian follicle, is still unclear. The goal of this study was to investigate the expression of LPA in theca cells originating from different bovine ovarian follicle types. Theca cells were separated from healthy, transitional and atretic ovarian follicles, based on intrafollicular estradiol: progesterone ratios. LPA concentration in the follicular fluid (FF) in different follicle types was measured, and expression of the enzymes responsible for LPA synthesis (autotaxin [AX], phospholipase A2 [PLA2]) and receptors for LPA (LPAR1-4) were determined. The obtained results confirmed the follicle-type dependent presence of LPA in the FF of the bovine ovarian follicles. The highest concentration of LPA was detected in follicles classified as healthy and dominant. LPAR1-4, PLA2 and AX expression in theca cells in all of the types of follicles examined were detected at mRNA and protein level. These results suggest that theca cells can be a source of LPA synthesis other than granulosa cells and COCs, as well as the target for its action in the bovine ovarian follicle, with PLA2 and LPAR4 playing major roles in LPA synthesis and action. © 2016 Blackwell Verlag GmbH.

  8. Cytokine (IL16) and tyrphostin actions on ovarian primordial follicle development.

    PubMed

    Feeney, Amanda; Nilsson, Eric; Skinner, Michael K

    2014-09-01

    An ovarian follicle is composed of an oocyte and surrounding theca and granulosa cells. Oocytes are stored in an arrested state within primordial follicles until they are signaled to re-initiate development by undergoing primordial-to-primary follicle transition. Previous gene bionetwork analyses of primordial follicle development identified a number of critical cytokine signaling pathways and genes potentially involved in the process. In the current study, candidate regulatory genes and pathways from the gene network analyses were tested for their effects on the formation of primordial follicles (follicle assembly) and on primordial follicle transition using whole ovary organ culture experiments. Observations indicate that the tyrphostin inhibitor (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one increased follicle assembly significantly, supporting a role for the MAPK signaling pathway in follicle assembly. The cytokine interleukin 16 (IL16) promotes primordial-to-primary follicle transition as compared with the controls, where as Delta-like ligand 4 (DLL4) and WNT-3A treatments have no effect. Immunohistochemical experiments demonstrated the localization of both the cytokine IL16 and its receptor CD4 in the granulosa cells surrounding each oocyte within the ovarian follicle. The tyrphostin LDN193189 (LDN) is an inhibitor of the bone morphogenic protein receptor 1 within the TGFB signaling pathway and was found to promote the primordial-to-primary follicle transition. Observations support the importance of cytokines (i.e., IL16) and cytokine signaling pathways in the regulation of early follicle development. Insights into regulatory factors affecting early primordial follicle development are provided that may associate with ovarian disease and translate to improved therapy in the future. © 2014 Society for Reproduction and Fertility.

  9. Cytokine (IL16) and Tyrphostin Actions on Ovarian Primordial Follicle Development

    PubMed Central

    Feeney, Amanda; Nilsson, Eric; Skinner, Michael K.

    2014-01-01

    An ovarian follicle is composed of an oocyte and surrounding theca and granulosa cells. Oocytes are stored in an arrested state within primordial follicles until they are signaled to re-initiate development by undergoing primordial to primary follicle transition. Previous gene bionetwork analyses of primordial follicle development identified a number of critical cytokine signaling pathways and genes potentially involved in the process. In the current study, candidate regulatory genes and pathways from the gene network analyses were tested for their effects on the formation of primordial follicles (follicle assembly) and on primordial follicle transition using whole ovary organ culture experiments. Observations indicate that the tyrphostin inhibitor BCI increased follicle assembly significantly, supporting a role for the mitogen activated protein kinase (MAPK) signaling pathway in follicle assembly. The cytokine interleukin 16 (IL16) promotes primordial to primary follicle transition as compared to the controls, where as Delta-like 4 (DLL4) and WNT-3A treatment have no effect. Immunohistochemical experiments demonstrated for both the cytokine IL16 and its receptor CD4 localization to the granulosa cells surrounding each oocyte within the ovarian follicle. The tyrphostin LDN193189 (LDN) is an inhibitor of the bone morphogenic protein receptor 1 (BMPR1) within the TGFB signaling pathway and was found to promote the primordial to primary follicle transition. Observations support the importance of cytokines (i.e. IL16) and cytokine signaling pathways in regulating early follicle development. Insights into regulatory factors affecting early primordial follicle development are provided that may associate with ovarian disease and translate to improved therapy in the future. PMID:24970835

  10. mTOR Controls Ovarian Follicle Growth by Regulating Granulosa Cell Proliferation

    PubMed Central

    Yu, James; Yaba, Aylin; Kasiman, Corinna; Thomson, Travis; Johnson, Joshua

    2011-01-01

    We have shown that inhibition of mTOR in granulosa cells and ovarian follicles results in compromised granulosa proliferation and reduced follicle growth. Further analysis here using spontaneously immortalized rat granulosa cells has revealed that mTOR pathway activity is enhanced during M-phase of the cell cycle. mTOR specific phosphorylation of p70S6 kinase and 4E-BP, and expression of Raptor are all enhanced during M-phase. The predominant effect of mTOR inhibition by the specific inhibitor Rapamycin (RAP) was a dose-responsive arrest in the G1 cell cycle stage. The fraction of granulosa cells that continued to divide in the presence of RAP exhibited a dose-dependent increase in aberrant mitotic figures known as anaphase bridges. Strikingly, estradiol consistently decreased the incidence of aberrant mitotic figures. In mice treated with RAP, the mitotic index was reduced compared to controls, and a similar increase in aberrant mitotic events was noted. RAP injected during a superovulation regime resulted in a dose-dependent reduction in the numbers of eggs ovulated. Implications for the real-time regulation of follicle growth and dominance, including the consequences of increased numbers of aneuploid granulosa cells, are discussed. PMID:21750711

  11. Combination of a GnRH agonist with an antagonist prevents flare-up effects and protects primordial ovarian follicles in the rat ovary from cisplatin-induced toxicity: a controlled experimental animal study

    PubMed Central

    2013-01-01

    Background With the continuous improvement of surgery and chemotherapeutic treatments, many tumour patients increasingly achieve long-term survival and can even be completely cured. However, platinum-containing drugs, which are widely used to treat a variety of types of cancer, cause menstrual disorders and ovarian failure, which in turn lead to infertility. Thus far, gonadotropin releasing hormone (GnRH) agonist (GnRHa) and antagonist (GnRHant) are reported to act as protective agents of the ovary in chemotherapy through the inhibition of the female gonadal axis. Nevertheless, they both have disadvantages that limit their use. GnRHa causes a flare-up effect during the first week after administration, and no long-acting GnRHant agent is available. GnRHa combined with GnRHant may prevent the flare-up effect of GnRHa and rapidly inhibit the female gonadal axis. Several clinical studies with small sample sizes have reported controversial conclusions. In this strictly controlled animal study, we investigated the advantages of combination treatment with GnRHa and GnRHant. Methods Rats aged 12 weeks were divided into six groups: Control, cisplatin (CDDP), GnRHa, GnRHant, Combination (sht, short-term) and Combination (lng, long-term) of GnRHa and GnRHant. The last four groups received Triptorelin (1 mg/kg·d, for 14 days), Cetrorelix (0.5 mg/kg·d, for 10 days), a combination of Triptorelin (1 mg/kg·d, for 10 days) and Cetrorelix (0.5 mg/kg·d, for 10 days) in the long-term group and for 3 days in the short-term group. The Control and CDDP groups received saline (1 ml/kg·d, for 10 day). Then, all groups apart from the Control group received cisplatin (1 mg/kg·d, for 10 days), and the Control group received another 10 days of saline as described above. Blood samples were collected to detect the serum levels of E2, LH and FSH. Observation of oestrous cyclicity was also performed after drug administration. Finally, bilateral ovaries were collected for

  12. Vasoactive intestinal peptide enhanced aromatase activity in the neonatal rat ovary before development of primary follicles or responsiveness to follicle-stimulating hormone

    SciTech Connect

    George, F.W.; Ojeda, S.R.

    1987-08-01

    The authors have investigated the factors that regulate aromatase activity in fetal-neonatal rat ovaries. Ovarian aromatase activity (assessed by measuring the amount of /sup 3/H/sub 2/O formed from (1..beta..-/sup 3/H)testosterone) is low prior to birth and increases to values greater than 30 pmol/hr per mg of protein between days 8 and 12 after birth. The appearance of ovarian aromatase coincides with the development of primordial follicles. Fetal-neonatal ovaries maintained in serum-free organ culture do not develop aromatase activity at the expected time. Ovine follicle-stimulating hormone, ovine luteinizing hormone, or their combination failed to induce the enzyme activity in cultured fetal ovaries, whereas follicle-stimulating hormone is effective in preventing the decline in aromatase activity when postnatal day 8 ovaries are placed in culture. In contrast to follicle-stimulating hormone, dibutyryl-cAMP markedly enhances ovarian aromatase in cultured fetal ovaries. Likewise, enhancement of endogenouse cAMP formation with forskolin or cholera toxin caused an increase in enzyme activity within 24 hr. Vasoactive intestinal peptide, a peptide known to occur in ovarian nerves, caused a dose-dependent increase in aromatase activity in fetal ovaries prior to folliculogenesis. Of related peptides tested, only the peptide having N-terminal histidine and C-terminal isoleucine amide was capable of inducing aromatase activity in fetal ovaries. The fact that VIP can induce aromatase activity in fetal rat ovaries prior to follicle formation and prior to responsiveness to follicle-stimulating hormone suggests that this neuropeptide may play a critical role in ovarian differentiation.

  13. Vasoactive intestinal peptide enhances aromatase activity in the neonatal rat ovary before development of primary follicles or responsiveness to follicle-stimulating hormone.

    PubMed Central

    George, F W; Ojeda, S R

    1987-01-01

    We have investigated the factors that regulate aromatase activity in fetal-neonatal rat ovaries. Ovarian aromatase activity (assessed by measuring the amount of 3H2O) formed from [1 beta-3H]testosterone) is low prior to birth (less than 0.5 pmol/hr per mg of protein) and increases to values greater than 30 pmol/hr per mg of protein between days 8 and 12 after birth. The appearance of ovarian aromatase (postnatal days 2-4) coincides with the development of primordial follicles. Fetal-neonatal ovaries maintained in serum-free organ culture do not develop aromatase activity at the expected time. Ovine follicle-stimulating hormone (0.1-1 microgram/ml), ovine luteinizing hormone (0.1 microgram/ml), or their combination failed to induce the enzyme activity in cultured fetal ovaries, whereas follicle-stimulating hormone is effective in preventing the decline in aromatase activity when postnatal day 8 ovaries are placed in culture. In contrast to follicle-stimulating hormone, dibutyryl-cAMP markedly enhances ovarian aromatase in cultured fetal ovaries. Likewise, enhancement of endogenous cAMP formation with forskolin or cholera toxin caused an increase in enzyme activity within 24 hr. Vasoactive intestinal peptide, a peptide known to occur in ovarian nerves, caused a dose-dependent increase in aromatase activity in fetal ovaries prior to folliculogenesis. Of related peptides tested, only the peptide having N-terminal histidine and C-terminal isoleucine amide was capable of inducing aromatase activity in fetal ovaries. The fact that VIP can induce aromatase activity in fetal rat ovaries prior to follicle formation and prior to responsiveness to follicle-stimulating hormone suggests that this neuropeptide may play a critical role in ovarian differentiation. Images PMID:3039508

  14. Exosome-mediated communication in the ovarian follicle.

    PubMed

    Di Pietro, C

    2016-03-01

    Cells are able to produce and release different types of vesicles, such as microvesicles and exosomes, in the extracellular microenvironment. According to the scientific community, both microvesicles and exosomes are able to take on and transfer different macromolecules from and to other cells, and in this way, they can influence the recipient cell function. Among the different macromolecule cargos, the most studied are microRNAs. MicroRNAs are a large family of non-coding RNAs involved in the regulation of gene expression. They control every cellular process and their altered regulation is involved in human diseases. Their presence in mammalian follicular fluid has been recently demonstrated, and here, they are enclosed within microvesicles and exosomes or they can also be associated to protein complexes. The presence of microvesicles and exosomes carrying microRNAs in follicular fluid could represent an alternative mechanism of autocrine and paracrine communication inside the ovarian follicle. The outcomes from these studies could be important in basic reproductive research but could also be useful for clinical application. In fact, the characterization of extracellular vesicles in follicular fluid could improve reproductive disease diagnosis and provide biomarkers of oocyte quality in ART (Assisted Reproductive Treatment).

  15. Follicle development in cryopreserved bitch ovarian tissue grafted to immunodeficient mouse.

    PubMed

    Commin, L; Buff, S; Rosset, E; Galet, C; Allard, A; Bruyere, P; Joly, T; Guérin, P; Neto, V

    2012-01-01

    The present study evaluated: (1) in vivo follicular development in canine ovarian tissue after slow freezing and xenotransplantation; and (2) the use of erythropoietin (EPO) as an angiogenic factor to optimise the transplantation procedure. Frozen-thawed ovarian tissue from five bitches was grafted into severe combined immunodeficient (SCID) mice (n=47) treated with or without EPO (500 IU kg(-1), once daily for 3 days) (Groups A and B, respectively) and analysed after 0, 1, 8 or 16 weeks. Follicle grade, follicle density, follicle morphology and stromal cells density were assessed by histological analysis, whereas vascularisation of the graft was quantified by immunohistochemistry with anti-α-smooth muscle actin antibody. Despite a massive loss of follicles after grafting, secondary follicle density was higher at 8 and 16 weeks than at 1 week regardless of EPO treatment. EPO significantly improved early follicle morphology and stromal cell density after 8 weeks and blood vessel density at 16 weeks after transplantation (P<0.05). Intact secondary follicles with more than three granulosa cells layers were observed 16 weeks after transplantation. The results suggest that canine ovarian tissue can be successfully preserved by our slow-freezing protocol because the tissue showed follicular growth after xenotransplantation. EPO treatment did not lessen the massive loss of follicles after transplantation.

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

    PubMed

    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.

  17. In vitro growth and development of isolated secondary follicles from vitrified caprine ovarian cortex.

    PubMed

    Leal, Érica S S; Vieira, Luis A; Sá, Naíza A R; Silva, Gerlane M; Lunardi, Franciele O; Ferreira, Anna C A; Campello, Cláudio C; Alves, Benner G; Cibin, Francielli W S; Smitz, Johan; Figueiredo, José R; Rodrigues, Ana P R

    2017-08-03

    The aim of this study was to evaluate the viability, antrum formation and in vitro development of isolated secondary follicles from vitrified caprine ovarian cortex in a medium previously established for fresh isolated secondary follicles, in the absence (α-minimum essential medium (α-MEM+) alone) or presence of FSH and vascular endothelial growth factor (VEGF; α-MEM++FSH+VEGF). Ovarian fragments were distributed among five treatments (T1 to T5): fresh follicles were fixed immediately (T1), follicles from fresh tissue were cultured in vitro in α-MEM+ (T2) or α-MEM++FSH+VEGF (T3) and follicles from vitrified tissue were cultured in vitro in α-MEM+ (T4) or α-MEM++FSH+VEGF (T5). After 6 days of culture, treated follicles (T2, T3, T4 and T5) were evaluated for morphology, viability and follicular development (growth, antrum formation and proliferation of granulosa cells by Ki67 and argyrophilic nucleolar organiser region (AgNOR) staining). The levels of reactive oxygen species (ROS) in the culture media were also assessed. Overall, morphology of vitrified follicles was altered (P<0.05) compared with the fresh follicles. Follicular viability, antrum formation and ROS were similar between treatments (P>0.05). The average overall and daily follicular growth was highest (P<0.05) in T3. Granulosa cells in all treatments (T1, T2, T3, T4 and T5) stained positive for Ki67. However, fresh follicles from T3 had significantly higher AgNOR staining (P<0.05) compared with follicles of T1, T2, T4 and T5. In conclusion, secondary follicles can be isolated from vitrified and warmed ovarian cortex and survive and form an antrum when growing in an in vitro culture for 6 days.

  18. 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.

  19. Comparative proteomic analysis of primordial follicles from ovaries of immature and aged rats.

    PubMed

    Govindaraj, Vijayakumar; Rao, A Jagannadha

    2015-01-01

    Age related decline in reproductive performance in women is well documented and apoptosis has been considered as one of the reasons for the decline of primordial follicle reserve. Recently we observed a decline in the efficiency of DNA repair ability in aged rat primordial follicles as demonstrated by decreased mRNA levels of DNA repair genes BRCA1 and H2AX. In the present study, a two-dimensional electrophoresis (2DE) proteomic approach was employed to identify differentially expressed proteins in primordial follicles isolated from ovaries of immature (∼20 days) and aged (∼400-450 days) rats. Using MALDI-TOF/TOF MS, we identified 13 differentially expressed proteins (p < 0.05) which included seven up-regulated and six down-regulated proteins in aged primordial follicles. These proteins are involved in a wide range of biological functions including apoptosis, DNA repair, and the immune system. Interestingly, the differentially expressed proteins such as FIGNL1 (DNA repair) and BOK (apoptotic protein) have not been previously reported in the rat primordial follicles and these proteins can be related to some common features of ovarian aging such as loss of follicle reserve and genome integrity. The quantitative differences of two important proteins BOK and FIGNL1 observed by the proteomic analysis were correlated with the transcript levels, as determined by semi-quantitative RT-PCR. Our results improve the current knowledge about protein factors associated with molecular changes in rat primordial follicles as a function of aging and our understanding of the proteomic processes involved in degenerative changes observed in aging primordial follicles.

  20. 3D culture of ovarian follicles: a system towards their engineering?

    PubMed

    Zuccotti, Maurizio; Merico, Valeria; Rebuzzini, Paola; Belli, Martina; Vigone, Giulia; Mulas, Francesca; Fassina, Lorenzo; Wruck, Wasco; Adjaye, James; Bellazzi, Riccardo; Garagna, Silvia

    2015-01-01

    Infertility in women is a health priority. Designing a robust culture protocol capable of attaining complete follicle growth is an exciting challenge, for its potential clinical applications, but also as a model to observe and closely study the sequence of molecular events that lie behind the intricate relationship existing between the oocyte and surrounding follicle cells. Here, we describe the procedures used to maintain the ovarian follicle 3D architecture employing a variety of in vitro systems and several types of matrices. Collagen and alginate are the matrices that led to better results, including proof-of-concept of full-term development. Pioneer in its kind, these studies underlie the drawbacks encountered and the need for a culture system that allows more quantitative analyses and predictions, projecting the culture of the ovarian follicle into the realm of tissue engineering.

  1. Phosphoinositide 3-kinase p110δ mediates estrogen- and FSH-stimulated ovarian follicle growth.

    PubMed

    Li, Qian; He, Hui; Zhang, Yin-Li; Li, Xiao-Meng; Guo, Xuejiang; Huo, Ran; Bi, Ye; Li, Jing; Fan, Heng-Yu; Sha, Jiahao

    2013-09-01

    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.

  2. Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container.

    PubMed

    Marques, Lis S; Bos-Mikich, Adriana; Godoy, Leandro C; Silva, Laura A; Maschio, Daniel; Zhang, Tiantian; Streit, Danilo P

    2015-12-01

    Cryopreservation of ovarian tissue has been studied for female germline preservation of farm animals and endangered mammalian species. However, there are relatively few reports on cryopreservation of fish ovarian tissue and especially using vitrification approach. Previous studies of our group has shown that the use of a metal container for the cryopreservation of bovine ovarian fragments results in good primordial and primary follicle morphological integrity after vitrification. The aim of this study was to assess the viability and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the same metal container. In Experiment 1, we tested the follicular viability of five developmental stages following vitrification in four vitrification solutions using fluorescein diacetate and propidium iodide fluorescent probes. These results showed that the highest viability rates were obtained with immature follicles (Stage I) and VS1 (1.5 M methanol + 4.5 M propylene glycol). In Experiment 2, we used VS1 to vitrify different types of ovarian tissue (fragments or whole ovaries) in two different carriers (plastic cryotube or metal container). In this experiment, Stage I follicle survival was assessed following vitrification by vital staining after 24 h in vitro culture. Follicular morphology was analyzed by light microscopy after vitrification. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P < 0.05) in vitrified fragments, when compared to whole ovaries. There were no significant differences in follicular survival and growth when the two vitrification devices were compared.

  3. In vivo effect of growth hormone on the expression of connexin-43 in bovine ovarian follicles.

    PubMed

    Kaiser, Germán G; Kölle, Sabine; Boie, Gudrun; Sinowatz, Fred; Palma, Gustavo A; Alberio, Ricardo H

    2006-05-01

    This study assessed the in vivo effects of recombinant growth hormone (rGH) administration on the expression of connexin-43 (Cx43) in bovine ovarian follicles. Two independent experiments were carried out using either estrous unsynchronized or synchronized multiparous Aberdeen Angus cows. rGH-treated animals were inoculated with a single dose of hormone (500 mg, intramuscular) while control animals were inoculated with hormone diluent. Five and 14 days after treatment (Experiments 1 and 2, respectively), ovarian Cx43 and apoptosis expression were assessed using immunohistochemistry. In both experiments primary, secondary, and tertiary follicles from rGH-treated and control groups distinctly expressed Cx43 protein. Primordial and atretic follicles were Cx43-negative. Interestingly, the number of Cx43 dots per granulosa cell did not show significant variation at different folliculogenesis stages neither in the rGH-treated nor in the control group. In unsynchronized animals, Cx43-positive follicles per total number of follicles ratio showed an interaction between stage of folliculogenesis and treatment due to significant differences between treatment groups in the early secondary follicle stage. In synchronized animals, there were significant differences between treatment groups and folliculogenesis stage. In both experiments, atretic follicles showed apoptosis-related DNA-fragmentation as determined by terminal uridin nick end labeling (TUNEL) assay. Tertiary follicles presented moderate TUNEL staining. Our results show significant increment in the number of ovarian follicles expressing the gap junction subunit Cx43 after in vivo rGH treatment. Therefore, we conclude that growth hormone can modulate in vivo gap junction assembly at early stages of folliculogenesis. Mol. Reprod. Dev. (c) 2006 Wiley-Liss, Inc.

  4. Ovarian development in athymic nude mice. II. The growth of the oocyte and follicle.

    PubMed

    Lintern-Moore, S; Pantelouris, E M

    1975-01-01

    Congenitally athymic mice homozygous for the Mendelian recessive mutation "nude" develop well defined morphological and quantitative changes in the ovarian follicle population. A decline in follicle numbers at 2 months of age is preceded by a retardation in follicle growth at 1 month of age. The growth of the oocyte and its nucleus are not affected by the nude mutation. However, the rate of growth and maximum size of the oocyte nucleolus are reduced in nudes. These developmental events are discussed in relation to the genetic activity of the oocyte, the role of pituitary gonadotrophins in follicular and oocyte growth and the possible role of the thymus gland in these processes.

  5. Hydrogel Based 3-Dimensional (3D) System for Toxicity and High-Throughput (HTP) Analysis for Cultured Murine Ovarian Follicles.

    PubMed

    Zhou, Hong; Malik, Malika Amattullah; Arab, Aarthi; Hill, Matthew Thomas; Shikanov, Ariella

    2015-01-01

    Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in

  6. Microscopic and molecular characterization of ovarian follicle atresia in Rhodnius prolixus Stahl under immune challenge.

    PubMed

    Medeiros, Marcelo N; Ramos, Isabela B; Oliveira, Danielle M P; da Silva, Rodrigo C B; Gomes, Fabio M; Medeiros, Luciano N; Kurtenbach, Eleonora; Chiarini, Luciana B; Masuda, Hatisaburo; de Souza, Wanderley; Machado, Ednildo A

    2011-07-01

    In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. An injection of A. niger conidia directly into the hemocoel of adult R. prolixus females at the onset of vitellogenesis caused no effect on host lifespan but elicited a net reduction in egg batch size. Direct inspection of ovaries from the mycosed insects revealed that fungal challenge led to atresia of the vitellogenic follicles. Light microscopy and DAPI staining showed follicle shrinkage, ooplasm alteration and disorganization of the monolayer of follicle cells in the atretic follicles. Transmission electron microscopy of thin sections of follicle epithelium also showed nuclei with condensed chromatin, electron dense mitochondria and large autophagic vacuoles. Occurrence of apoptosis of follicle cells in these follicles was visualized by TUNEL labeling. Resorption of the yolk involved an increase in protease activities (aspartyl and cysteinyl proteases) which were associated with precocious acidification of yolk granules and degradation of yolk protein content. The role of follicle atresia in nonspecific host-pathogen associations and the origin of protease activity that led to yolk resorption are discussed.

  7. Number and density of equine preantral follicles in different ovarian histological section thicknesses.

    PubMed

    Alves, K A; Alves, B G; Rocha, C D; Visonná, M; Mohallem, R F F; Gastal, M O; Jacomini, J O; Beletti, M E; Figueiredo, J R; Gambarini, M L; Gastal, E L

    2015-04-01

    Regardless of species, advances in preantral follicle culture and cryopreservation and transplant of ovarian tissue techniques are dependent on the number and density of preantral follicles in the ovary. This study tested the effect of different histological section thicknesses on number, classification, and density of equine preantral follicles. An ovarian fragment was obtained from 5- to 10-year-old mares (n = 14) after slaughter, and each fragment was submitted to three histological section thickness treatments: 3, 5, and 7 μm. The area (cm(2)) of each ovarian fragment was measured, and the sections were evaluated by light microscopy. The percentage of morphologically normal follicles (89%) was similar (P > 0.05) among primordial, transitional, and primary follicles and also among histological section thicknesses. A greater (P < 0.05) number of preantral follicles per histological section were seen in the 7-μm (8.0 ± 2.2) than that in the 3-μm (3.4 ± 0.7) treatment. Furthermore, a linear regression analysis reported that the number of preantral follicles increased (P < 0.05) when a thicker section treatment was used. However, no association (P > 0.05) between follicular density and treatment was observed. The mean number of preantral follicles per fragment (45.3 ± 18.8) and the follicular density (3.0 ± 0.5 follicles per cm(2)) were different (P < 0.05) among mares. In conclusion, this study on equine preantral follicles reported that (1) a 7-μm histological section thickness might be recommended because it allowed identification of a greater number of preantral follicles per sample, (2) a large individual variation in follicle population and density was detected regardless of histological section thickness, and (3) mares have a low number and density of preantral follicles when compared with those reported for other species. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. How Is the Number of Primordial Follicles in the Ovarian Reserve Established?

    PubMed

    Findlay, John K; Hutt, Karla J; Hickey, Martha; Anderson, Richard A

    2015-11-01

    The number of primordial follicles in the ovarian reserve is an important determinant of the length of the ovarian lifespan, and therefore the fertility of an individual. This reserve contains all of the oocytes potentially available for fertilization throughout the fertile lifespan. The maximum number is set during pregnancy or just after birth in most mammalian species; current evidence does not support neofolliculogenesis after the ovarian reserve is established, although this is increasingly being reexamined. Under physiological circumstances, this number will be influenced by the number of primordial germ cells initially specified in the epiblast of the developing embryo, their proliferation during and after migration to the developing gonads, and their death during oogenesis and formation of primordial follicles at nest breakdown. Death of germ cells during the establishment of the ovarian reserve occurs principally by autophagy or apoptosis, although the triggers that initiate these remain elusive. This review outlines the regulatory steps that determine the number of primordial follicles and thus the number of oocytes in the ovarian reserve at birth, using the mouse as the model, interspersed with human data where available. This information has application for understanding the variability in duration of fertility that occurs between normal individuals and with age, in premature ovarian insufficiency, and after chemotherapy or radiotherapy. © 2015 by the Society for the Study of Reproduction, Inc.

  9. 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

  10. Expression of follicle-stimulating hormone receptor (FSHR) in goat ovarian follicles and the impact of sequential culture medium on in vitro development of caprine preantral follicles.

    PubMed

    Saraiva, M V A; Celestino, J J H; Araújo, V R; Chaves, R N; Almeida, A P; Lima-Verde, I B; Duarte, A B G; Silva, G M; Martins, F S; Bruno, J B; Matos, M H T; Campello, C C; Silva, J R V; Figueiredo, J R

    2011-08-01

    This study evaluated the expression of FSH receptors (FSHR) in the different stages of goat follicle development and investigated whether the addition of increasing concentrations of FSH throughout the culture period influences the survival, growth and antral formation of in vitro-cultured caprine preantral follicles. The expression of FSHR was analysed before and after culturing follicles using real-time RT-PCR. For the culture, preantral follicles (≥150 μm) were isolated from ovarian fragments and cultured for 18 days in α-MEM+ alone or associated with recombinant FSH (rFSH: 100 or 1000 ng/ml), or in α-MEM+ supplemented with increasing concentrations of FSH throughout culture periods as follows: (a) sequential medium 1: FSH 100 ng/ml (from day 0 to 6), FSH 500 ng/ml (from day 6 to 12) and FSH 1000 ng/ml (from day 12 to 18); and (b) sequential medium 2: FSH 500 ng/ml (from day 0 to 9) and 1000 ng/ml (from day 9 to 18). Follicle development was evaluated on the basis of antral cavity formation, follicular and oocyte growth, and cumulus-oocyte complex health. The expression of FSHR in isolated caprine follicles increased from the preantral to antral phase. Regarding the culture, after 18 days, sequential medium 1 promoted follicular survival, antrum formation and a reduction in oocyte extrusion. Both sequential media promoted a higher rate of meiotic resumption compared with the other treatments. In conclusion, the addition of increased concentrations of FSH (sequential medium) has a significant impact on the in vitro development of caprine preantral follicles.

  11. 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.

  12. Ultrastructural localisation of calcium deposits in pig ovarian follicles.

    PubMed

    Rozinek, J; Rajmon, R; Petr, J; Rohlík, J; Jeseta, M; Sedmíková, M; Rehák, D; Jílek, F

    2006-01-01

    Calcium intracellular signaling regulates many intracellular events including oocyte maturation. This signaling is strongly dependent on the influx of calcium ions from extracellular spaces and on the state of intracellular calcium stores. In this study, intracellular calcium deposits were detected in follicle-enclosed pig oocytes using the combined oxalate-pyroantimonate method. These deposits were observed in the nucleus, the mitochondria, the cytoplasm, and on the surface of lipid droplets. The amount of calcium deposits was expressed as a percentage of the area of the respective cellular compartment, which is covered with calcium deposits on ultrathin sections. The distribution of calcium deposits in oocytes changed during folliculogenesis. The amount of calcium deposits in nuclei (1.11% of the area of oocyte nuclei) and cytoplasm (1.02%) in oocytes from secondary and early antral follicles (0.90% nuclei; 0.99% cytoplasm) is significantly lower (P < 0.05) than the amount of calcium deposits in these compartments in oocytes from primary follicles (2.51% nuclei; 2.34% cytoplasm) or antral follicles with growing oocyte (2.91% nuclei; 2.21% cytoplasm). The amount of calcium deposits in mitochondria of oocytes from primary follicles (1.27%) or antral follicles with growing oocyte (1.14%) is significantly lower (P < 0.05) than in the nucleus (2.51% in oocytes from primary follicles; 2.91% in growing oocytes from antral follicles) or cytoplasm (2.34% in oocytes from primary follicles; 2.21% in growing oocytes from antral follicles). The amount of calcium deposits in the cytoplasm of fully-grown oocytes (1.46%) dropped to levels significantly lower (P < 0.05) than those observed in the oocyte nucleus (2.29%). On the basis of these data, we can conclude that the population of follicles on pig ovaries differs in the distribution and concentration of calcium deposits in oocytes, and these changes may be involved in the regulation of the meiotic competence of oocytes.

  13. Altered state of primordial follicles in neonatal and early infantile rats due to maternal hypothyroidism: Light and electron microscopy approach.

    PubMed

    Danilović Luković, Jelena; Korać, Aleksandra; Milošević, Ivan; Lužajić, Tijana; Puškaš, Nela; Kovačević Filipović, Milica; Radovanović, Anita

    2016-11-01

    Thyroid hormones (TH) are one of the key factors for normal prenatal development in mammals. Previously, we showed that subclinical maternal hypothyroidism leads to premature atresia of ovarian follicles in female rat offspring in the pre-pubertal and pubertal periods. The influence of decreased concentration of TH on primordial follicles pool formation during neonatal and early infantile period of rat pups was not investigated previously. Maternal hypothyroidism during pregnancy has irreversible negative influence on primordial follicles pool formation and population of resting oocytes in female rat offspring. The study was done on neonatal and early infantile control (n-10) and hypothyroid (n-10) female rat pups derived from control (n-6) and propylthiouracil (PTU) treated pregnant dams (n-6), respectively. Ovaries of all pups were removed and processed for light and transmission electron microscopy (TEM). Number of nests, oogonia and oocytes per nest, primordial, primary, secondary and preantral follicles were determined. Screening for overall calcium presence in ovarian tissue was done using Alizarin red staining. Morphology and volume density of nucleus, mitochondria and smooth endoplasmic reticulum (sER) in the oocytes in primordial follicles was also assessed. Caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), both markers for apoptosis, and proliferating cell nuclear antigen (PCNA) for proliferation were determined in oocytes and granulosa cells in different type of follicles. In neonatal period, ovaries of hypothyroid pups had a decreased number of oogonia, oocytes and nests, an increased number of primordial follicles and a decreased number of primary and secondary follicles, while in early infantile period, increased number of primary, secondary and preantral follicles were found. Alizarin red staining was intense in hypothyroid neonatal rats that also had the highest content of dilated sER. Number of mitochondria with

  14. Lipopolysaccharide reduces the primordial follicle pool in the bovine ovarian cortex ex vivo and in the murine ovary in vivo.

    PubMed

    Bromfield, John J; Sheldon, I Martin

    2013-04-01

    Infections of the uterus or mammary gland with Gram-negative bacteria cause infertility in cattle, not only during disease but also for some time afterward. Even though these infections are in organs distant from the ovary, metritis and mastitis perturb antral follicle development and function in vivo. Although granulosa cells from antral follicles express toll-like receptor 4 (TLR4), and detect and mount an inflammatory response to lipopolysaccharide (LPS) from Gram-negative bacteria, it is not known whether LPS impacts preantral follicle development. The present study tested the hypothesis that LPS perturbs the development of primordial ovarian follicles. Exposure of bovine ovarian cortex ex vivo to LPS reduced the primordial follicle pool associated with increased primordial follicle activation. Ovarian cortex culture supernatants accumulated the inflammatory mediators IL-1beta, IL-6, and IL-8 in an LPS concentration-dependent manner. In addition, LPS exposure modulated key intracellular regulators of follicle activation with loss of the primordial follicle PTEN and cytoplasmic translocation of FOXO3. Acute exposure of mice in vivo to LPS also reduced the primordial follicle pool associated with increased follicle atresia. The increased follicle atresia was TLR4-dependent as Tlr4-deficient mice were insensitive to LPS-mediated follicle atresia. However, LPS did not affect the diameter of individually cultured bovine secondary follicles or their enclosed oocytes. In conclusion, LPS reduced the primordial ovarian follicle pool in the bovine ovarian cortex ex vivo and in the murine ovary in vivo. These observations provide an insight into how bacterial infections distant from the ovary have long term effects on fertility.

  15. Altered expression of Bcl-2 and Bax in follicles within dehydroepiandrosterone-induced polycystic ovaries in rats.

    PubMed

    Bas, Diana; Abramovich, Dalhia; Hernandez, Fatima; Tesone, Marta

    2011-05-01

    PCOS (polycystic ovary syndrome) is a heterogeneous disease characterized by hyperandrogenaemia, hirsutism, oligo- or amenorrhea, insulin resistance and anovulation. The aim of the present study was to evaluate if the balance between the ovarian expression of Bax (proapoptotic protein) and Bcl-2 (antiapoptotic protein) is altered in a PCOS model developed in rats by DHEA (dehydroepiandrosterone) administration. In addition, the ovarian morphology and the circulating progesterone levels were evaluated. Histological studies confirmed the presence of follicular cysts, atretic follicles and the absence of corpora lutea in the ovaries from the PCOS group and a significant decrease in circulating progesterone levels. Immunohistochemical studies showed that the expression of Bcl-2 and Bax were mainly localized in granulosa cells of AFs (antral follicles) in both groups. Bax expression was greater in preantral and AFs from PCOS ovarian sections than in the controls. In contrast, intense Bcl-2 immunostaining was observed in the control AFs, while Bcl-2 protein was either absent in PFs (preantral follicles) or weakly expressed in AFs from PCOS rats. These results were partially confirmed by Western studies. Data revealed that the ovarian level of Bcl-2 protein was lower in PCOS than in the control and that there were no differences in Bax ovarian levels between groups. However, Bax/Bcl-2 ratio was significantly higher in PCOS group than in the control group. In conclusion, an increase in ovarian apoptosis through an imbalance among the Bcl-2 family members may be involved in the transformation of growing follicles in cystic follicles in the ovaries from DHEA-induced PCOS rats.

  16. 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

  17. In Vitro Activation of Follicles and Fresh Tissue Auto-transplantation in Primary Ovarian Insufficiency Patients

    PubMed Central

    Zhai, Jun; Yao, Guidong; Dong, Fangli; Bu, Zhiqin; Cheng, Yuan; Sato, Yorino; Hu, Linli; Zhang, Yingying; Wang, Jingyuan; Dai, Shanjun; Li, Jing; Sun, Jing; Hsueh, Aaron J.; Kawamura, Kazuhiro

    2016-01-01

    Context: Recently, two patients with primary ovarian insufficiency (POI) delivered healthy babies after in vitro activation (IVA) treatment followed by auto-transplantation of frozen-thawed ovarian tissues. Objective: This study sought to report the first case of live birth after IVA treatment following fresh ovarian tissue grafting in patients with POI, together with monitoring of follicle development and serum hormonal changes. Design: This was a prospective observational cohort study. Setting: We performed IVA treatment in 14 patients with POI with mean age of 29 years, mean duration since last menses of 3.8 years, and average basal FSH level of 94.5 mIU/mL. Interventions: Prior to IVA treatment, all patients received routine hormonal treatments with no follicle development. We removed one ovary from patients with POI and treated them with Akt stimulators. We improved upon early procedures by grafting back fresh tissues using a simplified protocol. Main Outcome Measures: In six of the 14 patients (43%), a total of 15 follicle development waves were detected, and four patients had successful oocyte retrieval to yield six oocytes. For two patients showing no spontaneous follicle growth, human menopausal gonadotropin treatment induced follicle growth at 6–8 months after grafting. After vitro fertilization of oocyte retrieved, four early embryos were derived. Following embryo transfer, one patient became pregnant and delivered a healthy baby boy, with three other embryos under cryopreservation. Conclusion: IVA technology can effectively activate residual follicles in some patients with POI and allow them to conceive their own genetic offspring. IVA may also be useful for treating patients with ovarian dysfunction including aging women and cancer survivors. PMID:27571179

  18. In vitro interaction between resistin and peroxisome proliferator-activated receptor γ in porcine ovarian follicles.

    PubMed

    Rak-Mardyła, Agnieszka; Drwal, Eliza

    2016-03-01

    In the present study, using real-time polymerase chain reaction and immunoblotting methods, we quantified the expression of peroxisome proliferator-activated receptor (PPAR) γ, PPARα and PPARβ in different sized ovarian follicles (small (SF), medium (MF) and large (LF) follicles) in prepubertal and adult pigs. In prepubertal pigs, PPARγ and PPARα expression was highest in LF; however, PPARβ expression did not differ among SF, MF and LF. In mature pigs, only protein expression of PPARγ and PPARα increased during ovarian follicle development. Following identification of very high levels of PPARγ expression in LF in prepubertal and adult pigs, using in vitro culture of ovarian follicles, we determined the effect of resistin at 0.1, 1 and 10ngmL(-1) on PPARγ mRNA and protein expression and the effect of rosiglitazone at 25 and 50µM (a PPARγ agonist) on resistin mRNA and protein expression. Resistin increased PPARγ expression in ovarian follicles in both prepubertal and adult pigs, whereas rosiglitazone had an inhibitory effect on resistin expression. The role of PPARγ in regulating the effects of resistin on ovarian steroidogenesis was investigated using GW9662 (a PPARγ antagonist at dose of 1μM). In these studies, GW9662 reversed the effect of resistin on steroid hormone secretion. The data suggest that there is local cooperation between resistin and PPARγ expression in the porcine ovary. Resistin significantly increased the expression of PPARγ, whereas PPARγ decreased resistin expression; thus, PPARγ is a new key regulator of resistin expression and function.

  19. Ovarian ageing: the role of mitochondria in oocytes and follicles.

    PubMed

    May-Panloup, Pascale; Boucret, Lisa; Chao de la Barca, Juan-Manuel; Desquiret-Dumas, Valérie; Ferré-L'Hotellier, Véronique; Morinière, Catherine; Descamps, Philippe; Procaccio, Vincent; Reynier, Pascal

    2016-11-01

    There is a great inter-individual variability of ovarian ageing, and almost 20% of patients consulting for infertility show signs of premature ovarian ageing. This feature, taken together with delayed childbearing in modern society, leads to the emergence of age-related ovarian dysfunction concomitantly with the desire for pregnancy. Assisted reproductive technology is frequently inefficacious in cases of ovarian ageing, thus raising the economic, medical and societal costs of the procedures. Ovarian ageing is characterized by quantitative and qualitative alteration of the ovarian oocyte reserve. Mitochondria play a central role in follicular atresia and could be the main target of the ooplasmic factors determining oocyte quality adversely affected by ageing. Indeed, the oocyte is the richest cell of the body in mitochondria and depends largely on these organelles to acquire competence for fertilization and early embryonic development. Moreover, the oocyte ensures the uniparental transmission and stability of the mitochondrial genome across the generations. This review focuses on the role played by mitochondria in ovarian ageing and on the possible consequences over the generations. PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews concerning mitochondria and ovarian ageing, in animal and human species. Searches were performed using keywords belonging to three groups: 'mitochondria' or 'mitochondrial DNA'; 'ovarian reserve', 'oocyte', 'ovary' or 'cumulus cells'; and 'ageing' or 'ovarian ageing'. These keywords were combined with other search phrases relevant to the topic. References from these articles were used to obtain additional articles. There is a close relationship, in mammalian models and humans, between mitochondria and the decline of oocyte quality with ageing. Qualitatively, ageing-related mitochondrial (mt) DNA instability, which leads to the accumulation of mtDNA mutations in the oocyte, plays a key role in

  20. Granulosa cell responsiveness to follicle stimulating hormone during early growth of hen ovarian follicles.

    PubMed

    Johnson, A L; Lee, Jeeyoung

    2016-01-01

    In the laying hen ovary, the cyclic recruitment of a follicle represents a process in which a single follicle is selected to enter the rapid growth phase and undergo final maturation prior to ovulation. Published data support the proposal that final differentiation of the granulosa cell (GC) layer commences at the time of follicle selection. This process is characterized by the enhanced capacity for FSH-induced cell signaling via the protein kinase A/cyclic adenosine monophosphate (cAMP) pathway. One consequence of such signaling within the GC layer is the initial capacity for steroidogenesis (predominantly progesterone production) mediated by increased expression of mRNA encoding steroidogenic acute regulatory protein (STAR) and the cholesterol side-chain cleavage enzyme (CYP11A). Prior to selection, the GC layer remains minimally responsive to a 3 h challenge with FSH (10 ng/mL), in vitro, compared to that from the most recently selected 9- to 12-mm follicle. By comparison, when the duration of the cell culture prior to FSH challenge is increased to 18 h, GCs collected from 1- to 2-mm, 3- to 5-mm, and 6- to 8-mm follicles respond to a 3 h FSH challenge by increasing STAR expression and progesterone production, with the greatest response from GCs collected from 6- to 8-mm follicles. Culture with Bone Morphogenetic Protein 6 (BMP6) enhances both CYP11A expression and FSH responsiveness at each stage of development, with the greatest response again occurring in GCs from 6- to 8-mm follicles. Significantly, factors that activate mitogen activated protein kinase (MAPK) or protein kinase C (PKC) signaling prevent the ability of prolonged culture or culture with BMP6 to induce FSH-responsiveness and the initiation of GC differentiation at each stage of development. Collectively, these results provide further support for the hypothesis that prior to follicle selection, inhibitory cell signaling (e.g., MAPK, PKC) maintains the GC layer in an undifferentiated state in

  1. Ovarian innervation develops before initiation of folliculogenesis in the rat.

    PubMed

    Malamed, S; Gibney, J A; Ojeda, S R

    1992-10-01

    Sympathetic neurotransmitters have been shown to be present in the ovary of the rat during early postnatal development and to affect steroidogenesis before the ovary becomes responsive to gonadotropins, and before the first primordial follicles are formed. This study was undertaken to determine if development of the ovarian innervation is an event that antedates the initiation of folliculogenesis in the rat, Rattus norvegicus. Serial sections of postnatal ovaries revealed a negligible frequency of follicles 24 h after birth (about 1 primordial follicle per ovary). Twelve hours later there were about 500 follicles per ovary, a number that more than doubled to about 1300 during the subsequent 12 h, indicating that an explosive period of follicular differentiation occurs between the end of postnatal days 1 and 2. Electron microscopy demonstrated that before birth the ovaries are already innervated by fibers containing clear and dense-core vesicles. Immunohistochemistry performed on either fetal (day 19) or newborn (less than 15h after birth) ovaries showed the presence of catecholaminergic nerves, identified by their content of immunoreactive tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. While some of these fibers innervate blood vessels, others are associated with primordial ovarian cells, thereby suggesting their participation in non-vascular functions. Since prefollicular ovaries are insensitive to gonadotropins, the results suggest that the developing ovary becomes subjected to direct neurogenic influences before it acquires responsiveness to gonadotropins.

  2. Effects of resistin on porcine ovarian follicle steroidogenesis in prepubertal animals: an in vitro study

    PubMed Central

    2013-01-01

    Background Resistin was first reported to be an adipocyte-specific hormone, but recent studies have indicated a connection between resistin and reproductive function. However, it is not yet known if resistin is expressed by the ovary and if it can affect steroidogenesis in ovarian follicles from prepubertal pigs. Methods In this study, using real time PCR, immunoblotting, and ELISA, we quantified resistin expression and concentration in maturing ovarian follicles (small, 3–4 mm; medium, 4–5 mm; large, 6–7 mm) collected from prepubertal pigs. In addition, the dose-responsive effects of recombinant human resistin (0.1, 1, 10, and 100 ng/ml) on steroid hormone (i.e., progesterone [P4], androstendione [A4], testosterone [T], and estradiol [E2]) secretion in culture medium and steroidogenic enzyme (i.e., CYP11A1, 3betaHSD, CYP17A1, 17betaHSD, and CYP19A1) expression in ovarian follicles were determined. Results We observed that resistin gene and protein expression increased significantly (P < 0.05) during follicular growth, with large follicles expressing the highest level of this adipokine. Recombinant resistin also increased P4, A4, and T secretion by up-regulating the steady state levels of CYP11A1, 3betaHSD, CYP17A1, and 17betaHSD. Recombinant resistin had no effects on E2 secretion and CYP19A1 expression in ovarian follicles. Conclusion Our results show resistin expression in ovarian follicles from prepubertal pigs for the first time. We also show that recombinant resistin stimulates steroidogenesis in ovarian follicles by increasing the expression of CYP11A1, 3betaHSD, CYP17A1, and 17betaHSD. The presence of resistin in the porcine ovary and its direct effects on steroidogenesis suggest that resistin is a new regulator of ovary function in prepubertal animals. PMID:23680257

  3. Ovarian tissue culture in the presence of VEGF and fetuin stimulates follicle growth and steroidogenesis.

    PubMed

    Asadi, Ebrahim; Najafi, Atefeh; Moeini, Ashraf; Pirjani, Reihaneh; Hassanzadeh, Gholamreza; Mikaeili, Saideh; Salehi, Ensieh; Adutwum, Emmanuel; Soleimani, Mansoureh; Khosravi, Fariba; Barati, Mahmood; Abolhassani, Farid

    2017-02-01

    Ovarian tissue cryopreservation together with follicle culture provides a promising technique for fertility preservation in cancer patients. The study aimed to evaluate follicle parameters in a culture medium supplemented with VEGFA165 and/or fetuin. Vitrified-warmed ovarian cortical pieces were divided randomly into four culture groups consisting of basic culture medium (control), and the basic culture medium supplemented with VEGFA165, fetuin or both. After six days of culture, we evaluated the following: percentage of resting, primary and secondary growing follicles; survival rate; steroid hormones production; levels of reactive oxygen species, lipid peroxidation and total antioxidant capacity; and developmental and antioxidant gene expression. The addition of VEGFA165 alone or in combination with fetuin to the culture medium caused resting follicle activation and increased the number of growing follicles. In the VEGFA165 group, we found a significant increase in the concentrations of 17β-estradiol at day 6 and progesterone from 4th day of the culture period. In the VEGFA165 + fetuin group, the concentration of 17β-estradiol rose at day 4 of the culture period. The levels of BMP15, GDF9 and INHB mRNAs were increased in all treated groups. In the fetuin and fetuin + VEGFA165 groups, we observed a high level of total antioxidant capacity and expression of SOD1 and CAT genes, low reactive oxygen species and lipid peroxidation levels and increased number of viable follicles. In conclusion, the present study provides useful evidence that supplementation of culture medium with VEGFA165 + fetuin leads to primordial follicle activation and development and increased percentage of healthy secondary growing follicles. © 2017 Society for Endocrinology.

  4. Changes in ovarian protein expression during primordial follicle formation in the hamster.

    PubMed

    Mukherjee, Anindit; Reisdorph, Nichole; Guda, Chttibabu; Pandey, Sanjit; Roy, Shyamal K

    2012-01-02

    Although many proteins have been shown to affect the transition of primordial follicles to the primary stage, factors regulating the formation of primordial follicles remains sketchy at best. Differentiation of somatic cells into early granulosa cells during ovarian morphogenesis is the hallmark of primordial follicle formation; hence, critical changes are expected in protein expression. We wanted to identify proteins, the expression of which would correlate with the formation of primordial follicles as a first step to determine their biological function in folliculogenesis. Proteins were extracted from embryonic (E15) and 8-day-old (P8) hamster ovaries and fractionated by two-dimensional gel electrophoresis. Gels were stained with Proteosilver, and images of protein profiles corresponding to E15 and P8 ovaries were overlayed to identify protein spots showing altered expression. Some of the protein spots were extracted from SyproRuby-stained preparative gels, digested with trypsin, and analyzed by mass spectrometry. Both E15 and P8 ovaries had high molecular weight proteins at acidic, basic, and neutral ranges; however, we focused on small molecular weight proteins at 4-7 pH range. Many of those spots might represent post-translational modification. Mass spectrometric analysis revealed the identity of these proteins. The formation of primordial follicles on P8 correlated with many differentially and newly expressed proteins. Whereas Ebp1 expression was downregulated in ovarian somatic cells, Sfrs3 expression was specifically upregulated in newly formed granulosa cells of primordial follicles on P8. The results show for the first time that the morphogenesis of primordial follicles in the hamster coincides with altered and novel expression of proteins involved in cell proliferation, transcriptional regulation, and metabolism. Therefore, formation of primordial follicles is an active process requiring differentiation of somatic cells into early granulosa cells and

  5. Abnormal in vitro development of ovarian follicles explanted from mice exposed to tetrachlorvinphos.

    PubMed

    Nayudu, P L; Kiesel, P S; Nowshari, M A; Hodges, J K

    1994-01-01

    A system of mouse ovarian follicle culture in which follicles can be grown from a preantral stage of development through antral formation has been developed and modified recently by Nayudu and colleagues. Follicles have been shown to grow in this culture system at a relatively constant rate and show responsiveness to LH at the end of the culture by ovulation of mature oocytes. Reported here are the distinctly different in vitro growth patterns of follicles explanted from 22- to 24-day-old mice during a period when the colony was being treated for skin parasites with tetrachlorvinphos (TCVP) (Rabond). There is to date no information on the effects of this compound on the mammalian female reproductive system. For follicles from the TCVP treated group, the duration of growth as intact follicles was markedly reduced in comparison to mice of the same strain and source not treated with TCVP. In the treated group, premature termination of follicular growth was also associated with the spontaneous expulsion of oocytes with immature nuclei and without cumulus cells. For those follicles from treated mice that did remain in culture until the day luteinizing hormone was given, the ovulatory response was poor and the maturation response of the oocytes was low in comparison with the follicles from untreated mice. The effect of the treatment on the follicles was further characterized by obvious differences in the patterns of growth. Follicles in the untreated group grew in a linear pattern at around 25 microns/day; a single phase, fast pattern for the whole culture period.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    SciTech Connect

    Wang, Wei Craig, Zelieann R. Basavarajappa, Mallikarjuna S. Gupta, Rupesh K. Flaws, Jodi A.

    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). 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

  7. Ovarian Primordial and Non-Growing Follicle Counts According to the Stages of Reproductive Aging Workshop (STRAW) Staging System

    PubMed Central

    Hansen, Karl R.; Craig, LaTasha B.; Zavy, Michael T.; Klein, Nancy A.; Soules, Michael R.

    2011-01-01

    Objective To characterize the ovarian primordial and non-growing follicle number according to the Stages of Reproductive Aging Workshop (STRAW) staging system as defined by menstrual cycle characteristics. Methods Normal ovaries were collected from 63 women (age 26-52 years) undergoing oophorectomy for benign indications. Prior to surgery, each participant completed a detailed questionnaire collecting information regarding menstrual cycle characteristics and were classified by bleeding patterns into STRAW stages -4, -3, -2, and -1. A single ovary was selected for determination of the ovarian primordial and total non-growing follicle number utilizing a validated fractionator/optical disector method. A subset of the participants (n = 43) underwent transvaginal ultrasound examination for the determination of the ovarian antral follicle count and serum measurements of FSH, estradiol, anti-müllerian hormone and inhibin B. All measurements were obtained within two weeks of surgery, irrespective of cycle day. Results Significant differences were identified in ovarian primordial (p <0.0001) and non-growing follicle (p <0.0001) counts across the STRAW stages. In post-hoc testing, the differences in primordial follicle counts were significant between each of the STRAW stages. Significant differences were also identified in serum levels of anti-müllerian hormone, FSH and the ovarian antral follicle count across the STRAW stages. Conclusions Progression through the STRAW stages as defined by menstrual cycle characteristics is associated with progressive and significant decreases in the ovarian primordial follicle number. PMID:22189385

  8. Overexpression of Uromodulin-like1 accelerates follicle depletion and subsequent ovarian degeneration

    PubMed Central

    Wang, W; Tang, Y; Ni, L; Kim, E; Jongwutiwes, T; Hourvitz, A; Zhang, R; Xiong, H; Liu, H-C; Rosenwaks, Z

    2012-01-01

    Murine Uromodulin-like 1 (Umodl1) encodes Ca2+-dependent EGF-like membrane-bound proteins. This study presents its novel expression in the immune and female reproductive systems. Upon stimulation by CD3/CD28 antibodies, Umodl1 showed a prompt and robust response in the proliferating CD4+ T cells, suggesting its implication in immune defense against pathogens. In ovary, Umodl1 is regulated by gonadotropins. Mice carrying extra copies of functional Umodl1 were generated by BAC transgenesis. Defects in the female reproductive system became evident from 4 months of age, manifested by reduced or diminished fertility. Histology revealed that the ovaries contained very few discernible follicles in the cortical region, and were devoid of distinguishable corpus lutea (CL). Among the multilayered preantral follicles, elevated apoptosis was observed in both the oocytes and surrounding granulosa cells (GCs). Furthermore, a high level of PPARγ indicated an abnormal adipogenesis in the mutant ovaries, which resulted in the conversion of GCs into adipocytes. By 6 months of age, all mutant mice became anovulatory. Ovarian tissues including CL, follicles of various stages and associated stromal cells were degenerated. Altered expression of AMH, follicle-stimulating hormone and other ovary-specific marker genes such as Gdf-9, Rnf35, NOHLH and Gcx-1 further demonstrated that the molecular properties of the mutant ovaries have been severely disturbed. This work presents a novel animal model for investigating the pathogenesis of premature ovarian failure or early ovarian ageing. PMID:23190605

  9. Single-chain bifunctional vascular endothelial growth factor (VEGF)-follicle-stimulating hormone (FSH)-C-terminal peptide (CTP) is superior to the combination therapy of recombinant VEGF plus FSH-CTP in stimulating angiogenesis during ovarian folliculogenesis.

    PubMed

    Trousdale, Rhonda K; Pollak, Susan V; Klein, Jeffrey; Lobel, Leslie; Funahashi, Yasuhiro; Feirt, Nikki; Lustbader, Joyce W

    2007-03-01

    Infertility technologies often employ exogenous gonadotropin therapy to increase antral follicle production. In an effort to enhance ovarian response, several long-acting FSH therapies have been developed including an FSH-C-terminal peptide (CTP), where the FSH subunits are linked by the CTP moiety from human chorionic gonadotropin, which is responsible for the increased half-life of human chorionic gonadotropin. We found that administration of FSH-CTP for ovarian hyperstimulation in rats blunted ovarian follicle vascular development. In women, reduced ovarian vasculature has been associated with lower pregnancy rates. We were interested in determining whether vascular endothelial growth factor (VEGF) therapy could enhance ovarian angiogenesis in FSH-CTP-treated rats. Coadministration of systemic FSH-CTP plus recombinant VEGF was compared with treatment with a novel, single-chain bifunctional VEGF-FSH-CTP (VFC) analog. For VFC, the FSH portion targets the protein to the ovary and stimulates follicle growth, whereas VEGF enhances local vascular development. Both in vitro and in vivo studies confirm the dual FSH and VEGF action of the VFC protein. Evaluation of ovarian follicle development demonstrates that administration of combination therapy using VEGF and FSH-CTP failed to increase follicle vasculature above levels seen with FSH-CTP monotherapy. However, treatment with VFC significantly increased follicle vascular development while concurrently increasing the number of large antral follicles produced. In conclusion, we report the production and characterization of a long-acting, bifunctional VEGF-FSH-CTP protein that is superior to combination therapy for enhancing VEGF activity in the ovary and stimulating follicular angiogenesis in rats.

  10. In vitro development of primordial follicles after long-term culture of goat ovarian tissue.

    PubMed

    Matos, M H T; Bruno, J B; Rocha, R M P; Lima-Verde, I B; Santos, K D B; Saraiva, M V A; Silva, J R V; Martins, F S; Chaves, R N; Báo, S N; Figueiredo, J R

    2011-06-01

    This study aims to investigate the effects of follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on the survival and growth of caprine preantral follicles. Ovarian tissues were cultured for 1, 7, 14, 21 or 28 days in medium supplemented with FSH (FSH-2d or FSH-7d, i.e., with replacement of the culture medium every 2 or 7 days, respectively) or FSH+FGF-2 (replacement of the medium every 2 days). Non-cultured (control) and cultured ovarian fragments were processed for histological and ultrastructural analysis. After 28 days of culture, the media supplemented with FSH-2d was the most effective in maintaining the percentage of normal follicles and in promoting follicular growth. Furthermore, both treatments with FSH increased the percentage of the primary follicles. However, ultrastructural studies did not confirm follicular integrity from 14 days of culture onward. In conclusion, culturing tissue for up to 7 days in medium containing FSH alone or combined with FGF-2 maintains caprine preantral follicle integrity and promotes their growth in vitro.

  11. Expression and function of fibroblast growth factor 18 in the ovarian follicle in cattle.

    PubMed

    Portela, Valerio M; Machado, Mariana; Buratini, Jose; Zamberlam, Gustavo; Amorim, Renee L; Goncalves, Paulo; Price, Christopher A

    2010-09-01

    Fibroblast growth factors (FGF) are involved in paracrine signaling between cell types in the ovarian follicle. FGF8, for example, is secreted by oocytes and controls cumulus cell metabolism. The closely related FGF18 is also expressed in oocytes in mice. The objective of this study was to assess the potential role of FGF18 in follicle growth in a monovulatory species, the cow. Messenger RNA encoding FGF18 was detected primarily in theca cells, and in contrast to the mouse, FGF18 was not detected in bovine oocytes. Addition of FGF18 protein to granulosa cell cultures inhibited estradiol and progesterone secretion as well as the abundance of mRNA encoding steroidogenic enzymes and the follicle-stimulating hormone receptor. In vivo, onset of atresia of the subordinate follicle was associated with increased thecal FGF18 mRNA levels and FGF18 protein in follicular fluid. In vitro, FGF18 altered cell cycle progression as measured by flow cytometry, resulting in increased numbers of dead cells (sub-G1 peak) and decreased cells in S phase. This was accompanied by decreased levels of mRNA encoding the cell cycle checkpoint regulator GADD45B. Collectively, these data point to a unique role for this FGF in signaling from theca cells to granulosa cells and suggest that FGF18 influences the process of atresia in ovarian follicles.

  12. The correlation between the number of antral follicles and ovarian reserves (preantral follicles) in purebred Bos indicus and Bos taurus cows.

    PubMed

    Silva-Santos, Katia Cristina; Santos, Gustavo Martins Gomes Dos; Siloto, Letícia Schmidt; Santos, Joabel Tonellotto Dos; Oliveira, Eduardo Raele de; Machado, Fernanda Zandonadi; Rosa, Camila Oliveira; Seneda, Marcelo Marcondes

    2014-12-30

    The objectives of this study were to compare populations of preantral follicles between purebred Bos indicus and Bos taurus cows with high or low antral follicle counts (AFC) and to correlate the number of preantral follicles with the population of antral follicles. Nelore (Bos indicus, n=100) and Angus (Bos taurus, n=100) cow ovaries were collected at abattoirs and examined using ultrasonography. Antral follicles ≥3mm were counted, and the cows ovaries were assigned to high (G-High) or low (G-Low) AFC groups based on the mean number (±1 SD) of ovarian antral follicles: Bos indicus with high AFC (≥57 follicles, n=8) or low AFC (≤21 follicles, n=8) and Bos taurus with high (≥45 follicles, n=10) or low AFC (≤13 follicles, n=10). The ovaries were processed, and the number of preantral follicles was estimated. Between-groups comparisons were performed using a Kruskal-Wallis test, and the correlation between preantral and antral follicles was evaluated using a Pearson's correlation test (P≤0.05). A large variation in the number of preantral follicles was observed among the animals. Although there was a correlation between the population of preantral follicles and the number of antral follicles, there was no difference between the mean number of preantral follicles in the Bos indicus G-High (48,349±30,149) and G-Low groups (33,037±31,710) or between the Bos taurus G-High (35,050±36,060) and G-Low groups (30,481±43,360). Therefore, the preantral follicle population did not differ between purebred Bos indicus and Bos taurus cattle with high or low AFC but was correlated with the number of antral follicles. In addition to the large within-groups variation in the number of preantral follicles, some cows with high AFC had lower populations of preantral follicles compared to the low AFC group, and the highest population of preantral follicles was observed in both Bos indicus and Bos taurus with low AFC. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Influence of metabolic hormones and nutrition on ovarian follicle development in cattle: practical implications.

    PubMed

    Gong, J G

    2002-07-01

    Nutrition has long been known to have a profound influence on reproductive performance of female cattle, but the underlying mechanism remains poorly understood. Whilst early investigations focused on the modulation of nutrition on hypothalamic-pituitary axis, more recent studies have tested the hypothesis that metabolic hormones as nutritional signals exert a direct effect at the ovarian level. In cattle, treatment with recombinant bovine somatotrophin (rGH) significantly increases the population of small ovarian follicles. This is associated with increases in circulating concentrations of insulin and insulin-like growth factor-I (IGF-I). Subsequent studies, both in vitro and in vivo, have highlighted the importance of IGF-I and/or insulin acting in synergy with FSH and LH. More recently, we demonstrated that feeding heifers with 200% maintenance requirements for a short period significantly increases circulating insulin concentrations and population of small ovarian follicles. Based on these findings, our recent work has aimed at addressing some practical problems in cattle production. Firstly, we showed that both rGH pretreatment and increased dietary intake significantly enhance the response to standard superovulatory regimes. Secondly, we have demonstrated that feeding a diet to increase circulating insulin concentrations during the early lactation can advance the first ovulation postpartum and increase conception rate to the first service in dairy cows. In summary, nutrition influences ovarian follicle development in cattle possibly through changes in metabolic hormones. These interactions can be manipulated to improve reproductive performance.

  14. Leptin receptor signaling inhibits ovarian follicle development and egg laying in chicken hens

    PubMed Central

    2014-01-01

    Background Nutrition intake during growth strongly influences ovarian follicle development and egg laying in chicken hens, yet the underlying endocrine regulatory mechanism is still poorly understood. The relevant research progress is hindered by difficulties in detection of leptin gene and its expression in the chicken. However, a functional leptin receptor (LEPR) is present in the chicken which has been implicated to play a regulatory role in ovarian follicle development and egg laying. The present study targeted LEPR by immunizing against its extracellular domain (ECD), and examined the resultant ovarian follicle development and egg-laying rate in chicken hens. Methods Hens that have been immunized four times with chicken LEPR ECD were assessed for their egg laying rate and feed intake, numbers of ovarian follicles, gene expression profiles, serum lipid parameters, as well as STAT3 signaling pathway. Results Administrations of cLEPR ECD antigen resulted in marked reductions in laying rate that over time eventually recovered to the levels exhibited by the Control hens. Together with the decrease in egg laying rate, cLEPR-immunized hens also exhibited significant reductions in feed intake, plasma concentrations of glucose, triglyceride, high-density lipoprotein, and low-density lipoprotein. Parallelled by reductions in feed intake, mRNA gene expression levels of AgRP, orexin, and NPY were down regulated, but of POMC, MC4R and lepR up-regulated in Immunized hen hypothalamus. cLEPR-immunization also promoted expressions of apoptotic genes such as caspase3 in theca and fas in granulosa layer, but severely depressed IGF-I expression in both theca and granulosa layers. Conclusions Immunization against cLEPR ECD in egg-laying hens generated antibodies that mimic leptin bioactivity by enhancing leptin receptor transduction. This up-regulated apoptotic gene expression in ovarian follicles, negatively regulated the expression of genes that promote follicular development

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

    PubMed Central

    Wang, Wei; Craig, Zelieann R.; Basavarajappa, Mallikarjuna S.; Gupta, Rupesh; Flaws, Jodi A.

    2011-01-01

    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 32–35 days) were cultured with vehicle control (dimethylsulfoxide [DMSO]) or DEHP (1–100μg/ml) ± N-acetyl cysteine (NAC, an antioxidant at 0.25–1mM). 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 (0.25–1mM) blocks the ability of DEHP to inhibit follicle growth. Furthermore, DEHP (10μg/ml) significantly increases ROS levels and reduces the expression and activity of SOD1 compared to DMSO controls, whereas NAC (0.5mM) 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. PMID:22155089

  16. Systemic signals in aged males exert potent rejuvenating effects on the ovarian follicle reserve in mammalian females

    PubMed Central

    Niikura, Yuichi; Niikura, Teruko; Wang, Ning; Satirapod, Chonthicha; Tilly, Jonathan L.

    2010-01-01

    Through the use of parabiosis in mice, aging-related deterioration of skeletal muscle and liver has been linked to a loss of systemic factors that support adult stem or progenitor cell activity. Since aging-related ovarian failure has recently been attributed, at least in part, to a loss of de-novo oocyte-containing follicle formation associated with declining oogonial stem cell activity, herein we tested in mice if aging-related changes in systemic factors influence the size of the ovarian follicle reserve. Ovaries of young (2-month-old) females parabiotically joined with young females for 5 weeks possess comparable numbers of healthy and degenerative (atretic) oocyte-containing follicles in their ovaries as those detected in non-parabiotic young females. Joining of young females with young males significantly increases follicle atresia without a net change healthy follicle numbers. Surprisingly, young females joined with aged (24-month-old) males exhibit a significant increase in the number of primordial follicles comprising the ovarian reserve, and this occurs without changes in follicle growth activation or atresia. Blood of aged males also induces ovarian expression of the germ cell-specific meiosis gene, Stimulated by retinoic acid gene 8 (Stra8), in ovaries of female parabionts, further supporting the conclusion that the observed changes in the follicle reserve of females joined with aged males reflect increased oocyte formation. Thus, factors in male blood exert dramatic effects on ovarian follicle dynamics, and aging males possess a beneficial systemic factor that significantly expands the ovarian follicle reserve in females through enhanced oogenesis. PMID:21212462

  17. Systemic signals in aged males exert potent rejuvenating effects on the ovarian follicle reserve in mammalian females.

    PubMed

    Niikura, Yuichi; Niikura, Teruko; Wang, Ning; Satirapod, Chonthicha; Tilly, Jonathan L

    2010-12-01

    Through the use of parabiosis in mice, aging-related deterioration of skeletal muscle and liver has been linked to a loss of systemic factors that support adult stem or progenitor cell activity. Since aging-related ovarian failure has recently been attributed, at least in part, to a loss of de-novo oocyte-containing follicle formation associated with declining oogonial stem cell activity, herein we tested in mice if aging-related changes in systemic factors influence the size of the ovarian follicle reserve. Ovaries of young (2-month-old) females parabiotically joined with young females for 5 weeks possess comparable numbers of healthy and degenerative (atretic) oocyte-containing follicles in their ovaries as those detected in non-parabiotic young females. Joining of young females with young males significantly increases follicle atresia without a net change healthy follicle numbers. Surprisingly, young females joined with aged (24-month-old) males exhibit a significant increase in the number of primordial follicles comprising the ovarian reserve, and this occurs without changes in follicle growth activation or atresia. Blood of aged males also induces ovarian expression of the germ cell-specific meiosis gene,Stimulated by retinoic acid gene 8 (Stra8), in ovaries of female parabionts, further supporting the conclusion that the observed changes in the follicle reserve of females joined with aged males reflect increased oocyte formation. Thus, factors in male blood exert dramatic effects on ovarian follicle dynamics, and aging males possess a beneficial systemic factor that significantly expands the ovarian follicle reserve in females through enhanced oogenesis.

  18. Hedgehog signaling pathway in small bovine ovarian follicles

    USDA-ARS?s Scientific Manuscript database

    The hedgehog signaling pathway is involved in the regulation of cell proliferation, differentiation, and turnover in a variety of mammalian embryonic and adult tissues including bovine ovarian granulosa and theca cells. Binding of hedgehog to the patch receptor derepresses smoothened resulting in t...

  19. 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.

  20. Involvement of miRNAs and Cell-Secreted Vesicles in Mammalian Ovarian Antral Follicle Development.

    PubMed

    da Silveira, Juliano C; de Andrade, Gabriella M; Nogueira, Marcelo F G; Meirelles, Flávio V; Perecin, Felipe

    2015-12-01

    Ovarian follicular development is a controlled series of events culminating with an ovulatory or atretic follicle. MicroRNAs (miRNAs) are small noncoding RNAs involved in translational regulation of genes in different developmental processes. Deletion of Dicer in mice ovaries demonstrated the importance of miRNAs in reproduction, which led to infertility. The miRNAs were thought to act only within host cells; however, these molecules are also present in cell-secreted vesicles. These vesicles are present in body fluids such as milk, serum, and ovarian follicular fluid. Vesicles are secreted in extracellular fluids and travel from donor to target cells, mediating transfer of bioactive material. Herein we discuss the role of hormonal-regulated miRNAs within different ovarian follicular cells as well as cell-secreted vesicles participation in mammalian ovarian follicular fluid. Furthermore, we discuss the possibility of miRNAs transference mediated by cell-secreted vesicles present in ovarian follicular fluid, increasing the versatility of miRNA functions during antral follicle development. © The Author(s) 2015.

  1. Relationship of antral follicle counts to fertility and ovarian AMH mRNA levels in beef cows

    USDA-ARS?s Scientific Manuscript database

    In mammalian females, reproductive senescence is associated with decreased numbers of follicles in the ovary. Anti-Mullerian Hormone (AMH) of ovarian origin has been proposed to be a biomarker of the number of follicles in the ovary and of fertility in women, as well as to control activation of pri...

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

    SciTech Connect

    Yamoto, M.; Nakano, R.; Iwasaki, M.; Ikoma, H.; Furukawa, K.

    1986-08-01

    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 that the increase and decrease in receptors for hLH during the menstrual cycle might play an important role in the regulation of the ovarian cycle.

  3. 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

  4. Induction of multiple ovulation via modulation of angiotensin II receptors in in vitro ovarian follicle culture models.

    PubMed

    Kim, Yong Jin; Kim, Yoon Young; Kang, Byeong-Cheol; Kim, Moon Suk; Ko, In Kap; Liu, Hung Ching; Rosenwaks, Zev; Ku, Seung-Yup

    2016-09-15

    In vitro culture of ovarian follicles is a promising bioengineering technique for retrieving fertilizable oocytes from preserved ovarian tissues of cancer survivors. However, current in vitro follicle culture techniques are labour-intensive and of low efficiency, as only single follicle culture (SFC) has been possible to date. The present study investigated the feasibility of multifollicular cluster culture (MFCC) system using angiotensin II receptor (ATII-Rc) analogues. Ovarian pre-antral follicles isolated from 2-week-old C57BL6 mice were cultured with ATII-Rc agonist or antagonist and their maturation outcomes were compared with control group. When single follicles were cultured, the ovulation and maturation rates were similar in all three groups. When three-follicle clusters were cultured, up to three follicles were ovulated in the ATII-Rc agonist group while none or one follicle ovulated in control or antagonist groups (p < 0.0001). Significantly higher numbers of mature oocytes were obtained in the agonist group (three-follicle 28.2 ± 4.9 vs. SFC 11.0 ± 1.3, per 25 cultured droplets) (p < 0.0001), and the development of each fertilized oocytes was comparable to those from SFC. It is therefore concluded that this novel MFCC system can significantly improve the efficiency of in vitro mature oocyte retrieval via ATII-Rc modulation. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Immunohistochemical detection of a very high density lipoprotein (VHDL) in ovarian follicles of Triatoma infestans.

    PubMed

    González, M S; Ronderos, J R; Rimoldi, O J; Brenner, R R

    2001-04-01

    The ability of Triatoma infestans ovarian follicles to synthesize a very high-density lipoprotein (VHDL) has been examined by immunohistochemical methods. This kind of lipoprotein can be envisaged as a storage hexameric protein present in the hemolymph of some insect species. VHDL immunoreactivity is observed in oocytes at different stages of maturation. The antigen is present in the oocyte cytoplasm as well as in the follicular epithelial cells. The immunopositive reaction in the apical surface of follicle cells suggests both a VHDL synthesis and a secretion process. Furthermore, VHDL seems to be stored into oocyte in yolk granules. On the contrary, no immunopositive reaction is observed in the intracellular spaces between follicle cells, suggesting that VHDL is not incorporated from hemolymph into the oocyte.

  6. Analysis of LH receptor in canine ovarian follicles throughout the estrous cycle.

    PubMed

    De Los Reyes, Monica; Palomino, Jaime; Parraguez, Victor H; Ramirez, Fernando

    2017-04-15

    The aim of this study was to determine the mRNA LHR and LHR protein expression pattern in the canine ovarian follicles at different stage of development throughout the estrous cycle. Dog ovaries were obtained from 1-6y bitches at proestrus/estrus, anestrus and diestrus stages following ovariohysterectomy. Follicular cells were mechanically recovered from follicles distributed into four types (preantral, small antral, medium antral and large antral). Total RNA extraction was performed and the evaluation of gene expression levels was achieved by relative quantification q-PCR analysis. Intrafollicular amounts of LHR were assessed by western blot method. All results were evaluated by ANOVA. The expression levels of mRNA LHR in follicular cells were observed in every stage of development, however this gene expression varied over the estrous cycle. LHR transcripts increased (P < 0.05) from preantral to antral stage. There were not differences in LHR gene expression among follicles at preantral stages; however, at antral stages the lowest (P < 0.05) LHR mRNA expression was found at anestrus and the highest (P < 0.05) at proestrus/estrus. The LHR protein was also detected in dog follicles in all reproductive phases with patterns varying with stage of follicular development over the reproductive cycle. The antibody against human LHR revealed two bands at ∼90 and ∼67 kDa, probably representing the matured protein and its precursor respectively. Both bands LHR appeared already at preantral follicles increasing (P < 0.05) with growth. A high proportion of LHR was presented as immature forms in all follicles stages during different phases of the estrous cycle. In conclusion, the gene and protein of LHR are differentially expressed in dog follicles over the estrous cycle, increasing with growth and the precursor protein is the most predominant LHR form present in canine follicles. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Follicular development in cryopreserved Common Wombat ovarian tissue xenografted to Nude rats.

    PubMed

    Wolvekamp, M C; Cleary, M L; Cox, S L; Shaw, J M; Jenkin, G; Trounson, A O

    2001-01-31

    The Northern Hairy-nosed Wombat (Lasiorhinus krefftii) is a highly endangered marsupial species and every possible option for sustaining the species needs to be explored. One important approach may be the development of assisted reproductive technologies in the non-endangered Common Wombat (Vombatus ursinus) and Southern Hairy-nosed Wombat (Lasiorhinus latifrons) for application in breeding the Northern Hairy-nosed Wombat. In this study, it was examined whether cryopreserved Wombat ovarian tissue would develop following xenografting to immunologically deficient rats. Ovarian tissue was collected from Common Wombats (n = 3) and cryopreserved as small cortical pieces. After thawing the cortical pieces were grafted underneath the kidney capsule of Nude rats (n = 16). The grafts were recovered at 2, 4, and 10 weeks after transplantation and their gross and histological appearance investigated. Two weeks after grafting (n = 2), the tissue was revascularized and healthy primordial follicles were present. At week 4 (n = 2), some follicular development was present. At week 10, six rats received human chorionic gonadotrophin (hCG) to trigger follicle and oocyte maturation while another six rats were not given any treatment. The administration of hCG did not induce preovulatory follicles and oocyte maturation although type 5 follicles were present in ovarian tissue collected 10 weeks posttransplantation in both treated and untreated groups. This study demonstrates for the first time that Wombat ovarian tissue can survive and function when grafted into immunocompromized rats and that Wombat ovarian follicles can be recruited to growth and development in an ovarian xenograft. This model system has the potential to produce mature oocytes from endangered species for use in assisted reproductive technologies such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and mature oocytes from non-endangered species for nuclear transfer which may be necessary for

  8. Comparison of antral and preantral ovarian follicle populations between Bos indicus and Bos indicus-taurus cows with high or low antral follicles counts.

    PubMed

    Silva-Santos, K C; Siloto, L S; Santos, G M G; Morotti, F; Marcantonio, T N; Seneda, M M

    2014-02-01

    The objective was to compare populations of antral and pre-antral ovarian follicles in Bos indicus and Bos indicus-taurus cows with high and low antral follicle counts. Nelore (Bos indicus, n = 20) and Nelore X Angus (1/2 Bos indicus-taurus, n = 20) cows were subjected to follicular aspiration without regard to the stage of their oestrous cycle (day of aspiration = D0) to remove all follicles ≥3 mm and induce growth of a new follicular wave. Ovaries were examined by ultrasonography on D4, D19, D34, D49 and D64, and antral follicles ≥3 mm were counted. Thereafter, cows were assigned to one of two groups: high or low antral follicular count (AFC, ≥30 and ≤15 antral follicles, respectively). After D64, ovaries were collected after slaughter and processed for histological evaluation. There was high repeatability in the numbers of antral follicles for all groups (range 0.77-0.96). The mean (±SD) numbers of antral follicles were 35 ± 9 (Bos indicus) and 38 ± 6 (Bos indicus-taurus) for the high AFC group and 10 ± 3 (Bos indicus) and 12 ± 2 (Bos indicus-taurus) follicles for the low AFC. The mean number of preantral follicles in the ovaries of Bos indicus-taurus cows with high AFC (116 226 ± 83 156 follicles) was greater (p < 0.05) than that of Bos indicus cows (63 032 ± 58 705 follicles) with high AFC. However, there was no significant correlation between numbers of antral and preantral follicles. © 2013 Blackwell Verlag GmbH.

  9. 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.

  10. Protein and messenger RNA expression of interleukin 1 system members in bovine ovarian follicles and effects of interleukin 1β on primordial follicle activation and survival in vitro.

    PubMed

    Passos, J R S; Costa, J J N; da Cunha, E V; Silva, A W B; Ribeiro, R P; de Souza, G B; Barroso, P A A; Dau, A M P; Saraiva, M V A; Gonçalves, P B D; van den Hurk, R; Silva, J R V

    2016-01-01

    This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows and to evaluate the effects of IL-1β on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM(+) supplemented with IL-1β (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1β, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1β (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1β promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Alphafetoprotein and atretic follicles in the ovary of the pregnant rat.

    PubMed

    Seralini, G E; Lafaurie, M; Krebs, B; Stora, C

    1986-01-01

    Previous experiments have been conducted concerning the role of alphafetoprotein in genital system blockade in several cases: during adult rat N2-fluorenylacetamide hepatocarcinogenesis, after alphafetoprotein injections into normal adult female rats, during fetal life, and during postnatal and prepuberal development. In these conditions, alphafetoprotein is present at high plasma levels, and the normal cyclic ovarian function is stopped or nonexistent. The degenerating oocytes observed in the ovaries are often AFP-positive by histo-immunolocalization. Pregnancy corresponds to a physiological state in which alphafetoprotein levels are high while the gonadal activity is not characterized by ovulatory cycles. In order to assess our hypothesis, alpha-fetoprotein was studied in the ovary of pregnant rats from day 18 to 21 of gestation by an immunofluorescent technique, and alpha-fetoprotein was assayed in plasma samples. The results of this work show that, during pregnancy, follicular maturation is blocked at the antral stage, and the follicles contain degenerating oocytes that are AFP-positive in immunofluorescence. In conclusion, we suggest that the alpha-fetoprotein produced by the fetal liver and the yolk sac is disseminated in the amniotic fluid and passes through the placenta, and then reaches the ovarian follicles and the oocytes. The possible role of alphafetoprotein in follicular atresia is discussed.

  12. Effects of lowered temperatures and media on short-term preservation of zebu (Bos indicus) preantral ovarian follicles.

    PubMed

    Lucci, Carolina M; Kacinskis, Mirella A; Rumpf, Rodolfo; Báo, Sônia N

    2004-01-15

    The maintenance of follicle quality during the transportation of ovaries is essential for the successful cryopreservation and in vitro development of preantral follicles. The objective of this study was to evaluate the effect of cooling ovarian tissue on the conservation of zebu cow preantral follicles. Ovarian pieces were immersed in saline or coconut water (CW) solutions and maintained at 4 or 20 degrees C for 6, 12, or 18 h. Preantral follicles were evaluated by histology and transmission electron microscopy. Storage of ovarian pieces at 20 degrees C for 12 or 18 h significantly reduced the percentage of morphologically normal follicles compared to controls. In contrast, conservation at 4 degrees C for up to 18 h and at 20 degrees C for up to 6 h kept the percentage of normal follicles similar to controls. However, the type of solution that the ovaries were immersed in had little effect on the results. Decreased cellular metabolism probably accounted for better preservation of preantral follicles at 4 degrees C. In conclusion, zebu cow ovaries were successfully stored at 4 degrees C for up to 18 h with no morphological damage to preantral follicles. However, at 20 degrees C, ovaries could only be stored for 6 h.

  13. Steroid metabolism by ovarian follicles and extrafollicular tissue of the guppy (Poecilia reticulata) during oocyte growth and gestation.

    PubMed

    Venkatesh, B; Tan, C H; Kime, D E; Loy, G L; Lam, T J

    1992-06-01

    In the viviparous guppy, fertilization and gestation are intrafollicular. Fully developed embryos are ovulated at the end of gestation just prior to parturition. The metabolism in vitro of various radiolabeled steroid precursors by isolated ovarian follicles at various stages of the reproductive cycle and extrafollicular (EF) tissue of the guppy was investigated. While estradiol-17 beta was one of the end products of metabolism in vitellogenic follicles, 17 alpha, 20 beta-P and several 5-reduced metabolites were synthesized by postvitellogenic follicles. The yield of 17 alpha, 20 beta-P, however, was much lower than some 5 beta-reduced metabolites synthesized by postvitellogenic follicles. Gestation stage follicles rapidly converted the precursors into 5-reduced and polar 7-hydroxylated steroids, and their glucuronides. Although postpartum follicles showed very poor potential for steroid metabolism, they synthesized estradiol-17 beta from testosterone. These results demonstrate distinct changes occurring in the steroidogenic potential of the follicles during the reproductive cycle. Unlike in other viviparous vertebrates, no particular steroid seems to be involved in maintaining gestation in the guppy; all the steroid precursors are converted into highly polar metabolites and their conjugates during gestation, thereby facilitating their excretion. The EF ovarian tissue also synthesized 7-hydroxylated steroids and their glucuronides, providing evidence for the first time that the teleost ovarian EF tissue plays a role in steroidogenesis. The possible physiological significance of the synthesis of the novel polar steroids by the follicles and the EF tissue is discussed.

  14. Competitive inhibition of amino acid transport in human preovulatory ovarian follicles.

    PubMed

    Jóźwik, Maciej; Jóźwik, Marcin; Milewska, Anna Justyna; Battaglia, Frederick C; Jóźwik, Michał

    2017-10-01

    To date we have yet to examine whether amino acid (AA) transport in human ovarian follicles is affected by competitive inhibition. In contrast, transplacental transfer of AAs in late-gestation sheep is characterized by reciprocal competition. This phenomenon has been described by algebraic equations of umbilical uptake of AAs based on maternal arterial concentrations. In the present translational study at a university teaching hospital, we verified whether these equations apply to the transport of AAs from blood to follicular fluid (FF) in human preovulatory follicles. For this purpose we used our data on AA concentrations in blood and FF measured earlier by high-performance liquid chromatography in specimens from 14 patients undergoing oocyte retrieval for in vitro fertilization after controlled ovarian stimulation. The main outcome measure was statistical significance of Spearman correlation coefficients for measured versus calculated concentrations of 8 AAs: isoleucine, leucine, valine, phenylalanine, methionine, threonine, lysine, and arginine. Equations for umbilical uptake provided a highly accurate description of blood-to-FF transport for 7 AAs with the exception of lysine: R ≥ 0.899 (p < 0.0001) for the branched-chain AAs, R = 0.829 (p = 0.0003) for threonine, R = 0.754 (p = 0.0019) for arginine, and R = 0.631 (p = 0.0156) for phenylalanine and methionine. We conclude that these equations indicate competitive inhibition between the AAs studied. Our study strongly suggests that many AA transport systems operating in the placenta should also be active in the cells of the preovulatory follicle. Future studies on AA fluxes in human ovarian follicles must consider possible competitive inhibition. AA: amino acid; FF: follicular fluid; HPLC: high-performance liquid chromatography.

  15. Characterization of parameters for in vitro culture of isolated ovarian follicles of greenback flounder Rhombosolea tapirina.

    PubMed

    Pankhurst, N W; Riple, G

    2000-10-01

    Isolated ovarian follicles of greenback flounder Rhombosolea tapirina were incubated with a variety of gonadotropins (GtHs) and steroid precursors for periods of up to 42 h, and levels of free and glucuronated testosterone (T) and 17beta-estradiol (E(2)) in the medium, and free T and E(2) from inside follicles were measured by RIA. Short incubations (6 h) generated increases in T and E(2) in response to steroid precursors, but not human chorionic GtH (hCG), or salmon or carp GtH. At incubation times of 18 h, all GtHs stimulated T and, or E(2) production, whereas after 42-h incubation, GtH effects on E(2) production had disappeared. Steroid precursors remained effective at 18 and 42 h. T and E(2) glucuronides were formed in small quantities but did not account for loss of treatment effects at long incubation times. Instead, this could be explained by accumulation of E(2) in controls as a result of continued basal steroid production. Follicles absorbed substantial amounts of both endogenous and exogenous steroid from the medium, however, this did not appear to have any influence on changes in treatment effects with incubation time. Flounder follicles were most sensitive to hCG, followed by salmon and carp GtH at approximately 10-fold higher concentrations. Ovarian segments were not sensitive to any GtH but did convert exogenous steroid precursors indicating that tissue access by GtH may be a limiting factor under certain in vitro conditions. HCG augmented the conversion of 17-hydroxyprogesterone (17P) to T but not T to E(2), consistent with the relative GtH-insensitivity of aromatase in other species. Follicles converted a range of steroid precursors with equal competence, indicating that no step in the cleavage pathway is strongly rate-limited, and that choice of precursor is unlikely to affect the assessment of steroidogenic activity.

  16. POLARIZED INTERCELLULAR BRIDGES IN OVARIAN FOLLICLES OF THE CECROPIA MOTH

    PubMed Central

    Woodruff, Richard I.; Telfer, William H.

    1973-01-01

    Fluorescein-labeled rabbit serum globulin was injected into vitellogenic oocytes of the cecropia moth. Though the label spread throughout the ooplasm in less than 30 min, it was unable even after 2 h to cross the complex of intercellular bridges connecting the oocyte to its seven nurse cells. After injection into a single nurse cell, fluorescence was detected in the oocyte adjacent to the bridge complex within 3 min and had spread throughout the ooplasm in 30 min. Here also, the cell bodies of the six uninjected nurse cells remained nonfluorescent. Four of the nurse cells are not bridged directly to the oocyte but only through the apical ends of their siblings. Unidirectional movement must therefore occur in the apical cytoplasm of the nurse cells, as well as in the intercellular bridges. The nurse cells of healthy follicles had an intracellular electrical potential -40 mV relative to blood or dissecting solution, while oocytes measured -30 mV. A mV difference was also detected by direct comparison between a ground electrode in one cell and a recording electrode in the other. Three conditions were found in which the 10 mV difference was reduced or reversed in polarity. In all three cases fluorescent globulin was able in some degree to cross the bridges from the oocyte to the nurse cells. PMID:4125369

  17. The effect of androgens on ovarian follicle maturation: Dihydrotestosterone suppress FSH-stimulated granulosa cell proliferation by upregulating PPARγ-dependent PTEN expression.

    PubMed

    Chen, Mei-Jou; Chou, Chia-Hung; Chen, Shee-Uan; Yang, Wei-Shiung; Yang, Yu-Shih; Ho, Hong-Nerng

    2015-12-17

    Intraovarian hyperandrogenism is one of the determining factors of follicular arrest in women with polycystic ovary syndrome (PCOS). Using androgenized rat models, we investigated the effects of androgens on metabolism, as well as on factors involved in follicular arrest and the reduced number of estrus cycles. The dihydrotestosterone (DHT)-treated rats had fewer estrus cycles, higher numbers of large arrested follicles and an increased in body weight gain compared with the dehydroepiandrostenedione (DHEA)- and placebo-treated rats. In cultured rat granulosa cells, DHT suppressed follicle stimulating hormone (FSH)-induced granulosa cell proliferation and increased the accumulation of cells in the G2/M phase. DHT decreased phosphorylated Akt (p-Akt) and cyclin D1 levels through increasing PTEN. DHT-promoted PTEN expression was regulated by peroxisome proliferator-activated receptor gamma (PPARγ) in granulosa cells. Meanwhile, in the large follicles of the DHT-treated rats, the expressions of PPARγ and PTEN were higher, but the expression of p-Akt and proliferating cell nuclear antigen (PCNA) were lower. Conclusively, DHT and DHEA produced differential effects on metabolism in prepubertal female rats like clinical manifestations of women with PCOS. DHT treatment may affect ovarian follicular maturation by altering granulosa cell proliferation through the regulation of enhancing PPARγ dependent PTEN/p-Akt expression in the granulosa cells.

  18. The effect of androgens on ovarian follicle maturation: Dihydrotestosterone suppress FSH-stimulated granulosa cell proliferation by upregulating PPARγ-dependent PTEN expression.

    PubMed Central

    Chen, Mei-Jou; Chou, Chia-Hung; Chen, Shee-Uan; Yang, Wei-Shiung; Yang, Yu-Shih; Ho, Hong-Nerng

    2015-01-01

    Intraovarian hyperandrogenism is one of the determining factors of follicular arrest in women with polycystic ovary syndrome (PCOS). Using androgenized rat models, we investigated the effects of androgens on metabolism, as well as on factors involved in follicular arrest and the reduced number of estrus cycles. The dihydrotestosterone (DHT)-treated rats had fewer estrus cycles, higher numbers of large arrested follicles and an increased in body weight gain compared with the dehydroepiandrostenedione (DHEA)- and placebo-treated rats. In cultured rat granulosa cells, DHT suppressed follicle stimulating hormone (FSH)-induced granulosa cell proliferation and increased the accumulation of cells in the G2/M phase. DHT decreased phosphorylated Akt (p-Akt) and cyclin D1 levels through increasing PTEN. DHT-promoted PTEN expression was regulated by peroxisome proliferator-activated receptor gamma (PPARγ) in granulosa cells. Meanwhile, in the large follicles of the DHT-treated rats, the expressions of PPARγ and PTEN were higher, but the expression of p-Akt and proliferating cell nuclear antigen (PCNA) were lower. Conclusively, DHT and DHEA produced differential effects on metabolism in prepubertal female rats like clinical manifestations of women with PCOS. DHT treatment may affect ovarian follicular maturation by altering granulosa cell proliferation through the regulation of enhancing PPARγ dependent PTEN/p-Akt expression in the granulosa cells. PMID:26674985

  19. Chronic intermittent cold stress activates ovarian sympathetic nerves and modifies ovarian follicular development in the rat.

    PubMed

    Dorfman, M; Arancibia, S; Fiedler, J L; Lara, H E

    2003-06-01

    We studied the effects of a chronic intermittent cold stress regime on sympathetic nerve activation and ovarian physiology. This paradigm (4 degrees C for 3 h/day, Monday-Friday, for 3 or 4 wk) does not affect basal plasma levels of corticosterone. After 3 wk of stress, we detected a decrease in noradrenaline (NA) in the ovary, but after 4 wk, this ovarian neurotransmitter concentration increased over that of unstressed control rats. To analyze whether this effect on NA is preceded by an activation of the neurotrophic factor system responsible for growth and survival of sympathetic neurons, we measured both nerve growth factor (NGF) (by enzyme immunoassay) and the intraovarian levels of its low affinity receptor mRNA (by reverse transcription-polymerase chain reaction). The activation of sympathetic nerves was followed by an increase in NGF concentration without affecting the ovarian levels of either NGF or the mRNA of its receptor. Interestingly, follicular development changed during the stress procedure; after 3 or 4 wk of stress, we found a decrease in preantral healthy follicles without a compensatory increase in atresia. Concomitantly with the increase in NA and NGF in the ovary, we observed that a new population of follicles with hypertrophied thecal cell layers appeared after 4 wk of stress. These results suggest that chronic stress, through an intraovarian neurotrophin-mediated sympathetic activation, produces changes in follicular development that could lead to an impairment of reproductive function.

  20. Hydrogel network design using multifunctional macromers to coordinate tissue maturation in ovarian follicle culture

    PubMed Central

    Shikanov, Ariella; Smith, Rachel M; Xu, Min; Woodruff, Teresa K.; Shea, Lonnie D.

    2011-01-01

    Synthetic hydrogels with tunable properties are appealing for regenerative medicine. A critical limitation in hydrogel design at low solids concentration is the formation of defects, which increase gelation times and swelling, and reduce elasticity. Here, we report that tri-functional crosslinking peptides applied to 4-arm poly-(ethylene glycol) (PEG) hydrogels decreased swelling and gelation time relative to bi-functional crosslinkers. In contrast to bi-functional peptides, the third cross-linking site on the peptide created a branch point if an intramolecular crosslink formed, which prevented non-functional “dangling-ends” in the hydrogel network and enhanced the number of elastically active cross-links. The improved network formation enabled mouse ovarian follicle encapsulation and maturation in vitro. Hydrogels with bi-functional crosslinkers resulted in cellular dehydration, likely due to osmosis during the prolonged gelation. For tri-functional crosslinkers, the hydrogels supported a 17-fold volumetric expansion of the tissue during culture, with expansion dependent on the ability of the follicle to rearrange its microenvironment, which is controlled through the sensitivity of the cross-linking peptide to the proteolytic activity of plasmin. The improved network design enabled ovarian follicle culture in a completely synthetic system, and can advance fertility preservation technology for women facing premature infertility from anticancer therapies. PMID:21247629

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

    SciTech Connect

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

    1987-05-01

    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 3 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.

  2. Gonadotropin ratio affects the in vitro growth of rhesus ovarian preantral follicles

    PubMed Central

    Kim, Yoon Young; Yun, Jun-Won; Kim, Jong Min; Park, Chung Gyu; Rosenwaks, Zev; Liu, Hung Ching; Kang, Byeong-Cheol; Ku, Seung-Yup

    2016-01-01

    In vitro follicle growth (IVFG) strategy is critical in the fertility preservation of cancer survivors; however, its optimal protocol needs to be developed using primate models since the availability of human samples is limited. Only a few previous studies have reported the successful IVFG of rhesus monkey ovaries using low-dose follicle-stimulating hormone (FSH) (0.3 or 3 ng/mL) and long-term culture (up to 5 weeks) and it is still uncertain in regard to the optimal culture duration and effective dose of treated gonadotropins applicable to the IVFG of rhesus preantral follicles. Recently, we have reported that the FSH to luteinizing hormone (LH) ratio affects the in vitro growth of murine ovarian follicles. We aimed to investigate whether gonadotropin ratios affect the efficiency of rhesus follicular growth in vitro. Ovaries were collected from six necropsied rhesus macaques (4–9 years) and preantral follicles were retrieved and cultured for 14 days using 200 mIU/mL FSH. The characteristics of follicular growth were compared between the FSH:LH=1:1 (n=24) and FSH:LH=2:1 (n=24) groups. High concentration gonadotropin treatment shortened the duration required for in vitro maturation of rhesus preantral follicles. The FSH:LH=2:1 group showed a faster follicular growth and enabled the acquisition of mature oocytes, although the expression of growth differentiation factor (GDF)-9 and anti-Müllerian hormone (AMH) did not differ significantly between the two groups. Taken together, high dose gonadotropin treatment can shorten the duration of IVFG and the gonadotropin ratio is important in the IVFG of rhesus monkey ovaries. PMID:26980777

  3. The ameliorative effect of propolis against methoxychlor induced ovarian toxicity in rat.

    PubMed

    El-Sharkawy, Eman E; Kames, Amany O G; Sayed, S M; Nisr, Neveen A E L; Wahba, Nahed M; Elsherif, Walaa M; Nafady, Allam M; Abdel-Hafeez, M M; Aamer, A A

    2014-12-01

    A study was designed to evaluate ameliorative effect of propolis against methoxychlor (MXC) induced ovarian toxicity in rat. The organochlorine pesticide (MXC) is a known endocrine disruptor with estrogenic, anti-estrogenic, and anti-androgenic properties. To investigate whether chronic exposure to MXC could cause ovarian dysfunction, two groups of Sprague-Dawley adult female rats were exposed to MXC alone in a dose of 200mg/kg, twice/weekly, orally or MXC dose as previous plus propolis in a dose of 200mg/l/day, in drinking water for 10 months. Another two groups of rat were given corn oil (control) or propolis. Multiple reproductive parameters, ovarian weight, serum hormone levels, ovarian oxidative status and ovarian morphology were examined. In MXC-exposed group, there is a significant decrease in body and ovarian weight vs. control. MXC decreases serum estradiol and progesterone levels. A significant increase in the levels of lipid peroxidation was obtained while a significant decrease of the total antioxidant was recorded. Ovarian histopathology showed primary, secondary and vesicular follicles displaying an atretic morphology. Increase in the ovarian surface epithelium height accompanied with vacuolated, pyknotic oocytes were obtained. The previous toxic effects were neutralized by the administration of propolis in MXC+propolis group. The present results suggest that propolis may be effective in decreasing of MXC-induced ovarian toxicity in rat.

  4. LKB1 acts as a critical gatekeeper of ovarian primordial follicle pool

    PubMed Central

    Ma, Xue-Shan; Schatten, Heide; Fan, Heng-Yu; Wang, Zhen-Bo; Sun, Qing-Yuan

    2016-01-01

    Liver Kinase b1 (LKB1/STK11)is a tumor suppressor responsible for the Peutz-Jeghers syndrome, an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. Besides, the C allele of a SNP in the Lkb1 gene impedes the likelihood of ovulation in polycystic ovary syndrome (PCOS) in women treated with metformin, a known LKB1-AMPK activator. It is very likely that LKB1 plays roles in female fertility. To identify the physiological functions of LKB1 in the mouse ovary, we selectively disrupted LKB1 in oocytes by the Cre-LoxP conditional knockout system and found that Lkb1fl/fl; Gdf9-Cre mice were severely subfertile with significantly enlarged ovaries compared to Lkb1fl/fl mice. Interestingly, without Lkb1 expression in oocytes from the primordial follicle stage, the entire primordial follicle pool was activated but failed to mature and ovulate, subsequently causing premature ovarian failure (POF). Further investigation demonstrated that elevated mTOR signaling regulated by an AKT-independent LKB1-AMPK pathway was responsible for the excessive follicle activation and growth. Our findings reveal the role of LKB1 as an indispensable gatekeeper for the primordial follicle pool, offer new functional understanding for the tumor suppressor genes in reproductive organs, and might also provide valuable information for understanding POF and infertility. PMID:26745759

  5. Follicle-stimulating hormone regulates expression and activity of epidermal growth factor receptor in the murine ovarian follicle.

    PubMed

    El-Hayek, Stephany; Demeestere, Isabelle; Clarke, Hugh J

    2014-11-25

    Fertility depends on the precise coordination of multiple events within the ovarian follicle to ensure ovulation of a fertilizable egg. FSH promotes late follicular development, including expression of luteinizing hormone (LH) receptor by the granulosa cells. Expression of its receptor permits the subsequent LH surge to trigger the release of ligands that activate EGF receptors (EGFR) on the granulosa, thereby initiating the ovulatory events. Here we identify a previously unknown role for FSH in this signaling cascade. We show that follicles of Fshb(-/-) mice, which cannot produce FSH, have a severely impaired ability to support two essential EGFR-regulated events: expansion of the cumulus granulosa cell layer that encloses the oocyte and meiotic maturation of the oocyte. These defects are not caused by an inability of Fshb(-/-) oocytes to produce essential oocyte-secreted factors or of Fshb(-/-) cumulus cells to respond. In contrast, although expression of both Egfr and EGFR increases during late folliculogenesis in Fshb(+/-) females, these increases fail to occur in Fshb(-/-) females. Remarkably, supplying a single dose of exogenous FSH activity to Fshb(-/-) females is sufficient to increase Egfr and EGFR expression and to restore EGFR-dependent cumulus expansion and oocyte maturation. These studies show that FSH induces an increase in EGFR expression during late folliculogenesis and provide evidence that the FSH-dependent increase is necessary for EGFR physiological function. Our results demonstrate an unanticipated role for FSH in establishing the signaling axis that coordinates ovulatory events and may contribute to the diagnosis and treatment of some types of human infertility.

  6. Follicle-stimulating hormone regulates expression and activity of epidermal growth factor receptor in the murine ovarian follicle

    PubMed Central

    El-Hayek, Stephany; Demeestere, Isabelle; Clarke, Hugh J.

    2014-01-01

    Fertility depends on the precise coordination of multiple events within the ovarian follicle to ensure ovulation of a fertilizable egg. FSH promotes late follicular development, including expression of luteinizing hormone (LH) receptor by the granulosa cells. Expression of its receptor permits the subsequent LH surge to trigger the release of ligands that activate EGF receptors (EGFR) on the granulosa, thereby initiating the ovulatory events. Here we identify a previously unknown role for FSH in this signaling cascade. We show that follicles of Fshb−/− mice, which cannot produce FSH, have a severely impaired ability to support two essential EGFR-regulated events: expansion of the cumulus granulosa cell layer that encloses the oocyte and meiotic maturation of the oocyte. These defects are not caused by an inability of Fshb−/− oocytes to produce essential oocyte-secreted factors or of Fshb−/− cumulus cells to respond. In contrast, although expression of both Egfr and EGFR increases during late folliculogenesis in Fshb+/− females, these increases fail to occur in Fshb−/− females. Remarkably, supplying a single dose of exogenous FSH activity to Fshb−/− females is sufficient to increase Egfr and EGFR expression and to restore EGFR-dependent cumulus expansion and oocyte maturation. These studies show that FSH induces an increase in EGFR expression during late folliculogenesis and provide evidence that the FSH-dependent increase is necessary for EGFR physiological function. Our results demonstrate an unanticipated role for FSH in establishing the signaling axis that coordinates ovulatory events and may contribute to the diagnosis and treatment of some types of human infertility. PMID:25385589

  7. Ovarian toxicity of cyclophosphamide alone and in combination with ovarian irradiation in the rat

    SciTech Connect

    Jarrell, J.; Lai, E.V.; Barr, R.; McMahon, A.; Belbeck, L.; O'Connell, G.

    1987-05-01

    The effects of radiation and chemotherapy on gonadal function are relevant to the morbidity induced by such treatments. Cyclophosphamide given i.p. to rats on Day 30 of age delayed vaginal opening, prevented vaginal cyclicity, and caused a reduction in serum estradiol and progesterone. Antral follicular atresia increased in a dose-dependent fashion in response to cyclophosphamide (0 mg/kg, 53.5%; 1 mg/kg, 67.3%; 50 mg/kg, 65.7%; 100 mg/kg, 73.9%; 150 mg/kg, 92.2%). Despite such alterations in ovarian function, serum gonadotrophins did not rise. The concurrent administration of 0, 20, 30, 40, 50, and 60 Gy of radiation to the exteriorized ovaries in rats receiving 50 mg/kg cyclophosphamide induced widespread loss of primordial, preantral, and healthy antral follicles associated with reduction in serum progesterone and estradiol. Such irradiation induced dose-related increases in serum follicle-stimulating hormone and luteinizing hormone. Parenteral cyclophosphamide and local irradiation appear to induce ovarian toxicity by different mechanisms.

  8. 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.

  9. Promoting extracellular matrix remodeling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels.

    PubMed

    Tagler, David; Makanji, Yogeshwar; Tu, Tao; Bernabé, Beatriz Peñalver; Lee, Raymond; Zhu, Jie; Kniazeva, Ekaterina; Hornick, Jessica E; Woodruff, Teresa K; Shea, Lonnie D

    2014-07-01

    The in vitro growth of ovarian follicles is an emerging technology for fertility preservation. Various strategies support the culture of secondary and multilayer follicles from various species including mice, non-human primate, and human; however, the culture of early stage (primary and primordial) follicles, which are more abundant in the ovary and survive cryopreservation, has been limited. Hydrogel-encapsulating follicle culture systems that employed feeder cells, such as mouse embryonic fibroblasts (MEFs), stimulated the growth of primary follicles (70-80 µm); yet, survival was low and smaller follicles (<70 µm) rapidly lost structure and degenerated. These morphologic changes were associated with a breakdown of the follicular basement membrane; hence, this study investigated ascorbic acid based on its role in extracellular matrix (ECM) deposition/remodeling for other applications. The selection of ascorbic acid was further supported by a microarray analysis that suggested a decrease in mRNA levels of enzymes within the ascorbate pathway between primordial, primary, and secondary follicles. The supplementation of ascorbic acid (50 µg/mL) significantly enhanced the survival of primary follicles (<80 µm) cultured in alginate hydrogels, which coincided with improved structural integrity. Follicles developed antral cavities and increased to diameters exceeding 250 µm. Consistent with improved structural integrity, the gene/protein expression of ECM and cell adhesion molecules was significantly changed. This research supports the notion that modifying the culture environment (medium components) can substantially enhance the survival and growth of early stage follicles. © 2013 Wiley Periodicals, Inc.

  10. Increases in norepinephrine release and ovarian cyst formation during ageing in the rat.

    PubMed

    Acuña, Eric; Fornes, Romina; Fernandois, Daniela; Garrido, Maritza P; Greiner, Monika; Lara, Hernan E; Paredes, Alfonso H

    2009-06-16

    Depletion of ovarian follicles is associated with the end of reproductive function in ageing females. Recently, it has been described that this process parallels increases in the concentration of norepinephrine (NE) in the rat ovary. In sexually mature rats, experimentally-induced increases in the sympathetic tone of the ovary is causally related to ovarian cyst formation and deranged follicular development. Thus, there is a possibility that increased ovarian NE concentrations represent changes in the activity of sympathetic nerves, which consequently participate in the process of ovarian cyst formation observed during ageing in the human and experimental animal models. Sprague-Dawley rats between 6 and 14 months old were used to analyse the capacity of the ovary to release 3H-NE recently incorporated under transmural depolarisation in relation to changes in the ovarian follicular population. Morphometric analysis of ovarian follicles and real time PCR for Bcl2 and Bax mRNA were used to assess follicular atresia. From 8 months old, the induced release of recently incorporated 3H-norepinephrine (3H-NE) from the ovary and ovarian NE concentrations increased, reaching their peak values at 12 months old and remained elevated up to 14 months old. Increases in sympathetic nerve activity paralleled changes in the follicular population, as well as disappearance of the corpus luteum. In contrast, luteinised follicles, precystic follicles, and cystic follicles increased. During this period, the relationship between Bax and Bcl2 mRNAs (the proapoptotic/antiapoptotic signals) increased, suggesting atresia as the principal mechanism contributing to the decreased follicular population. When NE tone was increased, the mRNA ratio favoured Bcl2 to Bax and antiapoptotic signals dominated this period of development. Thus, these changing ratios could be responsible for the increase in luteinised follicles, as well as precystic and cystic follicles. These data suggest that the ageing

  11. Increases in norepinephrine release and ovarian cyst formation during ageing in the rat

    PubMed Central

    Acuña, Eric; Fornes, Romina; Fernandois, Daniela; Garrido, Maritza P; Greiner, Monika; Lara, Hernan E; Paredes, Alfonso H

    2009-01-01

    Background Depletion of ovarian follicles is associated with the end of reproductive function in ageing females. Recently, it has been described that this process parallels increases in the concentration of norepinephrine (NE) in the rat ovary. In sexually mature rats, experimentally-induced increases in the sympathetic tone of the ovary is causally related to ovarian cyst formation and deranged follicular development. Thus, there is a possibility that increased ovarian NE concentrations represent changes in the activity of sympathetic nerves, which consequently participate in the process of ovarian cyst formation observed during ageing in the human and experimental animal models. Methods Sprague-Dawley rats between 6 and 14 months old were used to analyse the capacity of the ovary to release 3H-NE recently incorporated under transmural depolarisation in relation to changes in the ovarian follicular population. Morphometric analysis of ovarian follicles and real time PCR for Bcl2 and Bax mRNA were used to assess follicular atresia. Results From 8 months old, the induced release of recently incorporated 3H-norepinephrine (3H-NE) from the ovary and ovarian NE concentrations increased, reaching their peak values at 12 months old and remained elevated up to 14 months old. Increases in sympathetic nerve activity paralleled changes in the follicular population, as well as disappearance of the corpus luteum. In contrast, luteinised follicles, precystic follicles, and cystic follicles increased. During this period, the relationship between Bax and Bcl2 mRNAs (the proapoptotic/antiapoptotic signals) increased, suggesting atresia as the principal mechanism contributing to the decreased follicular population. When NE tone was increased, the mRNA ratio favoured Bcl2 to Bax and antiapoptotic signals dominated this period of development. Thus, these changing ratios could be responsible for the increase in luteinised follicles, as well as precystic and cystic follicles

  12. Pattern of vitellogenesis and follicle maturational competence during the ovarian follicular cycle of Fundulus heteroclitus.

    PubMed

    Cerdá, J; Calman, B G; LaFleur, G J; Limesand, S

    1996-07-01

    The patterns of vitellogenesis and follicle maturational competence were examined across the semilunar spawning cycle of Fundulus heteroclitus. Daily egg collection showed spawning cyclicity in six experimental groups, with a mean period between spawnings of 14.9 +/- 0.3 days, indicated by the nonlinear regression sine-curve matching analysis. Each cycle was then dated from Day -7 to Day 7, with Day 0 as the peak egg-collection day. Females from each group were sampled on selected days during two to three consecutive spawning cycles, and these days were each chronologically given a temporal relation to Day 0 to pool the data into a composite. The analysis of the size-frequency distribution of ovarian follicles > or = 0.5-mm diameter across the composite revealed a constant recruitment of small follicles (0.5- to 0.7-mm diameter) into vitellogenesis, which was supported by the continuous presence of vitellogenin (Vtg) I mRNA in the liver of the females. The plasma levels of Vtg were also essentially constant across the cycle, except for a progressive decrease from Day -7 through Day 3 that could be related to a more active Vtg uptake by a dominant population of follicles up to 1.7 mm in diameter. A second and more selective recruitment of full-grown follicles (1.3- to 1.4-mm diameter) toward maturation was noted at Days -5, -4, which appeared associated with high plasma levels of estradiol-17 beta. However, the responsiveness of those follicles undergoing oocyte maturation in vitro after gonadotropin and maturation-inducing steroid (MIS), 17, 20 beta-dihydroxy-4-pregnen-3-one, stimulation dramatically declined from Days -1, 0, 1 to Days 4, 5, concomitantly with an increase of the population of the largest follicles (1.8- to 2.1-mm diameter) in the ovary. These findings extend previous observations on the process of follicular recruitment in F. heteroclitus and indicate that full-grown follicles may be recruited into maturation by a mechanism that modulates the

  13. Effects of deletion of the transcription factor Nrf2 and benzo[a]pyrene treatment on ovarian follicles and ovarian surface epithelial cells in mice

    PubMed Central

    Lim, Jinhwan; Ortiz, Laura; Nakamura, Brooke N.; Hoang, Yvonne D.; Banuelos, Jesus; Flores, Victoria N.; Chan, Jefferson Y.; Luderer, Ulrike

    2015-01-01

    Polycyclic aromatic hydrocarbons, like benzo[a]pyrene (BaP), are ubiquitous environmental pollutants and potent ovarian toxicants. The transcription factor NRF2 is an important regulator of the cellular response to electrophilic toxicants like BaP and to oxidative stress. NRF2 regulates transcription of genes involved in the detoxification of reactive metabolites of BaP and reactive oxygen species. We therefore hypothesized that Nrf2−/− mice have accelerated ovarian aging and increased sensitivity to the ovarian toxicity of BaP. A single injection of BaP dose-dependently depleted ovarian follicles in Nrf2+/+ and Nrf2−/− mice, but the effects of BaP were not enhanced in the absence of Nrf2. Similarly, Nrf2−/− mice did not have increased ovarian BaP DNA adduct formation compared to Nrf2+/+ mice. Ovarian follicle numbers did not differ between peripubertal Nrf2−/− and Nrf2+/+ mice, but by middle age, Nrf2−/− mice had significantly fewer primordial follicles than Nrf2+/+ mice, consistent with accelerated ovarian aging. PMID:26247513

  14. Dienogest, a selective progestin, reduces plasma estradiol level through induction of apoptosis of granulosa cells in the ovarian dominant follicle without follicle-stimulating hormone suppression in monkeys.

    PubMed

    Sasagawa, S; Shimizu, Y; Nagaoka, T; Tokado, H; Imada, K; Mizuguchi, K

    2008-07-01

    Dienogest is a selective progestin that has been shown to arrest ovarian follicular development in women, without affecting gonadotropin secretion. As luteal progesterone or exogeneous progestins are known to suppress ovarian folliculogenesis via the inhibition of gonadotropin secretion, this action of dienogest on ovaries seems to be unique. To examine the underlying mechanism of the antifolliculogenic effect of dienogest, female cynomolgus monkeys were treated with a single oral dose of 0.1 mg/kg dienogest on day 7 of the menstrual cycle. Plasma FSH, estradiol (E2), and progesterone levels were measured up to 15 days after dosing. In an additional experiment, ovaries were excised 24 h after dosing for histological examinations. As a result, plasma E2 level declined within 24 h after dosing, while dienogest did not decreased FSH level prior to E2 decline. After decline of E2 level, the low level of E2 was sustained for more than 11 days. It is considered that a single oral dose of dienogest induced atresia of the dominant follicle. In the histological examination, two out of three animals showed decline in E2 level. The ovarian dominant follicles from these animals showed apoptotic changes in granulosa cells with scattered aromatase expression within 24 h after dosing. These results indicate that the induction of atresia of the ovarian dominant follicle by direct action would be a possible mechanism of dienogest to inhibit plasma E2 level.

  15. Developmental effects of imatinib mesylate on follicle assembly and early activation of primordial follicle pool in postnatal rat ovary.

    PubMed

    Asadi-Azarbaijani, Babak; Santos, Regiane R; Jahnukainen, Kirsi; Braber, Saskia; van Duursen, Majorie B M; Toppari, Jorma; Saugstad, Ola D; Nurmio, Mirja; Oskam, Irma C

    2017-03-01

    Imatinib mesylate is an anti-cancer agent that competitively inhibits several receptor tyrosine kinases (RTKs). RTKs play important roles in the regulation of primordial follicle formation, the recruitment of primordial follicles into the pool of growing follicles and maturation of the follicles. In the present study, we investigated the effects of the tyrosine kinase inhibitor imatinib on primordial follicle assembly and early folliculogenesis in postnatal rats. Female Sprague-Dawley rats were treated with either imatinib (150mg/kg) or placebo (water) on postnatal days 2-4. Bilateral ovariectomy was performed on postnatal day 2 and 5. Histology, immunohistochemistry, and mRNA analysis were performed. Imatinib treatment was associated with increased density of the multi-oocyte follicles (P<0.01), oogonia (p<0.01) and germline clusters (P<0.05), decreased activation of primordial follicles, increased expression of c-Kit and AMH, and decreased protein expression of Kit-ligand and GDF9 when compared to age-matched controls. In conclusion, imatinib affects folliculogenesis in postnatal rat ovaries by delaying the cluster breakdown, follicular assembly and early activation of the primordial follicle pool. Copyright © 2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  16. Long-Term Moderate Oxidative Stress Decreased Ovarian Reproductive Function by Reducing Follicle Quality and Progesterone Production

    PubMed Central

    Lai, Zhiwen; Tian, Yong; Fang, Li; Wu, Meng; Xiong, Jiaqiang; Qin, Xian; Luo, Aiyue; Wang, Shixuan

    2016-01-01

    Ovarian aging is a long-term and complex process associated with a decrease in follicular quantity and quality. The damaging effects of reactive oxygen species (ROS) in ovarian aging and ovarian aging-associated disorders have received relatively little attention. Thus, we assessed if the oxidative stress induced by long-term (defined by the Environmental Protection Agency as at least 30 days in duration) moderate ozone inhalation reduced ovarian reserves, decreased ovarian function and induced ovarian aging-associated disorders. The expression of oxidative stress markers and antioxidant enzymes was used to determine the degree of oxidative stress. Ultrastructural changes in ovarian cells were examined via electron microscopy. The ovarian reserve was assessed by measuring multiple parameters, such as the size of the primordial follicle pool and anti-Müllerian hormone (AMH) expression. The estrous cycle, hormone levels and fertility status were investigated to assess ovarian function. To investigate ovarian aging-associated disorders, we utilized bone density and cardiovascular ultrasonography in mice. The levels of oxidized metabolites, such as 8-hydroxy-2´-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE) and nitrotyrosine (NTY), significantly increased in ovarian cells in response to increased oxidative stress. The ultrastructural analysis indicated that lipid droplet formation and the proportion of mitochondria with damaged membranes in granulosa cells were markedly increased in ozone-exposed mice when compared with the control group. Ozone exposure did not change the size of the primordial follicle pool or anti-Müllerian hormone (AMH) expression. The estrogen concentration remained normal; however, progesterone and testosterone levels decreased. The mice exposed to ozone inhalation exhibited a substantial decrease in fertility and fecundity. No differences were revealed by the bone density or cardiovascular ultrasounds. These findings suggest that the

  17. How do ovarian follicles interact? A many-body problem with unusual symmetry and symmetry-breaking properties

    NASA Astrophysics Data System (ADS)

    Michael Lacker, H.; Percus, Allon

    1991-06-01

    The assumption that hormonal feedback regulates ovarian follicle growth is used to formulate a many-body problem in which interactions are spatially independent. This mechanism of interaction is shown to be sufficient to account for the regulation of ovulation number. A method is also developed to test if this assumption is consistent with the observed spatial distribution of follicles in the Rhesus monkey ovary.

  18. In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.

    PubMed

    Migone, Fernando F; Cowan, Robert G; Williams, Rebecca M; Gorse, Kiersten J; Zipfel, Warren R; Quirk, Susan M

    2016-02-23

    Rupture of the ovarian follicle releases the oocyte at ovulation, a timed event that is critical for fertilization. It is not understood how the protease activity required for rupture is directed with precise timing and localization to the outer surface, or apex, of the follicle. We hypothesized that vasoconstriction at the apex is essential for rupture. The diameter and blood flow of individual vessels and the thickness of the apical follicle wall were examined over time to expected ovulation using intravital multiphoton microscopy. Vasoconstriction of apical vessels occurred within hours preceding follicle rupture in wild-type mice, but vasoconstriction and rupture were absent in Amhr2(cre/+)SmoM2 mice in which follicle vessels lack the normal association with vascular smooth muscle. Vasoconstriction is not simply a response to reduced thickness of the follicle wall; vasoconstriction persisted in wild-type mice when thinning of the follicle wall was prevented by infusion of protease inhibitors into the ovarian bursa. Ovulation was inhibited by preventing the periovulatory rise in the expression of the vasoconstrictor endothelin 2 by follicle cells of wild-type mice. In these mice, infusion of vasoconstrictors (either endothelin 2 or angiotensin 2) into the bursa restored the vasoconstriction of apical vessels and ovulation. Additionally, infusion of endothelin receptor antagonists into the bursa of wild-type mice prevented vasoconstriction and follicle rupture. Processing tissue to allow imaging at increased depth through the follicle and transabdominal ultrasonography in vivo showed that decreased blood flow is restricted to the apex. These results demonstrate that vasoconstriction at the apex of the follicle is essential for ovulation.

  19. In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation

    PubMed Central

    Migone, Fernando F.; Cowan, Robert G.; Williams, Rebecca M.; Gorse, Kiersten J.; Zipfel, Warren R.; Quirk, Susan M.

    2016-01-01

    Rupture of the ovarian follicle releases the oocyte at ovulation, a timed event that is critical for fertilization. It is not understood how the protease activity required for rupture is directed with precise timing and localization to the outer surface, or apex, of the follicle. We hypothesized that vasoconstriction at the apex is essential for rupture. The diameter and blood flow of individual vessels and the thickness of the apical follicle wall were examined over time to expected ovulation using intravital multiphoton microscopy. Vasoconstriction of apical vessels occurred within hours preceding follicle rupture in wild-type mice, but vasoconstriction and rupture were absent in Amhr2cre/+SmoM2 mice in which follicle vessels lack the normal association with vascular smooth muscle. Vasoconstriction is not simply a response to reduced thickness of the follicle wall; vasoconstriction persisted in wild-type mice when thinning of the follicle wall was prevented by infusion of protease inhibitors into the ovarian bursa. Ovulation was inhibited by preventing the periovulatory rise in the expression of the vasoconstrictor endothelin 2 by follicle cells of wild-type mice. In these mice, infusion of vasoconstrictors (either endothelin 2 or angiotensin 2) into the bursa restored the vasoconstriction of apical vessels and ovulation. Additionally, infusion of endothelin receptor antagonists into the bursa of wild-type mice prevented vasoconstriction and follicle rupture. Processing tissue to allow imaging at increased depth through the follicle and transabdominal ultrasonography in vivo showed that decreased blood flow is restricted to the apex. These results demonstrate that vasoconstriction at the apex of the follicle is essential for ovulation. PMID:26842836

  20. Transcriptome Profiling of the Theca Interna from Bovine Ovarian Follicles during Atresia

    PubMed Central

    Hatzirodos, Nicholas; Irving-Rodgers, Helen F.; Hummitzsch, Katja; Rodgers, Raymond J.

    2014-01-01

    The theca interna is a specialized stromal layer that envelops each growing ovarian follicle. It contains capillaries, fibroblasts, immune cells and the steroidogenic cells that synthesize androgens for conversion to estradiol by the neighboring granulosa cells. During reproductive life only a small number of follicles will grow to a sufficient size to ovulate, whereas the majority of follicles will undergo regression/atresia and phagocytosis by macrophages. To identify genes which are differentially regulated in the theca interna during follicular atresia, we undertook transcriptome profiling of the theca interna from healthy (n = 10) and antral atretic (n = 5) bovine follicles at early antral stages (<5 mm). Principal Component Analyses and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. A total of 543 probe sets were differentially expressed between the atretic and healthy theca interna. Further analyses of these genes by Ingenuity Pathway Analysis and Gene Ontology Enrichment Analysis Toolkit software found most of the genes being expressed were related to cytokines, hormones and receptors as well as the cell cycle and DNA replication. Cell cycle genes which encode components of the replicating chromosome complex and mitotic spindle were down-regulated in atretic theca interna, whereas stress response and inflammation-related genes such as TP53, IKBKB and TGFB1 were up-regulated. In addition to cell cycle regulators, upstream regulators that were predicted to be inhibited included Retinoblastoma 1, E2 transcription factor 1, and hepatocyte growth factor. Our study suggests that during antral atresia of small follicles in the theca interna, arrest of cell cycle and DNA replication occurs rather than up- regulation of apoptosis-associated genes as occurs in granulosa cells. PMID:24956388

  1. 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.

  2. Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles

    PubMed Central

    Shuhaibar, Leia C.; Egbert, Jeremy R.; Norris, Rachael P.; Lampe, Paul D.; Nikolaev, Viacheslav O.; Thunemann, Martin; Wen, Lai; Feil, Robert; Jaffe, Laurinda A.

    2015-01-01

    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. PMID:25775542

  3. Stimulatory effects of fasting on vascular endothelial growth factor (VEGF) production by growing pig ovarian follicles.

    PubMed

    Galeati, G; Spinaci, M; Govoni, N; Zannoni, A; Fantinati, P; Seren, E; Tamanini, C

    2003-11-01

    The aim of this study was to investigate the effect of fasting on both vascular endothelial growth factor (VEGF) production and VEGF mRNA expression in growing ovarian follicles (>5 mm in diameter) from gilts at 48 h after equine chorionic gonadotrophin (eCG) treatment. The concentrations of VEGF and albumin were measured in the follicular fluid of single follicles, and VEGF mRNA was determined in the follicle wall. Fasting resulted in a significant increase in VEGF concentrations in follicular fluid (20.64+/-0.72 versus 10.79+/-0.86 ng ml(-1), P<0.001), but it did not affect the total amount of VEGF mRNA in the follicle wall compared with that of fed animals. However, VEGF mRNA in the theca and granulosa compartments increased and decreased, respectively, compared with that of fed animals. The concentrations of albumin measured in follicular fluid as an index of vessel permeability were higher in fasted than in animals fed normally, most likely as a result of the increased VEGF production. Follicular steroidogenesis was impaired in fasted animals. Progesterone was the most abundant steroid in the follicular fluid and oestradiol was present in lower concentrations, thus indicating an alteration in the steroidogenic enzymatic cascade. In conclusion, fasting induces an increase in both VEGF production and vessel permeability. Such a reaction is unable under severe food deprivation to preserve follicle function, but may represent a mechanism that regulates blood vessel extension and distribution in relation to tissue requirements and availability of systemic nutrient.

  4. 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

  5. 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.

  6. Comparison of automated and manual follicle monitoring in an unrestricted population of 100 women undergoing controlled ovarian stimulation for IVF.

    PubMed

    Ata, Baris; Seyhan, Ayse; Reinblatt, Shauna Leigh; Shalom-Paz, Einat; Krishnamurthy, Srinivasan; Tan, Seang Lin

    2011-01-01

    Ovarian response to gonadotrophin stimulation is monitored with serial ultrasound (US) examinations. Sonography-based Automated Volume Count (SonoAVC) is a relatively new three-dimensional (3D) US technology, which automatically generates a set of measurements including the mean follicular diameter (MFD) and a volume-based diameter (d(V)) for each follicle in the ovaries. The present study aimed to assess the applicability and reproducibility of this automated follicle measurement method in an IVF programme. For this prospective method comparison study, 100 women undergoing US monitoring of a controlled ovarian stimulation cycle were recruited. Each follicle was manually measured by taking the mean of maximal diameters on three orthogonal planes with two-dimensional (2D) US. A 3D volume of each ovary was then captured. The ovarian volumes were later analysed using SonoAVC. The agreement between the two methods for the numbers of follicles and the size of the leading follicle was assessed with the Bland-Altman method. The reproducibility of SonoAVC measurements was assessed with the intraclass correlation coefficient (ICC). Both SonoAVC-generated MFD and d(V)-based follicle counts, as well as the leading follicle diameter, had good agreement with conventional 2D US measurements. SonoAVC measurements had very good reproducibility, with ICC ≥0.8 for most evaluations. Automated follicle monitoring with SonoAVC can replace or be used interchangeably with conventional 2D measurements. Automated follicle monitoring can save time, provide a method of quality control and create opportunities for developing HCG criteria based on follicular volume or for monitoring patients from a distance.

  7. Modulation of GR activity does not affect the in vitro metabolism of cortisol by rainbow trout ovarian follicles.

    PubMed

    Li, Mao; Christie, Heather; Leatherland, John

    2014-12-01

    The goal of the study was to determine whether the metabolic clearance of cortisol from rainbow trout (Oncorhynchus mykiss) ovarian follicles is affected by the level of ovarian steroidogenesis, and whether it involves the activation of glucocorticoid receptors (GRs). Ovarian follicles were incubated in vitro; the adenylate cyclase activator, forskolin, was used to stimulate ovarian steroidogenesis, and the modulation of GR activity was brought about using GR agonists (cortisol and dexamethasone) or the GR antagonist, mifepristone (RU486). The follicles were co-incubated with [2, 4, 6, 7 (3)H] cortisol, and the tritium-labelled steroid products were separated by HPLC. In addition, the rates of expression of genes encoding for the two forms of GR (gr1 and gr2) were measured. Cortisone, cortisol sulphate, and cortisone sulphate were the major glucocorticoid products of cortisol metabolism, indicative of the action of 11β-hydroxysteroid dehydrogenase and glucocorticoid sulphotransferase in the follicular cells. There were no effects of RU486 or forskolin on the rates of [(3)H]cortisol metabolism suggesting that cortisol metabolism by ovarian follicles was independent of GR activation, and not influenced by increased activation of gonadal reproductive steroidogenesis.

  8. 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.

  9. 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.

  10. Development of sheep primordial follicles encapsulated in alginate or in ovarian tissue in fresh and vitrified samples.

    PubMed

    Sadeghnia, Samaneh; Akhondi, Mohammad Mehdi; Hossein, Ghamartaj; Mobini, Sahba; Hosseini, Laleh; Naderi, Mohammad Mehdi; Boroujeni, Sara Borjian; Sarvari, Ali; Behzadi, Bahareh; Shirazi, Abolfazl

    2016-04-01

    In vitro follicle growth is a promising strategy for female fertility preservation. This study was conducted to compare the development of ovine follicles either isolated or in the context of ovarian cortical pieces after short term (8 days) three-dimensional culture in fresh and vitrified samples. Four different experiments were conducted; I) culture of ovarian cortical pieces encapsulated in 0.5% and 1% alginate and without alginate encapsulation (CP-0.5%, CP-1% and CP, respectively), II) culture of isolated primordial and primary follicles encapsulated in 1% and 2% alginate (IF-1% and IF-2%, respectively), III) culture of fresh and vitrified-warmed cortical pieces (F-CP and Vit-CP, respectively), and IV) culture of fresh and vitrified-warmed encapsulated isolated follicles (F-IF and Vit-IF, respectively). The number of secondary follicles after culture was negatively influenced by encapsulation of ovarian cortical pieces (6.3 ± 3.3 and 10.6 ± 0.9 vs 21.5 ± 2.3 in CP-0.5% and CP-1% vs CP, respectively). The diameter of follicles in IF-2% was higher than IF-1% (54.06 ± 2 vs 41.9 ± 1.5) and no significant difference in follicular viability was observed between the two groups. The proportions of different follicular types and their viability after culture in vitrified-warmed cortical pieces were comparable with fresh ones. The viability of vitrified-warmed isolated follicles was lower than fresh counterparts. The growth rate of fresh follicles was higher than vitrified-warmed follicles after culture (47.9 ± 1 vs 44.6 ± 1). In conclusion, while encapsulation of ovarian cortical pieces decreased the follicles' development, it could better support the growth of isolated follicles. Moreover, the viability and growth rate of isolated-encapsulated follicles was decreased by vitrification. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Toxicological impact of technical imidacloprid on ovarian morphology, hormones and antioxidant enzymes in female rats.

    PubMed

    Kapoor, Upasana; Srivastava, M K; Srivastava, L P

    2011-12-01

    Technical imidacloprid was evaluated for its effect on ovarian morphology, hormones and antioxidant enzymes in female rats after 90 days oral exposure. Luteinizing hormone (LH), follicle stimulating hormone (FSH) and progesterone levels were estimated in serum of rats and activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and level of reduced glutathione (GSH) and lipid peroxidation (LPO) were estimated in ovary after oral administration of imidacloprid (5, 10, and 20mg/kg/day) for 90 days. Decreased ovarian weight together with significant patho-morphological changes in follicles, antral follicles and atretic follicles were observed at 20mg/kg/day. Imidacloprid at 5 and 10mg/kg/day has not produced any significant changes in ovarian morphology, hormones and antioxidant status of ovary. However 20mg/kg/day dose has produced significant alterations in the levels of LH, FSH and progesterone. Similarly significant changes in SOD, CAT, GPx, GSH, and LPO were observed at 20mg/kg/day dose level. Therefore, it is concluded that imidacloprid at 20mg/kg/day dose level has produced significant toxicological impact on ovary of female rats as evident by pathomorphological changes, hormonal imbalance and generating oxidative stress and can be considered primarily as Lowest Observed Effect Level (LOEL) for chronic study.

  12. Presence of growth hormone receptor (GH-R) mRNA and protein in goat ovarian follicles and improvement of in vitro preantral follicle survival and development with GH.

    PubMed

    Martins, F S; Saraiva, M V A; Magalhães-Padilha, D M; Almeida, A P; Celestino, J J H; Padilha, R T; Cunha, R M S; Silva, J R V; Campello, C C; Figueiredo, J R

    2014-07-01

    This study aimed to demonstrate the expression of growth hormone receptor (GH-R) mRNA and protein in goat ovarian follicles in order to investigate the effects of GH on the survival and development of preantral follicles. The ovaries were processed for the isolation of follicles to study GH-R mRNA expression or to localization of GH-R by immunohistochemical analysis. Pieces of ovarian cortex were cultured for 7 days in minimum essential medium(+) (MEM(+)) in the presence or absence of GH at different concentrations (1, 10, 50, 100, and 200 ng/mL). High expression levels of GH-R mRNA were observed in granulosa/theca cells from large antral follicles. However, preantral follicles do not express mRNA for GH-R. Immunohistochemistry demonstrated that the GH-R protein was expressed in the oocytes/granulosa cells of antral follicles, but any protein expression was observed in preantral follicles. The highest (P < 0.05) rate of normal follicles and intermediate follicles was observed after 7 days in MEM(+) plus 10 ng/mL GH (70%). In conclusion, GH-R mRNA and protein are expressed in caprine antral follicles, but not in preantral follicles. Moreover, GH maintains the survival of goat preantral follicles and promotes the development of primordial follicles. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. 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.

  14. 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.

  15. 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.

  16. Corticosterone metabolism by chicken follicle cells does not affect ovarian reproductive hormone synthesis in vitro

    PubMed Central

    Rettenbacher, Sophie; Henriksen, Rie; Groothuids, Ton G.; Lepschy, Michael

    2013-01-01

    Glucocorticoids affect reproductive hormone production in many species. In chickens, elevated plasma corticosterone down-regulates testosterone and progesterone concentrations in plasma, but also in egg yolk. This suppression could be mediated via the hypothalamic-pituitary system but also via local inhibition of gonadal activity by glucocorticoids. As the latter has not been tested in birds yet, we tested if corticosterone directly inhibits ovarian steroid synthesis under in vitro conditions. We hypothesized that degradation of corticosterone by follicular cells impairs their ability to synthesize reproductive hormones due to either inhibition of enzymes or competition for common co-factors. Therefore, we first established whether follicles degrade corticosterone. Follicular tissue was harvested from freshly euthanized laying hens and incubated with radiolabelled corticosterone. Radioactive metabolites were visualized and quantified by autoradiography. Follicles converted corticosterone in a time-dependent manner into metabolites with a higher polarity than corticosterone. The predominant metabolite co-eluted with 20β-dihydrocorticosterone. Other chicken tissues mostly formed the same metabolite when incubated with corticosterone. In a second experiment, follicles were incubated with either progesterone or dehydroepiandrosterone. Corticosterone was added in increasing dosages up to 1000 ng per ml medium. Corticosterone did not inhibit the conversion of progesterone and dehydroepiandrosterone into a number of different metabolites, including 17α-hydroxyprogesterone, androstenedione and testosterone. In conclusion, avian tissues degrade corticosterone mostly to 20β-dihydrocorticosterone and even high corticosterone dosages do not affect follicular hormone production under in vitro conditions. PMID:23333751

  17. Survival and growth of goat primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water.

    PubMed

    Silva, José R V; van den Hurk, Robert; Costa, Sonia H F; Andrade, Evelyn R; Nunes, Ana P A; Ferreira, Francisco V A; Lôbo, Raimundo N B; Figueiredo, José R

    2004-04-01

    The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. We investigated the effectiveness of five culture media, two supplements and three culture periods on the survival and growth of goat primordial follicles after culturing ovarian cortex. The media were based on minimal essential minimum (MEM) and coconut water solution (CWS) added in the proportion of 0, 25, 50, 75 or 100%. The two supplements were none versus supplemented with insulin-transferrin-selenium, pyruvate, glutamine, hypoxanthine, and BSA. Pieces of goat ovarian cortex were cultured in the media for 1, 3 or 5 days and representative samples were evaluated at day 0 as non-cultured controls. The replicates were the two ovaries of five mixed breed goats. The number of primordial, intermediate, primary and secondary follicles at each period of culture and the number of degenerated follicles were evaluated. Mitotic activity of granulosa cells was studied by immunolocalization of proliferating cell nuclear antigen (PCNA). The number of follicles in each stage and degenerated follicles were statistically analyzed by ANOVA using a factorial design and the significance of differences assessed using Tukey test. Chi-square test was used to compare the percentage of follicles with PCNA positive granulosa cells. As the culture period progressed, the number of primordial follicles fell and there was a significant increase in the number of primary follicles. The fall in the number of primordial follicles was particularly marked after 1 day culture. No effect of media on the number of primordial and primary follicles was observed after culture, but MEM as well as supplements increased the number of intermediate follicles. Follicular degeneration was kept at the same level after culture in the media tested, except for pure CWS that increased the number of degenerated follicles. In

  18. 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

  19. Assessment of ovarian reserve: Anti-Mullerian hormone versus follicle stimulating hormone

    PubMed Central

    Jamil, Zehra; Fatima, Syeda Sadia; Cheema, Zahra; Baig, Safia; Choudhary, Roha Ahmed

    2016-01-01

    Background: This study aimed to evaluate the strength of anti-Mullerian hormone (AMH) and follicle stimulating hormone (FSH) in reflecting the antral follicle count (AFC) in infertile females. Materials and Methods: This cross-sectional study was conducted on 160 females, visiting infertility clinic for assisted reproduction. Serum samples collected on the 3rd day of the cycle were assayed for FSH, luteinizing hormone, and AMH while AFC was assessed via transvaginal ultrasound. The study cohort was segregated into three groups based on AFC. Results: Chronological age and FSH was significantly high in females with very low AFC (P < 0.01 and 0.009, respectively), yet they failed to discriminate patients with normal and higher follicle count (P = 0.65 and 0.84). Conversely, AMH reported highly significant difference between very low AFC and with those having either normal AFC (P = 0.002) or higher AFC (P = 0.001). Moreover, a significant difference in AMH was observed between normal and higher AFC group (P = 0.04). Conclusion: Compared to female’s age and FSH, AMH is superior in clustering study cohort on the bases of antral follicular pool, especially in setups with nonavailability of technological expertise to assess AFC. Incorporation of AMH along with other biomarkers improves estimation of baseline ovarian reserve, required to standardize dose for optimum response; avoiding the risk of failure to retrieve oocyte or inappropriate stimulation leading to ovarian hyperstimulation syndrome. Further prospective studies are required to ascertain its role in predicting the outcomes of ART in such patients. PMID:28163746

  20. Exclusion of polymeric immunoglobulins and selective immunoglobulin Y transport that recognizes its Fc region in avian ovarian follicles.

    PubMed

    Kitaguchi, Kohji; Osada, Kenichi; Horio, Fumihiko; Murai, Atsushi

    2008-02-15

    In avian species, blood immunoglobulin (Ig) Y, the equivalent to mammalian IgG, is selectively incorporated into ovarian follicles, but other classes, IgA and IgM, are much less abundant in the follicles. Several mammalian Igs, including IgG and IgA, are also incorporated into ovarian follicles when administered to birds. To clarify the Ig structure required for incorporation into ovarian follicles, Ig uptakes were determined after the intravenous injection of chicken and human Igs. Three chicken Igs (cIgY, cIgA and cIgM) and two human IgAs (monomeric hIgA and polymeric hIgA) were labeled with digoxigenin, and their uptakes into quail (Coturnix japonica) egg yolks were determined by ELISA and SDS-PAGE. The uptake of cIgY was the highest among the three cIgs (22% of injected cIgY was recovered from egg yolks). Chicken IgA was efficiently incorporated into egg yolk when it formed a monomeric state. Pentameric IgM was untransportable into egg yolk. We also found that the uptake of monomeric hIgA was much more efficient than that of polymeric hIgA. These results suggest that the retention of the monomeric form contributes to the efficient transport of Igs into ovarian follicles. On the other hand, Ig uptakes among monomeric Igs nevertheless differed; for example, a time-course analysis showed that the rate of monomeric cIgY uptake was approximately eight times faster than that of monomeric hIgA. The injection of cIgY fragments Fc, Fab and F(ab')(2) resulted in the largest uptake of Fc fragment, with the same level as that of cIgY. These results suggest the presence of a selective IgY transport system that recognizes its Fc region in avian ovarian follicles.

  1. Follicle pool, ovarian surgery and the risk for a subsequent trisomic pregnancy.

    PubMed

    Honorato, T C; Henningsen, A A; Haadsma, M L; Land, J A; Pinborg, A; Lidegaard, Ø; Groen, H; Hoek, A

    2015-03-01

    Is there an association between trisomic pregnancy, a marker for decreased oocyte quality, and the reduced oocyte quantity that follows ovarian surgery? Previous ovarian surgery is not associated with an increased risk for a subsequent trisomic pregnancy. Ovarian surgery diminishes the number of oocytes. The risk for a trisomic pregnancy is suggested to be higher in women with fewer oocytes, independent of their chronological age. This is a matched case-control study. Cases are women with a confirmed trisomic pregnancy occurring between 1 January 2000 and 31 December 2010 regardless of pregnancy outcome and controls are women that had a live born child without a trisomy. In total, there were 8573 participants in the study; 1723 cases and 6850 controls. Data were obtained from Danish medical registries. Matching criteria were maternal age and year of conception. Number of controls matched per case ranged from one to four. Among cases and controls with a trisomic pregnancy, 2.7% (46/1723) versus 2.5% (172/6850) had undergone ovarian surgery before pregnancy. History of ovarian surgery is not associated with a higher risk for a subsequent trisomic pregnancy (odds ratio = 1.00, 95% confidence interval 0.99-1.01). Subgroup analyses by indication of surgery and interval between ovarian surgery and pregnancy do not show an effect on trisomic pregnancy risk. The medical registries used to select cases and controls did not contain information on surgical technique nor volume of ovarian tissue resected, previous trisomic pregnancy prior to the ovarian surgery or long-term use of oral contraceptives. Therefore, correction for these factors was not performed. We did not confirm the hypothesis that ovarian surgery, a marker for decreased oocyte quantity, is related to trisomic pregnancy, a marker for decreased oocyte quality. This suggests that ovarian surgery, which has a direct reductive effect on the size of the follicle pool, may affect oocyte quality differently when

  2. Short-term exposure of human ovarian follicles to cyclophosphamide metabolites seems to promote follicular activation in vitro.

    PubMed

    Lande, Yechezkel; Fisch, Benjamin; Tsur, Abraham; Farhi, Jacob; Prag-Rosenberg, Roni; Ben-Haroush, Avi; Kessler-Icekson, Gania; Zahalka, Muayad A; Ludeman, Susan M; Abir, Ronit

    2017-01-01

    How chemotherapy affects dormant ovarian primordial follicles is unclear. The 'burnout' theory, studied only in mice, suggests cyclophosphamide enhances primordial follicle activation. Using 4-hydroperoxycyclophosphamide (4hc) and phosphoramide mustard (PM), this study assessed how the active cyclophosphamide metabolites 4-hydroxycyclophosphamide (4-OHC) and PM, affect human primordial follicles. Frozen-thawed human ovarian samples were sliced and cultured with basic culture medium (cultured controls) or with 4hc/PM (3 µmol/l/10 µmol/l) (treated samples) for 24-48 h. Follicular counts and classification, Ki67 and anti-Müllerian hormone (AMH) immunohistochemistry and an apoptosis assay were used for evaluation, and 17β-oestradiol and AMH were measured in spent media samples. Generally, there was primordial follicle decrease and elevated developing follicle rates in treated samples compared with cultured (P = 0.04 to P < 0.0005) and uncultured controls (P < 0.05 to P < 0.0001). No traces of apoptosis were found. There were almost twicethe levels of AMH and 17β-oestradiol in treated compared with untreated samples (AMH with 4hc 3 µmol/l; P = 0.04). All follicles stained positively for AMHincluded treated samples. Ki67 positive staining was noted in all samples. Cyclophosphamide metabolites seem to enhance human primordial follicle activation to developing follicles, in vitro. Study findings support the 'burnout' theory as the mechanism of chemotherapy-induced ovarian toxicity. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  3. Neonatal exposure to 17α-ethynyl estradiol (EE) disrupts follicle development and reproductive hormone profiles in female rats.

    PubMed

    Zhang, Haolin; Taya, Kazuyoshi; Nagaoka, Kentaro; Yoshida, Midori; Watanabe, Gen

    2017-07-05

    Toxic effects induced by exposure to endocrine-disrupting chemicals during fetal and neonatal periods can be irreversible and exert effects throughout an animal's entire life. Our previous study showed that neonatal exposure to 17α-ethynyl estradiol (EE) induced irregular estrous cycle in adults. To uncover the reason for the delayed effect after neonatal exposure to EE, reproductive parameters including ovarian weight, ovarian steroidogenesis, and hormonal profiles were investigated in developing female rats. Ovarian weight decreased at postnatal days (PND) 14 and 21 after neonatal exposure to EE. Ovarian histology at PND21 showed that the ratio of follicles with a diameter >300μm decreased and the ratio of follicles with a diameter of 100-150μm increased in EE-treated ovaries, indicating that neonatal exposure to EE retarded follicular development. Moreover, the expression of P450arom increased at PND14 and the expressions of inhibin/activin subunits βA and βB decreased at PND21 in EE-treated ovaries. Consistent with the expression of P450arom, circulating levels of 17β-estradiol increased at PND14 in EE-treated animals. Furthermore, the circulating levels of luteinizing hormone (LH) also increased at PND14 in the treated animals. Although the expression of Kiss1 did not change in the anteroventral periventricular nucleus (AVPV) of the hypothalamus between controls and EE-treated rats, the expression of Kiss1 was reduced in the arcuate nucleus (ARC) of the hypothalamus at PND14. Based upon those results, we suggest that neonatal exposure to EE disrupted the system regulating the interactions between the reproductive hormones and follicle development in pre-pubertal rats, which may result in reproduction dysfunction in adulthood. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Expressed transcripts associated with high rates of egg production in chicken ovarian follicles.

    PubMed

    Yang, K T; Lin, C Y; Huang, H L; Liou, J S; Chien, C Y; Wu, C P; Huang, C W; Ou, B R; Chen, C F; Lee, Y P; Lin, E C; Tang, P C; Lee, W C; Ding, S T; Cheng, W T K; Huang, M C

    2008-02-01

    The purpose of this study was to characterize differentially expressed transcripts associated with varying rates of egg production in Taiwan country chickens. Ovarian follicles were isolated from two strains of chicken which showed low (B) or high (L2) rates of egg production, then processed for RNA extraction and cDNA library construction. Three thousand and eight forty clones were randomly selected from the cDNA library and amplified by PCR, then used in microarray analysis. Differentially expressed transcripts (P<0.05, log(2)> or = 1.75) were sequenced, and aligned using GenBank. This analysis revealed 20 non-redundant sequences which corresponded to known transcripts. Eight transcripts were expressed at a higher level in ovarian tissue prepared from chicken strain B, and 12 transcripts were expressed at a higher level in L2 birds. These differential patterns of expression were confirmed by semi-quantitative RT-PCR. We show that transcripts of cyclin B2 (cycB2), ferritin heavy polypeptide 1 (FTH1), Gag-Pol polyprotein, thymosin beta4 (TB4) and elongation factor 1 alpha1 (EEF1A1) were enriched in B strain ovarian follicles. In contrast, thioredoxin (TXN), acetyl-CoA dehydrogenase long chain (ACADL), inhibitor of growth family member 4 (ING4) and annexin II (ANXA2) were expressed in at higher levels in the L2 strain. We suggest that our approach may lead to the isolation of effective molecular markers that can be used in selection programs in Taiwan country chickens.

  5. The domestic dog and cat as models for understanding the regulation of ovarian follicle development in vitro.

    PubMed

    Songsasen, N; Comizzoli, P; Nagashima, J; Fujihara, M; Wildt, D E

    2012-12-01

    The culture of ovarian follicles is an important tool for understanding the mechanisms controlling follicle development and differentiation of the oocyte. The benefit of recovering meiotically and developmentally competent oocytes from early stage follicles (primordial, primary, pre-antral and early antral) also would be significant, ranging from rescue of genomes from endangered species to preserving fertility in women facing cancer treatments. This research field is at an early stage of scientific discovery. To-date, live offspring from cultured primordial follicles that produced fertilizable oocytes has occurred only in the mouse. Progress in other more complex species has been limited because larger animals have longer durations of natural folliculogenesis, thereby requiring more culture time to generate fully grown follicles and oocytes. We believe the dog and cat are excellent models for understanding more about folliculogenesis in vitro. This review highlights what is known about this topic for these two species as well as future priorities. We have discovered that it is more challenging to maintain viability of primordial follicles within ovarian tissues in vitro in the dog than the cat. Nonetheless, it is possible to grow both isolated cat and dog pre-antral follicles in culture. Although the follicles of both species have the capacity to increase in size and produce steroids, only cat oocytes appear morphologically normal. The reason for this striking difference between these two species is an area of high research priority. While much more fundamental data are required, we envision advanced technology that will allow harvesting oocytes from the vast, unused follicle stores sequestered within carnivore ovaries. These gametes have utility for reproducing genetically valuable dogs and cats that are 'companions' or biomedical models for investigating human disorders as well as for salvaging the genomes of rare canid and felid species that die before

  6. The domestic dog and cat as models for understanding the regulation of ovarian follicle development in vitro

    PubMed Central

    Songsasen, N.; Comizzoli, P.; Nagashima, J.; Fujihara, M.; Wildt, D.E

    2012-01-01

    The culture of ovarian follicles is an important tool for understanding of the mechanisms controlling follicle development and differentiation of its oocyte. The benefit of recovering meiotically and developmentally competent oocytes from early stage follicles (primordial, primary, preantral and early antral) also would be significant, ranging from rescue of genomes from endangered species to preserving fertility in women facing cancer treatments. This field of research is at an early stage of scientific discovery. To-date, live offspring from cultured primordial follicles that produced fertilizable oocytes has occurred only in the mouse. Progress in other more complex species has been limited because larger animals have longer durations of natural folliculogenesis, thereby requiring more culture time to generate fully grown follicles and oocytes. We believe the dog and cat are excellent models for understanding more about folliculogenesis in vitro. This review highlights what is known about this topic for these two species as well as future priorities. In brief, it is more challenging to maintain viability of primordial follicles within ovarian tissues in vitro in the dog than the cat. Nonetheless, it is possible to grow both isolated cat and dog preantral follicles in culture. Although the follicles of both species have the capacity to increase in size and produce steroids, only cat oocytes are morphologically normal. This striking difference between the dog and cat is an area of high research priority. While much more fundamental data are required, we envision advanced technology that will allow harvesting oocytes from the vast, unused follicle stores sequestered within carnivore ovaries. These gametes have utility for reproducing genetically valuable dogs and cats that are ‘companions’ or biomedical models for investigating human disorders or for salvaging the genomes of rare canid and felid species that die before contributing to genetic management

  7. Remarkable features of ovarian morphology and reproductive hormones in insulin-resistant Zucker fatty (fa/fa) rats

    PubMed Central

    2010-01-01

    Background Zucker fatty (fa/fa) rats are a well-understood model of obesity and hyperinsulinemia. It is now thought that obesity/hyperinsulinemia is an important cause of endocrinological abnormality, but to date there have been no reports on the changes in ovarian morphology or the ovarian androgen profile in rat models of obesity and insulin resistance. Methods In this study we investigated the effects of obesity and hyperinsulinemia on ovarian morphology and the hormone profile in insulin-resistant Zucker fatty rats (5, 8, 12 and 16 weeks of age, n = 6-7). Results Ovaries from 5-week-old fatty rats had significantly greater total and atretic follicle numbers, and higher atretic-to-total follicle ratios than those from lean rats. Ovaries from 12- and 16-week-old fatty rats showed interstitial cell hyperplasia and numerous cysts with features of advanced follicular atresia. In addition, serum testosterone and androstenedione levels significantly declined in fatty rats from age 8 to 16 weeks, so that fatty rats showed significantly lower levels of serum testosterone (12 and 16 weeks) and androstenedione (all weeks) than lean rats. This may reflect a reduction of androgen synthesis during follicular atresia. Serum adiponectin levels were high in immature fatty rats, and although the levels declined significantly as they matured, it remained significantly higher in fatty rats than in lean rats. On the other hand, levels of ovarian adiponectin and its receptors were significantly lower in mature fatty rats than in lean mature rats or immature fatty rats. Conclusions Our findings indicate that ovarian morphology and hormone profiles are significantly altered by the continuous insulin resistance in Zucker fatty rats. Simultaneously, abrupt reductions in serum and ovarian adiponectin also likely contribute to the infertility seen in fatty rats. PMID:20576113

  8. Effects of in vivo testosterone manipulation on ovarian morphology, follicular development, and follicle yolk testosterone in the homing pigeon.

    PubMed

    Goerlich, Vivian C; Dijkstra, Cor; Groothuis, Ton G G

    2010-07-01

    To date, our understanding of the function of testosterone in female reproductive physiology is only marginal although there are indications that testosterone is involved in modulating follicular recruitment, growth, atresia, and ovulation. Studies elevating testosterone in breeding female birds have, in most instances, found detrimental effects, such as delayed clutch initiation or decreased clutch size. In our previous study, testosterone treatment of female homing pigeons delayed clutch initiation without diminishing fecundity. In this study, we explore whether the observed effect might have been caused by testosterone influencing follicle maturation or ovulation. We implanted mature female pigeons with testosterone prior to pairing, which resulted in constant elevation of circulating testosterone concentrations within the physiological range. We killed females after they had laid the first egg and measured ovarian and follicular development. Ovarian mass and pre-hierarchical yolky follicles were not affected by the treatment; however, testosterone females produced smaller and lighter preovulatory follicles. High plasma testosterone levels at oviposition or a strong temporal increase in testosterone were negatively related to mass and diameter of second follicles. We proposed that sustained elevation of testosterone delays follicular maturation, potentially via negative feedback on the hypothalamo-pituitary-gonadal axis. Furthermore, to gain better insight into the regulation of yolk hormone acquisition, we measured testosterone concentrations in the preovulatory follicles. We found no differences between treatment groups but follicle yolk contained much higher levels of testosterone than yolk of un-incubated eggs, suggesting that hormone measurements performed after oviposition do not correctly reflect maternal allocation.

  9. Continuous cadmium exposure from weaning to maturity induces downregulation of ovarian follicle development-related SCF/c-kit gene expression and the corresponding changes of DNA methylation/microRNA pattern.

    PubMed

    Weng, Shaozheng; Wang, Wenxiang; Li, Yuchen; Li, Hong; Lu, Xiaoli; Xiao, Shihua; Wu, Tingting; Xie, Meimei; Zhang, Wenchang

    2014-03-21

    Cadmium (Cd) impairs ovary structure and function in mature animals. However, the influence of Cd on follicle development from weaning to maturity is obscure. In the current study, 21-day-old Wistar rats were administered Cd chloride at doses of 0, 0.5, 2.0 and 8.0 mg/kg body weight once a day for eight weeks by gavage. After administration, a significant decrease in ovarian wet weight, ovarian/body weight ratios, and primordial follicles, in addition to an increase in atresic follicles, were observed. Transmission electron microscopy and TUNEL assay confirmed the increase of follicle apoptosis as Cd concentration increased. Real-time quantitative PCR and Western blotting showed a significantly decreased expression of follicle development-related factors, stem cell factor (SCF) and c-kit. Bisulfite sequencing suggested that the total methylation percentages of SCF/c-kit promoter region were not obvious change after Cd exposure. Real-time quantitative PCR revealed a significantly increased expression of miR-193, miR-221 and miR-222, which regulate c-kit, in the 2.0 mg/kg and 8.0 mg/kg treatment groups. Overall, this study proved that Cd administration from weaning to maturity could damage follicle development, suggesting that SCF/c-kit might play an important role in this effect. In addition, microRNAs might play a role in c-kit protein downregulation.

  10. Pediatric and Teen Ovarian Tissue Removed for Cryopreservation Contains Follicles Irrespective of Age, Disease Diagnosis, Treatment History, and Specimen Processing Methods

    PubMed Central

    Duncan, Francesca E.; Pavone, Mary Ellen; Gunn, Alexander H.; Badawy, Sherif; Gracia, Clarisa; Ginsberg, Jill P.; Lockart, Barbara; Gosiengfiao, Yasmin

    2015-01-01

    Purpose: Fertility preservation in a pediatric and teen female population is challenging because standard technologies of egg and embryo freezing may not be possible due to premenarcheal status. Ovarian tissue cryopreservation (OTC) with the intent of future ovarian tissue transplantation or in vitro follicle growth may be the only option to preserve fertility. The purpose of this study was to add to the general understanding of primordial follicle dynamics in young patients. Methods: First, the unique infrastructure of the Oncofertility Consortium National Physicians Cooperative (OC-NPC) is described, which simultaneously drives clinical fertility preservation and basic research to explore and expand the reproductive options for those in need. Then, the OC-NPC research resource is used to perform a histological evaluation of ovarian tissue from 24 participants younger than 18 years of age. Results: Primordial follicles, which comprise the ovarian reserve, were observed in all participant tissues, irrespective of variables, including age, diagnosis, previous treatment history, tissue size, and tissue processing methods. Primordial follicles were present in ovarian tissue, even in participants who had a previous history of exposure to chemotherapy and/or radiation treatment regimens, which placed them at risk for iatrogenic infertility or premature ovarian failure. Conclusion: Primordial follicles were observed in ovarian tissue from all participants examined, despite population and tissue heterogeneity. These results increase the understanding of human follicle dynamics and support OTC as a promising fertility preservation modality in the young female population. Future studies to evaluate follicle quality within these tissues are warranted. PMID:26697267

  11. Pediatric and Teen Ovarian Tissue Removed for Cryopreservation Contains Follicles Irrespective of Age, Disease Diagnosis, Treatment History, and Specimen Processing Methods.

    PubMed

    Duncan, Francesca E; Pavone, Mary Ellen; Gunn, Alexander H; Badawy, Sherif; Gracia, Clarisa; Ginsberg, Jill P; Lockart, Barbara; Gosiengfiao, Yasmin; Woodruff, Teresa K

    2015-12-01

    Fertility preservation in a pediatric and teen female population is challenging because standard technologies of egg and embryo freezing may not be possible due to premenarcheal status. Ovarian tissue cryopreservation (OTC) with the intent of future ovarian tissue transplantation or in vitro follicle growth may be the only option to preserve fertility. The purpose of this study was to add to the general understanding of primordial follicle dynamics in young patients. First, the unique infrastructure of the Oncofertility Consortium National Physicians Cooperative (OC-NPC) is described, which simultaneously drives clinical fertility preservation and basic research to explore and expand the reproductive options for those in need. Then, the OC-NPC research resource is used to perform a histological evaluation of ovarian tissue from 24 participants younger than 18 years of age. Primordial follicles, which comprise the ovarian reserve, were observed in all participant tissues, irrespective of variables, including age, diagnosis, previous treatment history, tissue size, and tissue processing methods. Primordial follicles were present in ovarian tissue, even in participants who had a previous history of exposure to chemotherapy and/or radiation treatment regimens, which placed them at risk for iatrogenic infertility or premature ovarian failure. Primordial follicles were observed in ovarian tissue from all participants examined, despite population and tissue heterogeneity. These results increase the understanding of human follicle dynamics and support OTC as a promising fertility preservation modality in the young female population. Future studies to evaluate follicle quality within these tissues are warranted.

  12. Study origin of germ cells and formation of new primary follicles in adult human and rat ovaries.

    PubMed

    Bukovsky, Antonin; Gupta, Satish K; Virant-Klun, Irma; Upadhyaya, Nirmala B; Copas, Pleas; Van Meter, Stuart E; Svetlikova, Marta; Ayala, Maria E; Dominguez, Roberto

    2008-01-01

    The central thesis regarding the human ovaries is that, although primordial germ cells in embryonal ovaries are of extraovarian origin, those generated during the fetal period and in postnatal life are derived from the ovarian surface epithelium (OSE) bipotent cells. With the assistance of immune system-related cells, secondary germ cells and primitive granulosa cells originate from OSE stem cells in the fetal and adult human gonads. Fetal primary follicles are formed during the second trimester of intrauterine life, prior to the end of immune adaptation, possibly to be recognized as self-structures and renewed later. With the onset of menarche, a periodical oocyte and follicular renewal emerges to replace aging primary follicles and ensure that fresh eggs for healthy babies are always available during the prime reproductive period. The periodical follicular renewal ceases between 35 and 40 yr of age, and the remaining primary follicles are utilized during the premenopausal period until exhausted. However, the persisting oocytes accumulate genetic alterations and may become unsuitable for ovulation and fertilization. The human OSE stem cells preserve the character of embryonic stem cells, and they may produce distinct cell types, including new eggs in vitro, particularly when derived from patients with premature ovarian failure or aging and postmenopausal ovaries. Our observations also indicate that there are substantial differences in follicular renewal between adult human and rat ovaries. As part of this chapter, we present in detail protocols utilized to analyze oogenesis in humans and to study interspecies differences when compared to the ovaries of rat females.

  13. Kit ligand promotes the transition from primordial to primary follicles after in vitro culture of ovine ovarian tissue.

    PubMed

    Cavalcante, A Y P; Gouveia, B B; Barberino, R S; Lins, T L B G; Santos, L P; Gonçalves, R J S; Celestino, J J H; Matos, M H T

    2016-08-01

    This study evaluated the effects of kit ligand (KL) on the morphology and development of ovine preantral follicles (fresh control) and after 7 days of in vitro culture in α-Minimal Essential Medium (α-MEM; control medium) or the presence of KL (1, 10, 50, 100 or 200 ng/ml). There was an increase in the percentage of primary follicles at the concentration of 100 ng/ml KL, compared with the fresh control, control medium (α-MEM) and the other KL concentrations. Follicle diameter was significantly higher than the control medium only at concentrations of 50 and 100 ng/ml KL. In conclusion, 100 ng/ml KL promoted the transition from primordial to primary follicles (follicular activation) after in vitro culture of ovine ovarian tissue.

  14. The magnitude of gonadotoxicity of chemotherapy drugs on ovarian follicles and granulosa cells varies depending upon the category of the drugs and the type of granulosa cells.

    PubMed

    Yuksel, Aytac; Bildik, Gamze; Senbabaoglu, Filiz; Akin, Nazli; Arvas, Macit; Unal, Fehmi; Kilic, Yagmur; Karanfil, Isil; Eryılmaz, Baldan; Yilmaz, Pelin; Ozkanbaş, Can; Taskiran, Cagatay; Aksoy, Senai; Guzel, Yılmaz; Balaban, Basak; Ince, Umit; Iwase, Akira; Urman, Bulent; Oktem, Ozgur

    2015-12-01

    groups of 13 to receive a single IP injection of: saline (control), gemcitabine (200 mg/kg), cisplatin (50 mg/kg) or cyclophosphamide (200 mg/kg). The animals were euthanized 72 h later. Follicle counts and serum AMH levels were compared between the groups. In vitro cytotoxicity studies were performed using mitotic non-luteinized rat (SIGC) and human (COV434, HGrC1) granulosa cells, and non-mitotic luteinized human (HLGC) granulosa cells. The cells were plated at a density of 5000 cells/well using DMEM-F12 culture media supplemented with 10% FBS. Chemotherapy agents were used at their therapeutic blood concentrations. The growth of mitotic granulosa cells was monitored real-time using xCelligence system. Live/dead cell and apoptosis assays were also carried out using intravital Yo-Pro-1 staining and cleaved caspase-3 expression, respectively. Estradiol (E2), progesterone (P) and anti-Mullerian hormone (AMH) levels were assayed with ELISA. Cyclophosphamide and cisplatin caused massive atresia of both primordials and growing follicles in the rat ovary whereas gemcitabine impacted pre-antral/antral follicles only. Cyclophosphamide and cisplatin induced apoptosis of both mitotic non-luteinized and non-mitotic luteinized granulosa cells in vitro. By contrast, cytotoxicity of gemcitabine was confined to mitotic non-luteinized granulosa cells. This study tested only three chemotherapeutic agents. The experimental methodology described here could be applied to other drugs for detailed analysis of their ovarian cytotoxicity. These findings indicate that in vivo and in vitro cytotoxic actions of chemotherapy drugs on the ovarian follicles and granulosa cells vary depending upon the their mechanism of action and the nature of the granulosa cells. © 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.

  15. 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.

  16. Dysregulation of ovarian follicular development in female rat: LH decreases FSH sensitivity during preantral-early antral transition.

    PubMed

    Orisaka, Makoto; Hattori, Katsushige; Fukuda, Shin; Mizutani, Tetsuya; Miyamoto, Kaoru; Sato, Takashi; Tsang, Benjamin K; Kotsuji, Fumikazu; Yoshida, Yoshio

    2013-08-01

    Several clinical studies have shown a correlation of hypersecretion of LH and polycystic ovary syndrome (PCOS), infertility, and miscarriage in women, suggesting that chronically elevated LH impairs fertility. Growth arrest of small antral follicles in PCOS is also assumed to be associated with an abnormal endocrine environment involving increased LH stimulation, a hyperandrogenic milieu, and subsequent dysregulated FSH action in the ovarian follicles. In this study, we examined whether and how LH modulates follicular development and steroid production during preantral-early antral follicle transition by using a rat preantral follicle culture system. LH augments testosterone and estradiol production in preantral follicles via up-regulating mRNA abundance of CYP17A1 and CYP19A1. LH promotes rat preantral follicle growth, and the follicular size reaches that of early antral follicles in vitro, a response attenuated by the specific androgen receptor antagonist and a targeted disruption of androgen receptor gene. Sustained follicle stimulation by LH, but not by androgen, decreases FSH receptor mRNA levels and FSH receptor signaling and inhibits FSH-induced follicular growth. The data suggest that LH promotes preantral-early antral transition via the increased synthesis and growth-promoting action of androgen. However, chronic LH stimulation impairs FSH-dependent antral follicle growth by suppressing granulosa cell FSHR expression via the modulation of intraovarian regulators, including LH-induced thecal factors.

  17. Infertility and ovarian follicle reserve depletion are associated with dysregulation of the FSH and LH receptor density in human antral follicles.

    PubMed

    Regan, Sheena L P; Knight, Phil G; Yovich, John L; Stanger, James D; Leung, Yee; Arfuso, Frank; Dharmarajan, Arun; Almahbobi, Ghanim

    2017-02-07

    The low take-home baby rate in older women in Australia (5.8%) undergoing IVF (5.8%) is linked to the depletion of the ovarian reserve of primordial follicles. Oocyte depletion causes an irreversible change to ovarian function. We found that the young patient FSH receptor and LH receptor expression profile on the granulosa cells collected from different size follicles were similar to the expression profile reported in natural cycles in women and sheep. This was reversed in the older patients with poor ovarian reserve. The strong correlation of BMPR1B and FSH receptor density in the young was not present in the older women; whereas, the LH receptor and BMPR1B correlation was weak in the young but was strongly correlated in the older women. The reduced fertilisation and pregnancy rate was associated with a lower LH receptor density and a lack of essential down-regulation of the FSH and LH receptor. The mechanism regulating FSH and LH receptor expression appears to function independently, in vivo, from the dose of FSH gonadotrophin, rather than in response to it. Restoring an optimum receptor density may improve oocyte quality and the pregnancy rate in older women.

  18. A novel method for toxicology: in vitro culture system of a rat preantral follicle.

    PubMed

    Wan Xuying; Zhu Jiangbo; Zhu Yuping; Xili, Ma; Liu Zhen; Wang Fei; Xu Guifeng; Zhang Tianbao

    2011-08-01

    Preantral follicle in vitro culture systems have been successfully or nearly successfully established for sheep, pig and mouse, and applied on follicle development and regulation research on reproductive biology and physiology. However, there have been few studies concerning rat preantral follicle in vitro development. The objective is to establish an in vitro culture system for rat preantral follicles which can be used for reproductive biology and toxicology research. Rat preantral follicles are mechanically separated, cultured in vitro in single follicle mode for continuous 12 days using 96-well plates, and then administrated ovulation induction. The observation on follicle development, hormone level, and ovum formation are recorded and assessed. Taking in vivo growth and in vitro maturation of oocytes group as control group, in vitro growth and maturation of oocytes group is assessed to see whether this in vitro culture method is successful. The conditions for rat follicle culture are determined based on the mouse pre-antral follicle culture. The in vitro culture system for rat preantral follicles established in this study is feasible and successful, and can serve as model for reproductive biology and toxicology research.

  19. Androgens regulate ovarian follicular development by increasing follicle stimulating hormone receptor and microRNA-125b expression.

    PubMed

    Sen, Aritro; Prizant, Hen; Light, Allison; Biswas, Anindita; Hayes, Emily; Lee, Ho-Joon; Barad, David; Gleicher, Norbert; Hammes, Stephen R

    2014-02-25

    Although androgen excess is considered detrimental to women's health and fertility, global and ovarian granulosa cell-specific androgen-receptor (AR) knockout mouse models have been used to show that androgen actions through ARs are actually necessary for normal ovarian function and female fertility. Here we describe two AR-mediated pathways in granulosa cells that regulate ovarian follicular development and therefore female fertility. First, we show that androgens attenuate follicular atresia through nuclear and extranuclear signaling pathways by enhancing expression of the microRNA (miR) miR-125b, which in turn suppresses proapoptotic protein expression. Second, we demonstrate that, independent of transcription, androgens enhance follicle-stimulating hormone (FSH) receptor expression, which then augments FSH-mediated follicle growth and development. Interestingly, we find that the scaffold molecule paxillin regulates both processes, making it a critical regulator of AR actions in the ovary. Finally, we report that low doses of exogenous androgens enhance gonadotropin-induced ovulation in mice, further demonstrating the critical role that androgens play in follicular development and fertility. These data may explain reported positive effects of androgens on ovulation rates in women with diminished ovarian reserve. Furthermore, this study demonstrates mechanisms that might contribute to the unregulated follicle growth seen in diseases of excess androgens such as polycystic ovary syndrome.

  20. Assembly of ovarian follicles in the caecilians Ichthyophis tricolor and Gegeneophis ramaswamii: light and transmission electron microscopic study.

    PubMed

    Beyo, R S; Sreejith, P; Divya, L; Oommen, O V; Akbarsha, M A

    2007-08-01

    Though much is known about various aspects of reproductive biology of amphibia, there is little information on the cellular and mechanistic basis of assembly of ovarian follicles in this group. This is especially true of the caecilians. Therefore, taking advantage of the abundant distribution of caecilians in the Western Ghats of India, two species of caecilians, Ichthyophis tricolor and Gegeneophis ramaswamii, were subjected to light and transmission electron microscopic analysis to trace the sequential changes during the assembly of ovarian follicles. The paired ovaries of these caecilians are elongated sac-like structures each including numerous vitellogenic follicles. The follicles are connected by a connective tissue stroma. This stroma contains nests of oogonia, primary oocytes and pregranulosa cells as spatially separated nests. During assembly of follicles the oocytes increase in size and enter the meiotic prophase when the number of nucleoli in the nucleus increases. The mitochondrial cloud or Balbiani vitelline body, initially localized at one pole of the nucleus, disperses through out the cytoplasm subsequently. Synaptonemal complexes are prominent in the pachytene stage oocytes. The pregranulosa cells migrate through the connective tissue fibrils of the stroma and arrive at the vicinity of the meiotic prophase oocytes. On contacting the oocyte, the pregranulosa cells become cuboidal in shape, wrap the diplotene stage oocyte as a discontinuous layer and increase the content of cytoplasmic organelles and inclusions. The oocytes increase in size and are arrested in diplotene when the granulosa cells become flat and form a continuous layer. Soon a perivitelline space appears between the oolemma and granulosa cells, completing the process of assembly of follicles. Thus, the events in the establishment of follicles in the caecilian ovary are described.

  1. Positive impact of sucrose supplementation during slow freezing of cat ovarian tissues on cellular viability, follicle morphology, and DNA integrity.

    PubMed

    Tanpradit, Nae; Comizzoli, Pierre; Srisuwatanasagul, Sayamon; Chatdarong, Kaywalee

    2015-06-01

    The objectives of the study were to (1) examine and optimize the impact of sucrose during slow freezing and (2) compare the results of two freezing methods (slow freezing and vitrification) on cellular viability (germinal and stromal cells), follicle morphology, DNA integrity, and gap junction protein expression (connexin 43 [Cx 43]). Different sucrose supplementations (0, 0.1, and 0.3 M) in standard freezing medium were compared before and after slow freezing. Ovarian tissue slow frozen using 0.1- (4.0 ± 0.4) or 0.3-M sucrose (3.9 ± 0.5) yielded better follicular viability (number of positive follicles per 0.0625 mm(2)) than the group without sucrose (1.9 ± 0.2; P < 0.05). Morphologically normal primordial follicles were higher in the sucrose-treated groups (0.1 M, 47.4% and 0.3 M, 43.5%) than the group without sucrose (0 M, 33.8%; P < 0.05). Moreover, less apoptotic primordial follicles were found in both sucrose groups (0.1 M, 1.2% and 0.3 M, 1.9%) than the group without sucrose (7.7%; P < 0.05). However, their Cx 43 expression showed no difference among the groups of different sucrose concentrations. In terms of the freezing methods used, vitrified ovarian tissues had fewer viable follicles (3.2 ± 0.6) than the slow-freezing method (4.6 ± 0.6; P < 0.05). In addition, the slow freezing resulted in more postthawed morphologically normal primordial follicles (38.8% vs. 28.3%, P < 0.05) and less apoptotic primordial follicles (3.8% vs. 8.9%, P < 0.05) than vitrification. The Cx 43 expression showed no difference between slow freezing and vitrification. The present study reported the positive effects of sucrose supplementation and slow-freezing method on the follicular viability, follicular histologic appearances of follicles, and apoptosis of the follicles and stromal cells in cat ovarian tissues. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Lifestyle factors associated with histologically derived human ovarian non-growing follicle count in reproductive age women†

    PubMed Central

    Peck, Jennifer D.; Quaas, Alexander M.; Craig, LaTasha B.; Soules, Michael R.; Klein, Nancy A.; Hansen, Karl R.

    2016-01-01

    STUDY QUESTION Are lifestyle factors (smoking, BMI, alcohol use and oral contraceptive pill use) associated with the human ovarian reserve as determined by the total ovarian non-growing follicle number? SUMMARY ANSWER Light to moderate alcohol use was significantly associated with greater ovarian non-growing follicle (NGF) count, whereas other lifestyle factors were not significantly related. WHAT IS KNOWN ALREADY A single previous investigation has suggested that smoking and alcohol use are associated with lower ovarian follicle density. However, this investigation utilized follicle density as the outcome of interest rather than the estimated total ovarian NGF count. STUDY DESIGN, SIZE, DURATION This cross-sectional investigation included a convenience sample of premenopausal women from two different academic sites, the University of Washington (n = 37, from 1999–2004) and the University of Oklahoma (n = 73, from 2004–2013), undergoing incidental oophorectomy at the time of hysterectomy (total n = 110, age range 21–52 years). PARTICIPANTS/MATERIALS, SETTING, METHODS Prior to undergoing oophorectomy, participants completed detailed questionnaires regarding lifestyle exposures. Following surgery, total ovarian NGF counts were determined with systematic random sampling rules and a validated fractionator/optical dissector technique. Associations between lifestyle factors and log-transformed ovarian follicle counts were determined using multivariable linear regression. MAIN RESULTS AND THE ROLE OF CHANCE After controlling for age, BMI, oral contraceptive pill (OCP) use, tobacco use and site of collection, cumulative alcohol use (measured in alcoholic drinks per day multiplied by years of drinking) was associated with ovarian NGF count. Women reporting light (>0 to <1 drink-years) and moderate (1–3 drink-years) alcohol use had greater NGF counts (β = 0.75, P = 0.04, and β = 1.00, P = 0.03; light and moderate use, respectively) as compared with non

  3. 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.

  4. Comparison of vitrification and conventional slow freezing for cryopreservation of ovarian tissue with respect to the number of intact primordial follicles: A meta-analysis.

    PubMed

    Zhou, Xin-Hui; Zhang, Dan; Shi, Jin; Wu, Yi-Jun

    2016-09-01

    Vitrification is the standard method for cryopreserving human oocytes and embryos, but its effects on ovarian tissue are uncertain. The purpose of this meta-analysis was to compare the proportion of intact primordial follicles in ovarian tissue cryopreserved with vitrification versus slow freezing. Medline, Cochrane, EMBASE, and Google Scholar databases were searched until November 11, 2014 using combinations of the search terms: ovarian tissue, cryopreservation, vitrification, follicle, follicles. Inclusion criteria were randomized controlled trails, two-arm prospective studies, and retrospective studies in which ovarian tissues were preserved by vitrification or conventional slow freezing. The primary outcome was the proportion of intact primordial follicles. Six studies were included in the meta-analysis. The number of patients ranged from 3 to 20, and age ranged from 20 to 43 years. Total number of morphologically intact follicles ranged from 14 to 2058, among which 6 to 724 were primordial. The pooled odds ratio (OR) showed no significant difference in the proportion of intact primordial follicles after slow freezing or vitrification (OR = 1.228, 95% confidence interval [CI]: 0.769-1.961, P = 0.390). Sensitivity analysis using the leave-one-out approach indicated no considerable changes in the direction and magnitude of the pooled estimates when individual studies were excluded one at a time, indicating good reliability of the current analysis. Vitrification and slow freezing produce equivalent results with respect to intact primordial follicles for the cryopreservation of human ovarian tissue. However, the included studies varied in the cryopreservation protocols used.

  5. From primordial germ cells to primordial follicles: a review and visual representation of early ovarian development in mice.

    PubMed

    Wear, Hannah M; McPike, Matthew J; Watanabe, Karen H

    2016-06-21

    Normal development of reproductive organs is crucial for successful reproduction. In mice the early ovarian developmental process occurs during the embryonic and postnatal period and is regulated through a series of molecular signaling events. Early ovarian development in mice is a seventeen-day process that begins with the rise of six primordial germ cells on embryonic day five (E5) and ends with the formation of primordial follicles on postnatal day two (P2). We reviewed the current literature and created a visual representation of early ovarian development that depicts the important molecular events and associated phenotypic outcomes based on primary data. The visual representation shows the timeline of key signaling interactions and regulation of protein expression in different cells involved in ovarian development. The major developmental events were divided into five phases: 1) origin of germ cells and maintenance of pluripotency; 2) primordial germ cell migration; 3) sex differentiation; 4) formation of germ cell nests; and 5) germ cell nest breakdown and primordial follicle formation. This review and visual representation provide a summary of the current scientific understanding of the key regulation and signaling during ovarian development and highlights areas needing further study. The visual representation can be used as an educational resource to link molecular events with phenotypic outcomes; serves as a tool to generate new hypotheses and predictions of adverse reproductive outcomes due to perturbations at the molecular and cellular levels; and provides a comprehendible foundation for computational model development and hypothesis testing.

  6. Dual modality imaging of a novel rat model of ovarian carcinogenesis

    NASA Astrophysics Data System (ADS)

    Kanter, Elizabeth; Walker, Ross; Marion, Sam; Brewer, Molly A.; Hoyer, Patricia B.; Barton, Jennifer K.

    2006-07-01

    Ovarian cancer is the fifth leading cause of cancer death in women, in part because of the limited knowledge about early stage disease. We develop a novel rat model of ovarian cancer and perform a pilot study to examine the harvested ovaries with complementary optical imaging modalities. Rats are exposed to repeated daily dosing (20 days) with 4-vinylcyclohexene diepoxide (VCD) to cause early ovarian failure (model for postmenopause), and ovaries are directly exposed to 7,12-dimethylbenz(a)anthracene (DMBA) to cause abnormal ovarian proliferation and neoplasia. Harvested ovaries are examined with optical coherence tomography (OCT) and light-induced fluorescence (LIF) at one, three, and five months post-DMBA treatment. VCD causes complete ovarian follicle depletion within 8 months after onset of dosing. DMBA induces abnormal size, cysts, and neoplastic changes. OCT successfully visualizes normal and abnormal structures (e.g., cysts, bursa, follicular remnant degeneration) and the LIF spectra show statistically significant changes in the ratio of average emission intensity at 390:450 nm between VCD-treated ovaries and both normal cycling and neoplastic DMBA-treated ovaries. Overall, this pilot study demonstrates the feasibility of both the novel animal model for ovarian cancer and the ability of optical imaging techniques to visualize ovarian function and health.

  7. 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.

  8. Effect of activin A and insulin-like growth factor-I on in vitro development of preantral follicles isolated from cryopreserved ovarian tissues in the mouse.

    PubMed

    Choi, Jongyeob; Lee, BoEun; Lee, EunYoung; Yoon, Byung-Koo; Choi, DooSeok

    2008-12-01

    Cryopreservation of ovarian tissue has been reported to delay the development of preantral follicles by temporary suppression of granulosa cell proliferation during in vitro culture. This delay might be overcome by treatment with activin A and/or IGF-I, known to stimulate granulosa cell proliferation. However, the effects of these growth factors, on delayed follicle development induced by ovarian tissue cryopreservation, have not been evaluated. Therefore, we studied the effects of activin A and/or IGF-I on granulosa cell proliferation and follicle development in preantral follicles isolated from mouse cryopreserved ovarian tissues. The preantral follicles isolated from fresh ovarian tissues were cultured with control medium (CM) for 10 days. The preantral follicles isolated from cryopreserved ovarian tissues were cultured with CM and with CM+activin A (100 ng/ml), IGF-I (50 ng/ml) or activin A+IGF-I added for 10, 12 and 14 days. The follicles were stimulated with hCG at the end of culture. The granulosa cell proliferation was evaluated by measuring the PCNA expression and the follicle development assessed by comparing the follicle diameter and oocyte maturation. The expressed level of PCNA was significantly decreased in the cryopreserved preantral follicles cultured with CM, compared to the fresh group (p<0.05), but increased to the level of the fresh group by the addition of activin A, IGF-I or activin A and IGF-I. The maximum follicle diameter and oocyte maturation rate were obtained in the fresh group after 10 days of culture, while the diameter and oocyte maturation rate of cryopreserved preantral follicles reached similar levels after 14 days. Under conditions of CM with added activin A or activin A+IGF-I, both the diameter and oocyte maturation rate of the cryopreserved preantral follicles improved to the levels of the fresh group after 12 days. However, the stimulatory effect was not different in comparisons between activin A and activin A+IGF-I. In

  9. Obesity induced by cafeteria diet disrupts fertility in the rat by affecting multiple ovarian targets.

    PubMed

    Bazzano, M V; Torelli, C; Pustovrh, M C; Paz, D A; Elia, E M

    2015-11-01

    Obesity constitutes a health problem of increasing worldwide prevalence. Among the health detriments caused by obesity, reproduction is disrupted. However, the mechanisms involved in this disruption are not fully understood. Animals fed a cafeteria diet constitute the model for the study of obesity that most closely reflects Western diet habits. The aims of this study were to evaluate whether a cafeteria diet affects ovarian function and to contribute to the understanding of the mechanisms involved. For that purpose, 22-day-old female Wistar rats were fed ad libitum with a standard diet (control group; n = 20) or cafeteria diet (CAF group; n = 20). The cafeteria diet induced obesity and hyperglycaemia, without altering serum triglycerides, cholesterol or C-reactive protein concentrations. This diet also altered ovarian function: the rats showed prolonged dioestrous phases, decreased serum oestradiol concentrations and increased number of antral atretic follicles. Moreover, follicular cysts were detected in the CAF group, concomitantly with a decrease in the number of anti-Müllerian hormone immunoreactive pre-antral follicles and COX-2-positive antral and pre-ovulatory follicles. The authors conclude that a cafeteria diet reduces ovarian reserve, induces the presence of follicular cysts and disturbs the ovulatory process, leading to the delayed pregnancy observed in these animals. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  10. Transcriptome profiling of granulosa cells of bovine ovarian follicles during growth from small to large antral sizes

    PubMed Central

    2014-01-01

    Background At later stages of folliculogenesis, the mammalian ovarian follicle contains layers of epithelial granulosa cells surrounding an antral cavity. During follicle development granulosa cells replicate, secrete hormones and support the growth of the oocyte. In cattle, the follicle needs to grow > 10 mm in diameter to allow an oocyte to ovulate, following which the granulosa cells cease dividing and differentiate into the specialised cells of the corpus luteum. To better understand the molecular basis of follicular growth and granulosa cell maturation, we undertook transcriptome profiling of granulosa cells from small (< 5 mm; n = 10) and large (> 10 mm, n = 4) healthy bovine follicles using Affymetrix microarrays (24,128 probe sets). Results Principal component analysis for the first two components and hierarchical clustering showed clustering into two groups, small and large, with the former being more heterogeneous. Size-frequency distributions of the coefficient of variation of the signal intensities of each probe set also revealed that small follicles were more heterogeneous than the large. IPA and GO enrichment analyses revealed that processes of axonal guidance, immune signalling and cell rearrangement were most affected in large follicles. The most important networks were associated with: (A) Notch, SLIT/ROBO and PI3K signalling, and (B) ITGB5 and extracellular matrix signalling through extracellular signal related kinases (ERKs). Upstream regulator genes which were predicted to be active in large follicles included STAT and XBP1. By comparison, developmental processes such as those stimulated by KIT, IHH and MEST were most active in small follicles. MGEA5 was identified as an upstream regulator in small follicles. It encodes an enzyme that modifies the activity of many target proteins, including those involved in energy sensing, by removal of N-acetylglucosamine from serine and threonine residues. Conclusions Our data suggest that as

  11. Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation.

    PubMed

    Smitz, J; Dolmans, M M; Donnez, J; Fortune, J E; Hovatta, O; Jewgenow, K; Picton, H M; Plancha, C; Shea, L D; Stouffer, R L; Telfer, E E; Woodruff, T K; Zelinski, M B

    2010-01-01

    BACKGROUND Female cancer patients are offered 'banking' of gametes before starting fertility-threatening cancer therapy. Transplants of fresh and frozen ovarian tissue between healthy fertile and infertile women have demonstrated the utility of the tissue banked for restoration of endocrine and fertility function. Additional methods, like follicle culture and isolated follicle transplantation, are in development. METHODS Specialist reproductive medicine scientists and clinicians with complementary expertise in ovarian tissue culture and transplantation presented relevant published literature in their field of expertise and also unpublished promising data for discussion. As the major aims were to identify the current gaps prohibiting advancement, to share technical experience and to orient new research, contributors were allowed to provide their opinioned expert views on future research. RESULTS Normal healthy children have been born in cancer survivors after orthotopic transplantation of their cryopreserved ovarian tissue. Longevity of the graft might be optimized by using new vitrification techniques and by promoting rapid revascularization of the graft. For the in vitro culture of follicles, a successive battery of culture methods including the use of defined media, growth factors and three-dimensional extracellular matrix support might overcome growth arrest of the follicles. Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation. Large animals, including non-human primates, are essential working models. CONCLUSIONS Experiments on ovarian tissue from non-human primate models and from consenting fertile and infertile patients benefit from a multidisciplinary approach. The new discipline of oncofertility requires professionalization, multidisciplinarity and mobilization of funding for basic and translational research.

  12. Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation

    PubMed Central

    Smitz, J.; Dolmans, M.M.; Donnez, J.; Fortune, J.E.; Hovatta, O.; Jewgenow, K.; Picton, H.M.; Plancha, C.; Shea, L.D.; Stouffer, R.L.; Telfer, E.E.; Woodruff, T.K.; Zelinski, M.B.

    2010-01-01

    BACKGROUND Female cancer patients are offered ‘banking’ of gametes before starting fertility-threatening cancer therapy. Transplants of fresh and frozen ovarian tissue between healthy fertile and infertile women have demonstrated the utility of the tissue banked for restoration of endocrine and fertility function. Additional methods, like follicle culture and isolated follicle transplantation, are in development. METHODS Specialist reproductive medicine scientists and clinicians with complementary expertise in ovarian tissue culture and transplantation presented relevant published literature in their field of expertise and also unpublished promising data for discussion. As the major aims were to identify the current gaps prohibiting advancement, to share technical experience and to orient new research, contributors were allowed to provide their opinioned expert views on future research. RESULTS Normal healthy children have been born in cancer survivors after orthotopic transplantation of their cryopreserved ovarian tissue. Longevity of the graft might be optimized by using new vitrification techniques and by promoting rapid revascularization of the graft. For the in vitro culture of follicles, a successive battery of culture methods including the use of defined media, growth factors and three-dimensional extracellular matrix support might overcome growth arrest of the follicles. Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation. Large animals, including non-human primates, are essential working models. CONCLUSIONS Experiments on ovarian tissue from non-human primate models and from consenting fertile and infertile patients benefit from a multidisciplinary approach. The new discipline of oncofertility requires professionalization, multidisciplinarity and mobilization of funding for basic and translational research. PMID:20124287

  13. Functional heterologous gap junctions in Fundulus ovarian follicles maintain meiotic arrest and permit hydration during oocyte maturation.

    PubMed

    Cerdá, J L; Petrino, T R; Wallace, R A

    1993-11-01

    The physiological significance of heterologous gap junctions between granulosa cells and the oocyte was investigated in late vitellogenic ovarian follicles of the teleost Fundulus heteroclitus. Lucifer Yellow injected into the oocyte readily passed to the overlying granulosa cells, demonstrating effective dye-coupling. Passage of the fluorescent dye, and hence intercellular communication, was inhibited both by the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and by 1-octanol, known uncouplers of gap junctions in a variety of invertebrate and vertebrate cell types. Octanol alone also initiated resumption of meiosis in follicle-enclosed oocytes, indicating that granulosa cells normally maintain meiotic arrest, as apparently occurs in mammalian and amphibian follicles. Both PMA and octanol also consistently inhibited the hydration process that normally accompanies meiotic maturation. These results support a previously suggested hypothesis that K+, which is the primary osmotic effector for oocyte hydration, is translocated via gap junction from granulosa cells to the maturing oocyte.

  14. 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.

  15. Morphological and functional preservation of pre-antral follicles after vitrification of macaque ovarian tissue in a closed system

    PubMed Central

    Ting, A. Y.; Yeoman, R. R.; Campos, J. R.; Lawson, M. S.; Mullen, S. F.; Fahy, G. M.; Zelinski, M. B.

    2013-01-01

    STUDY QUESTION What are the appropriate conditions to vitrify the macaque ovarian cortex in a large-volume, closed system that will preserve functional pre-antral follicles? SUMMARY ANSWER The combination of glycerol, ethylene glycol (EG) and polymers with cooling in liquid nitrogen (LN2) vapor and a two-step warming procedure was able to preserve tissue and follicle morphology as well as function of a small population of secondary follicles in the macaque ovarian cortex following vitrification in a closed system. WHAT IS KNOWN ALREADY For prepubertal cancer patients or those who require immediate cancer therapy, ovarian tissue cryopreservation offers the only hope for future fertility. However, the efficacy of live birth from the transplantation of cryopreserved ovarian tissue is still unclear. In addition, live birth from cryopreserved ovarian tissue has only been demonstrated after tissue autotransplantation, which poses the risk of transmitting metastatic cancer cells back to the cancer survivor in certain cancers. STUDY DESIGN, SIZE, DURATION Non-human primate model, n = 4, randomized, control versus treatment. End-points were collected from tissue histology, tissue culture (48 h) and isolated secondary follicle culture (6 weeks). PARTICIPANTS/MATERIALS, SETTING, METHODS Two vitrification solutions (VSs) containing EG + glycerol (VEG) and EG + dimethylsulfoxide (VED) were examined for vitrification, devitrification and thermodynamic properties. Once the optimal VS was determined, macaque ovarian cortical pieces (3 × 3 × 0.5 mm3) were divided into fresh and two vitrified groups (VEG and VED). For the vitrification groups, tissues were exposed to 1/4, 1/2 and 1× VS for 5 min/step as well as 1× VS + polymers for 1 min at 37°C, loaded into high-security straws with 1 ml of VS + polymers, heat sealed and cooled in LN2 vapor. Samples were warmed in a 40°C water bath and cryoprotective agents were diluted with 1, 0.5, 0.25 and 0 M sucrose. Tissues were fixed

  16. Growth hormone suppression of apoptosis in preovulatory rat follicles and partial neutralization by insulin-like growth factor binding protein.

    PubMed

    Eisenhauer, K M; Chun, S Y; Billig, H; Hsueh, A J

    1995-07-01

    A growing body of evidence suggests that growth hormone (GH) plays a role in regulating ovarian function by augmenting gonadotropin stimulation of granulosa cell differentiation and folliculogenesis. The majority of follicles in the mammalian ovary do not ovulate, but instead undergo a degenerative process (atresia) involving apoptotic cell death. The objective of the present study was to investigate the role of GH in regulating follicle apoptosis and to determine whether or not insulin-like growth factor-I (IGF-I) mediates GH action in this process. Preovulatory follicles obtained from eCG-primed rats were cultured for 24 h in serum-free conditions with or without hormone treatments. After culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3' ends with [32P]dideoxy-ATP. Culture of preovulatory follicles resulted in a spontaneous onset of apoptotic DNA fragmentation that was suppressed by ovine GH (oGH) in a dose-dependent manner, reaching a maximum of 65% suppression. To rule out the effect of residual gonadotropin in the oGH preparation, follicles were also cultured with recombinant bovine growth hormone (rbGH). Like oGH, rbGH suppressed apoptotic DNA fragmentation. Our earlier study indicated that hCG and FSH treatment also suppress apoptosis in the present model system, but no additive effect of GH and either hCG or FSH on the suppression of apoptosis was observed. To determine whether the observed effect of GH action on follicle apoptosis is mediated by IGF-I, three types of studies were carried out.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Evaluation of Antral Follicle Count and Ovarian Morphology in Crossbred Beef Cows: Investigation of Influence of Stage of the Estrous Cycle, Age, and Birth Weight

    USDA-ARS?s Scientific Manuscript database

    Depletion of the ovarian reserve is associated with reproductive senescence in mammalian females, and there is a positive relationship between the size of the ovarian reserve and the number of antral follicles on the surface of the ovary. Therefore, we conducted a series of experiments to investiga...

  18. The angiogenic properties of the rat vibrissa hair follicle associate with the bulb.

    PubMed

    Stenn, K S; Fernandez, L A; Tirrell, S J

    1988-03-01

    As the hair follicle is one of the most rapidly growing tissues in the body, it must be nourished by a rich blood supply. Histological studies have indicated that the number of vessels about a growing follicle exceeds that about a resting follicle, so we postulated that the hair follicle might provide its own angiogenic stimulus during certain phases of its growth. Reported here are experiments testing the angiogenic properties of the growing (anagen) hair follicle. Using the rabbit corneal pocket angiogenesis assay and cycled anagen rat vibrissae hair follicles, we found that the mesenchymal dermal papilla had no angiogenic properties, but the anagen bulb was angiogenic. These findings suggest a mechanism for the cycling of hair follicles and an example of normal epithelium to mesenchyme interactions.

  19. Follicle-stimulating hormone polypeptide modified nanoparticle drug delivery system in the treatment of lymphatic metastasis during ovarian carcinoma therapy.

    PubMed

    Fan, Lingling; Chen, Jun; Zhang, Xiaoyan; Liu, Yingtao; Xu, Congjian

    2014-10-01

    Traditional chemotherapy drugs have an obvious drawback of nonspecific biodistribution in treating ovarian cancer. Follicle-stimulating hormone receptor (FSHR), a G-protein coupled receptor which is mainly expressed in reproductive system, is an important drug target in developing novel therapeutics. Using a polypeptide of follicle-stimulating hormone (named as FSHP), a conjugated nanoparticle, FSHP-NP was developed to target FSHR in lymphatic metastasis of ovarian cancer. FSHP-NP was tested for recognition specificity and uptake efficiency on FSHR-expressing cells. A paclitaxel (PTX)-loaded FSHP-NP (FSHP-NP-PTX) was further developed and its anti-tumor effect was determined in vivo and in vitro. Taking NuTu-19 cells as an example, FSHP-NP-PTX displayed significantly stronger anti-cell proliferative and anti-tumor effects in a dose- and time-dependent manner when compared with free PTX or naked PTX-loaded nanoparticles (NP-PTX) in vitro. In vivo examinations showed that the size and weight of the lymph nodes were reduced in the FSHP-NP-PTX group. FSHR as a novel therapeutic target in ovarian cancer and delivery of PTX via conjugated nanoparticle (FSHP-NP) might represent a new therapeutic approach in ovarian cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. 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.

  1. Effect of leptin on in vitro development of ovine preantral ovarian follicles.

    PubMed

    Kamalamma, P; Kona, S S R; Praveen Chakravarthi, V; Siva Kumar, A V N; Punyakumari, B; Rao, V H

    2016-01-15

    The influence of human or ovine leptin on in vitro culture of preantral follicles (PFs) isolated from sheep ovaries was investigated. Among the 12 different concentrations (0-1000 ng/mL) of human leptin tested, proportion of PFs exhibiting growth, mean increase in diameter, antrum formation, and maturation of the oocytes to MII stage were the best in 10 ng/mL. Culture of sheep ovarian PFs in TCM 199 supplemented with 10 ng/mL of human or ovine leptin FSH (2.5 μg/mL), thyroxine (1 μg/mL), insulinlike growth factor I (10 ng/mL), and GH (1 mIU/mL) resulted in significantly (P ≤ 0.05) greater average increase in diameter (11 and 9 vs. 6 μm), better proportions of PFs exhibiting growth (66% and 58% vs. 48%), antrum formation (51% and 51% vs. 34%), and maturation of oocytes to MII stage (24% and 22% vs. 7%) than the control medium. It is concluded that (1) the optimum dose of leptin for the growth of sheep PFs in vitro was 10 ng/mL, (2) human or ovine leptin supported similar development in vitro of PFs in sheep, (3) inclusion of leptin along with FSH, thyroxine, insulinlike growth factor I, and GH resulted in only a marginal further improvements in in vitro development of sheep PFs'.

  2. Cryopreservation of preantral ovarian follicles in situ from domestic cats (Felis catus) using different cryoprotective agents.

    PubMed

    Lima, Ana Kelen F; Silva, Alexandre R; Santos, Regiane R; Sales, Daniele M; Evangelista, Andreia F; Figueiredo, José R; Silva, Lúcia D M

    2006-10-01

    The objective of this investigation was to verify the structural characteristics of preantral follicles (PAF) of cat ovarian tissue after cryopreservation in 1.5 M glycerol or ethylene glycol, using a slow-freezing procedure. Ovaries (n = 10) from domestic cats were divided into fragments. One fragment was immediately preserved for classical histology (fresh control), and additional fragments were immersed in minimum essential medium plus 10% bovine fetal serum (MEM+BFS), or MEM+BFS supplemented with 1.5 M glycerol or ethylene glycol. The samples were frozen and plunged into liquid nitrogen. After 1 wk, the samples were thawed. A total of 600 PAF were evaluated. In the fresh control, there were 71.3% normal PAF. After thawing, the rates of normal PAF were 26.0, 39.3 and 58.0% for samples without cryoprotectant or with glycerol or ethylene glycol, respectively. We concluded that ethylene glycol was useful for the cryopreservation of feline PAF in situ.

  3. Acute 7,12-dimethylbenz[a]anthracene exposure causes differential concentration-dependent follicle depletion and gene expression in neonatal rat ovaries

    SciTech Connect

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

    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). After 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

  4. Molecular characterisation and expression profiling of the ENO1 gene in the ovarian follicle of the Sichuan white goose.

    PubMed

    Kang, Bo; Jiang, Dong Mei; Bai, Lin; He, Hui; Ma, Rong

    2014-01-01

    The ENO1 gene encodes a multifunctional enzyme that has been identified as a key component of the glycolytic pathway. Our previous studies demonstrated that ENO1 gene expression was higher in the ovaries of laying geese compared with prelaying geese. However, the molecular characterisation and expression profiling of the ENO1 gene in geese tissues and ovarian follicles remain to be determined. In this study, ENO1 cDNA (1,445 bp long) of the Sichuan white goose was cloned and characterised. The ORF of ENO1 cDNA is 1,305 bp in length and encodes a 434 amino acid protein with a molecular weight of 47.27 kDa. ENO1 expression in all of the examined tissues was the highest in spleen and the lowest in breast muscle. High expression of ENO1 appeared in the kidney, liver, adrenal gland, and retina. With increasing follicle growth, ENO1 gene expression began to decrease from the small white follicle to F5, which was followed by a sharp increase in expression in F4 and then a gradual decrease in expression from F3 to F1. Furthermore, in the postovulatory follicles (POF), the levels of ENO1 gene expression decreased gradually from POF1 to POF4. In conclusion, the ENO1 transcript was widely distributed in various tissues of the Sichuan white goose, but ENO1 expression was tissue-specific. Furthermore, the results of the ENO1 expression profiling of ovarian follicles suggest that ENO1 may play an important dual role in the progress of follicular development, where ENO1 acts as a glycolytic enzyme and also mediates apoptosis.

  5. Bone morphogenetic protein 1 is expressed in porcine ovarian follicles and promotes oocyte maturation and early embryonic development

    PubMed Central

    LEI, Xiaocan; CUI, Kuiqing; CAI, Xiaoyan; REN, Yanping; LIU, Qingyou; SHI, Deshun

    2016-01-01

    In the present study, we tried to determine whether bone morphogenetic protein 1 (BMP1) plays a role in ovarian follicular development and early embryo development. We systematically investigated the expression and influence of BMP1 during porcine follicle and early embryonic development. Immunohistochemistry demonstrated that the BMP1 protein is expressed in granular cells and oocytes during follicular development, from primary to pre-ovulatory follicles, including atretic follicles and the corpus luteum. The mRNA expression of BMP1 significantly increased as the porcine follicles grew. Immunofluorescence analysis indicated that BMP1 was expressed in cumulus-oocyte complexes (COCs), oocytes and porcine embryos during early in vitro culture. qPCR and western blot analysis showed that the expression of BMP1 was significantly up-regulated in mature porcine oocytes and COCs compared to immature oocytes and COCs. BMP1 is expressed in early porcine embryos, and its expression reaches a peak at the 8-cell stage. To determine the effect of BMP1 on the maturation of oocytes and the development of early embryos, various concentrations of BMP1 recombinant protein or antibody were added to the in vitro culture media, respectively. BMP1 significantly affected the porcine oocyte maturation rate, the cleavage rate and the blastocyst development rate of embryos cultured in vitro in a positive way, as well as the blastocyst cell number. In conclusion, BMP1 is expressed throughout porcine ovarian follicle development and early embryogenesis, and it promotes oocyte maturation and the developmental ability of embryos during early in vitro culture. PMID:27890905

  6. Embryonic stem cell-derived granulosa cells participate in ovarian follicle formation in vitro and in vivo.

    PubMed

    Woods, Dori C; White, Yvonne A R; Niikura, Yuichi; Kiatpongsan, Sorapop; Lee, Ho-Joon; Tilly, Jonathan L

    2013-05-01

    Differentiating embryonic stem cells (ESCs) can form ovarian follicle-like structures in vitro, consisting of an oocyte-like cell surrounded by somatic cells capable of steroidogenesis. Using a dual-fluorescence reporter system in which mouse ESCs express green fluorescent protein (GFP) under the control of a germ cell-specific Pou5f1 gene promoter and red fluorescent protein (Discosoma sp red [DsRed]) driven by the granulosa cell-specific Forkhead box L2 (Foxl2) gene promoter, we first confirmed in vitro formation of follicle-like structures containing GFP-positive cells surrounded by DsRed-positive cells. Isolated DsRed-positive cells specified from ECSs exhibited a gene expression profile consistent with granulosa cells, as revealed by the detection of messenger RNAs (mRNAs) for Foxl2, follistatin (Fst), anti-Müllerian hormone (Amh), and follicle-stimulating hormone receptor (Fshr) as well as by production of both progesterone and estradiol. In addition, treatment of isolated DsRed-expressing cells with follicle-stimulating hormone (FSH) significantly increased estradiol production over basal levels, confirming the presence of functional FSH receptors in these cells. Last, ESC-derived DsRed-positive cells injected into neonatal mouse ovaries became incorporated within the granulosa cell layer of immature follicles. These studies demonstrate that Foxl2-expressing ovarian somatic cells derived in vitro from differentiating ESCs express granulosa cell markers, actively associate with germ cells in vitro, synthesize steroids, respond to FSH, and participate in folliculogenesis in vivo.

  7. Embryonic Stem Cell–Derived Granulosa Cells Participate in Ovarian Follicle Formation In Vitro and In Vivo

    PubMed Central

    Woods, Dori C.; White, Yvonne A. R.; Niikura, Yuichi; Kiatpongsan, Sorapop; Lee, Ho-Joon

    2013-01-01

    Differentiating embryonic stem cells (ESCs) can form ovarian follicle-like structures in vitro, consisting of an oocyte-like cell surrounded by somatic cells capable of steroidogenesis. Using a dual-fluorescence reporter system in which mouse ESCs express green fluorescent protein (GFP) under the control of a germ cell–specific Pou5f1 gene promoter and red fluorescent protein (Discosoma sp red [DsRed]) driven by the granulosa cell–specific Forkhead box L2 (Foxl2) gene promoter, we first confirmed in vitro formation of follicle-like structures containing GFP-positive cells surrounded by DsRed-positive cells. Isolated DsRed-positive cells specified from ECSs exhibited a gene expression profile consistent with granulosa cells, as revealed by the detection of messenger RNAs (mRNAs) for Foxl2, follistatin (Fst), anti-Müllerian hormone (Amh), and follicle-stimulating hormone receptor (Fshr) as well as by production of both progesterone and estradiol. In addition, treatment of isolated DsRed-expressing cells with follicle-stimulating hormone (FSH) significantly increased estradiol production over basal levels, confirming the presence of functional FSH receptors in these cells. Last, ESC-derived DsRed-positive cells injected into neonatal mouse ovaries became incorporated within the granulosa cell layer of immature follicles. These studies demonstrate that Foxl2-expressing ovarian somatic cells derived in vitro from differentiating ESCs express granulosa cell markers, actively associate with germ cells in vitro, synthesize steroids, respond to FSH, and participate in folliculogenesis in vivo. PMID:23536570

  8. Effect of mancozeb on ovarian compensatory hypertrophy and biochemical constituents in hemicastrated albino rat.

    PubMed

    Mahadevaswami, M P; Jadaramkunti, U C; Hiremath, M B; Kaliwal, B B

    2000-01-01

    Mancozeb a carbamate fungicide was administered orally at doses of 500, 600, 700, and 800 mg/kg/day to normal hemicastrated virgin rats for 15 consecutive days to examine the effect on ovarian hypertrophy. Sham-operated and hemicastrated control rats were administered a similar quantity of olive oil. The vaginal smear and body weight of the rats were recorded daily and rats were sacrificed on the Day 16. The ovary, uterus, kidney, adrenal, spleen, liver, lungs, heart, thymus, and thyroid were removed and weighed. The left ovary from each animal was serially sectioned and stained for histologic studies. The hemicastrated control rats revealed a significant increase in relative ovarian weight with 66.3% hypertrophy. Treatment with 700 and 800 mg/kg/day mancozeb revealed a decrease in ovarian hypertrophy with 28.2 and 22.8% hypertrophy, respectively. There was no significant change in the number of estrous cycles and duration of each phase of the estrous cycle with 500 mg/kg/day mancozeb. However, there was a decrease in the number of estrous cycles, duration of proestrus, estrus, and metestrus with concomitant significant increase in the duration of the diestrus phase with 600, 700, and 800 mg/kg/day mancozeb treatment. There was a significant decrease in the number of healthy follicles with concomitant increase in the number of atretic follicles at higher doses of mancozeb. There were no significant changes in the body and organ weight with 500, 600, 700, and 800 mg/kg/day of mancozeb. The levels of protein, glycogen, total lipid, phospholipid, and neutral lipid were elevated in the liver, uterus, and ovary after hemicastration. Protein, glycogen, total lipid, phospholipid, and neutral lipid were not significantly changed in the liver, uterus, and ovary after 500 mg/kg/day mancozeb. However, treatment with 600, 700, and 800 mg/kg/day mancozeb showed a significant decrease in the levels of protein, glycogen, total lipid, phospholipid, and neutral lipid in the liver

  9. Effectiveness of a recombinant human follicle stimulating hormone on the ovarian follicles, peripheral progesterone, estradiol-17β, and pregnancy rate of dairy cows

    PubMed Central

    Ali, Mohamed; Moustafa M., Zeitoun

    2016-01-01

    Aims: This study aimed at elucidating the effects of recombinant human follicle stimulating hormone (r-hFSH) on the ovarian follicular dynamics, progesterone, estradiol-17β profiles, and pregnancy of dairy cows. Materials and Methods: Three groups (G, n=5 cows) of multiparous dairy cows were used. G1 (C) control cows were given controlled internal drug release (CIDR) and prostaglandin F2α; G2 (L) cows were given low dose (525 IU and G3 (H) cows were given high dose (1800 IU) of r-hFSH on twice daily basis at the last 3 days before CIDR removal. All cows were ultrasonically scanned for follicular growth and dynamics, and blood samples were collected every other day for two consecutive estrus cycles for the determination of estradiol-17β and progesterone. Results: Estrus was observed in all C and L but not in H cows. Dominant follicle was bigger in L compared to C and H cows. Dominant follicle in C (16.00±2.5 mm) and L cows (17.40±2.3 mm) disappeared at 72 h after CIDR removal. However, in H cows, no ovulation has occurred during 7 days post-CIDR removal. Progesterone was not different (p>0.10) among groups, whereas estradiol-17β revealed significant (p<0.01) reduction in H (15.96±2.5 pg/ml) cows compared to C (112.26±26.1 pg/ml) and L (97.49±15.9 pg/ml) cows. Pregnancy rate was higher in L cows (60%) compared with C cows (20%). However, H cows were not artificially inseminated due to non-ovulation. Only a cow of C group has calved one calf, however, 2 of the L cows gave birth of twins and a cow gave single calf. Conclusion: Administration of a low dose (525 IU) of r-hFSH resulted in an optimal size of dominant follicle, normal values of progesterone and estradiol-17β, and 40% twinning rate, howeverusing 1800 IU of r-hFSH, have adverse effects on ovarian follicular dynamics and hormonal profiles with non-pregnancy of dairy cows raised under hot climate. PMID:27536029

  10. The significance of antral follicle size prior to stimulation in predicting ovarian response in a multiple dose GnRH antagonist protocol.

    PubMed

    Lai, Qiaohong; Chen, Cai; Zhang, Zhijun; Zhang, Shu; Yu, Qilin; Yang, Ping; Hu, Jun; Wang, Cong-Yi

    2013-01-01

    Prediction of ovarian responses prior to stimulation is not only useful for patient counseling, but also important in tailoring the optimal dosage of gonadotrophin for individual patients. By prospectively study of 214 women undergoing in vitro fertilization and embryo transfer (IVF-ET) treatment, we obtained data supporting that antral follicle size could be an additional valuable predictive marker other than the antral follicle count (AFC) in predicting ovarian response. Our studies revealed that AFC achieved the best predictive value in relation to the number of oocyte obtained, followed by antral follicle size, basal follicle stimulating hormone (FSH) and body mass index (BMI). Unlike AFC, antral follicle size was noted to be negatively correlated with the dosage (R = -0.493) and duration (R = -0.465) of rFSH stimulation. Antral follicle size was also found with higher negative regression coefficient (B = -0.661) as compared with that of basal FSH concentration (B = -0.326) and BMI (b = -0.281). More importantly, women with antral follicle size 6-7 mm showed significantly higher AFC, oocytes retrieved, fertilized oocytes and grade I/II embryos along with much lower transfer cycle cancellation rate (7.5% vs. 16-17%). Together, our data suggest that basal antral follicle size could be a valued predictive marker in women with IVF-ET treatment, in which women with antral follicle size 6-7 mm are likely predisposed to better IVF-ET outcomes.

  11. 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

  12. 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

  13. Preoviposition activation of cathepsin-like proteinases in degenerating ovarian follicles of the mosquito Culex pipiens pallens.

    PubMed

    Uchida, K; Ohmori, D; Ueno, T; Nishizuka, M; Eshita, Y; Fukunaga, A; Kominami, E

    2001-09-01

    Within developing ovaries of many insects, some developing follicles or oocytes usually degenerate (follicular atresia or oosorption), while the others may continue to grow to maturity, thus maintaining the balance between the number of eggs and reproductive circumstances such as available nutrients. To help clarify the phenomenon of follicular atresia during ovarian development, we examined cysteine proteinases stored in mosquito Culex pipiens pallens ovaries. First, analysis using synthesized substrates showed that cathepsin B- and L-like proteinases gradually accumulated in the developing ovaries after a blood meal, which required more than 10 min of preincubation under acidic conditions to reach their maximum activities. However, homogenates of degenerating follicles 3 days after feeding showed proteolytic activities without acid treatment, suggesting that the proteinases had already been activated, while the extract of normally developing follicles collected from the same ovaries required more than 10 min of acid preincubation to reach the optimum activities, suggesting that the enzymes remained as inactive forms. Chemical and immunohistochemical analyses showed that more proteinases are located in the cytoplasm, rather than being associated with yolk granules. Ovarian proteinases, which are believed to become activated at the onset of embryogenesis, should also be activated during oogenesis, presumably to enhance oosorption.

  14. Zearalenone exposure impairs ovarian primordial follicle formation via down-regulation of Lhx8 expression in vitro.

    PubMed

    Zhang, Guo-Liang; Sun, Xiao-Feng; Feng, Yan-Zhong; Li, Bo; Li, Ya-Peng; Yang, Fan; Nyachoti, Charles Martin; Shen, Wei; Sun, Shi-Duo; Li, Lan

    2017-02-15

    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 30μM ZEA in vitro had significantly less germ cell numbers compared to the 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. Copyright © 2017. Published by Elsevier Inc.

  15. FSH and eCG impact follicles development and expression of ovarian FSHR and caspase-9 in mice.

    PubMed

    Wei, S; Gong, Z; Guo, H; Zhang, T; Ma, Z

    2017-01-01

    The study aimed to investigate the effects of FSH and eCG on the ovarian and follicular development, expression levels of FSHR and caspase-9 of ovaries in vivo. One hundred and five prepuberty mice were allocated into FSH-1, FSH-2, FSH-3, eCG-1, eCG-2, eCG-3 groups and control group (CG). Mice in FSH-1, FSH-2 and FSH-3 were intramuscularly injected with 5, 10 and 20 IU FSH twice (on day 0 and 4), respectively. Mice in eCG-1, eCG-2 and eCG-3 were intraperitoneally injected with 10, 20 and 40 IU eCG on day 0 and 4. Mice in the CG were injected with 0.5 ml normal saline on day 0 and 4. Left and right ovaries of each mouse were dissected aseptically on days 7, 14 and 21, respectively. The results showed that on days 14 and 21 the ovarian sizes and follicle numbers of FSH-3 and eCG-3 groups were greater than CG (P<0.05). FSHR mRNA of FSH-2 and eCG-1 were higher than CG on days 14 and 21 (P<0.05). FSHR proteins of FSH-3 were higher than CG on days 14 and 21 (P<0.05). Caspase-9 mRNA in FSH and eCG groups was less than CG. There were positive correlations between follicle numbers and FSH and eCG doses. FSHR protein expressions had positive correlations between ovarian weights and sizes of ovary and follicle numbers (r=0.971, P<0.05) in FSH-treated mice. Serum FSH concentrations of FSH-2, FSH-3, eCG-2 and eCG-3 groups were greater than that of CG. In conclusion, eCG and FSH promoted the ovarian development, follicle genesis, FSH secretion, FSHR mRNA and protein expressions in ovaries of mice. FSH and eCG inhibited the expression of ovarian caspase-9 mRNA.

  16. Human ovarian follicular development: from activation of resting follicles to preovulatory maturation.

    PubMed

    Gougeon, A

    2010-05-01

    By integrating morphometrical and endocrinological data, as well as biological effects of various molecules synthesized by the human follicle, we propose a dynamic view of the follicle growth within the human ovary. Folliculogenesis starts with entry of resting follicles into the growth phase, a process where the kit system plays a key role. Several months are required for a new growing follicle to reach the preantral stage (0.15mm), then 70 additional days to reach the size of 2mm. Early growing follicle growth is regulated by subtle interactions between follicle-stimulating hormone (FSH) and local factors produced by theca and granulosa cells (GCs), as well as the oocyte. From the time they enter the selectable stage during the late luteal phase, follicles become sensitive to cyclic changes of FSH in terms of granulosa cell proliferation. During the early follicular phase, the early selected follicle grows very quickly and estradiol is present in the follicular fluid. However, the total steroid production remains moderate. From the mid-follicular phase, the preovulatory follicle synthesizes high quantities of estradiol, then after the mid-cycle gonadotropin surge, very large amounts of progesterone. At this stage of development, the responsiveness of the follicle to gonadotropins is maximum, especially to luteinizing hormone (LH) that triggers granulosa wall dissociation and cumulus expansion as well as oocyte nuclear maturation. Thus, as the follicle develops, its responsiveness to gonadotropins progressively increases under the control of local factors acting in an autocrine/paracrine fashion.

  17. Perinatal exposure to mixtures of endocrine disrupting chemicals reduces female rat follicle reserves and accelerates reproductive aging.

    PubMed

    Johansson, Hanna Katarina Lilith; Jacobsen, Pernille Rosenskjold; Hass, Ulla; Svingen, Terje; Vinggaard, Anne Marie; Isling, Louise Krag; Axelstad, Marta; Christiansen, Sofie; Boberg, Julie

    2016-06-01

    Exposure to endocrine disrupting chemicals (EDCs) during development can have negative consequences later in life. In this study we investigated the effect of perinatal exposure to mixtures of human relevant EDCs on the female reproductive system. Rat dams were exposed to a mixture of phthalates, pesticides, UV-filters, bisphenol A, butylparaben, as well as paracetamol. The compounds were tested together (Totalmix) or in subgroups with anti-androgenic (AAmix) or estrogenic (Emix) potentials. Paracetamol was tested separately. In pre-pubertal rats, a significant reduction in primordial follicle numbers was seen in AAmix and PM groups, and reduced plasma levels of prolactin was seen in AAmix. In one-year-old animals, the incidence of irregular estrous cycles was higher after Totalmix-exposure and reduced ovary weights were seen in Totalmix, AAmix, and PM groups. These findings resemble premature ovarian insufficiency in humans, and raises concern regarding potential effects of mixtures of EDCs on female reproductive function.

  18. Ultrastructure of the basal lamina of bovine ovarian follicles and its relationship to the membrana granulosa.

    PubMed

    Irving-Rodgers, H F; Rodgers, R J

    2000-03-01

    Different morphological phenotypes of follicular basal lamina and of membrana granulosa have been observed. Ten preantral follicles (< 0. 1 mm), and 17 healthy and six atretic antral follicles (0.5-12 mm in diameter) were processed for light and electron microscopy to investigate the relationship the between follicular basal lamina and membrana granulosa. Within each antral follicle, the shape of the basal cells of the membrana granulosa was uniform, and either rounded or columnar. There were equal proportions of follicles follicles had only rounded basal cells. Conventional basal laminae of a single layer adjacent to the basal granulosa cells were observed in healthy follicles at the preantral and antral stages. However, at the preantral stage, the conventional types of basal lamina were enlarged or even partially laminated. A second type of basal lamina, described as 'loopy', occurred in about half the preantral follicles and in half the antral follicles follicles. 'Loopy' basal laminae were composed of basal laminae aligning the basal surface of basal granulosa cells, but with additional layers or loops often branching from the innermost layer. Each loop was usually < 1 microm long and had vesicles (20-30 nm) attached to the inner aspect. Basal cellular processes were also common, and vesicles could be seen budding off from these processes. In antral follicles, conventional basal laminae occurred in follicles with rounded basal granulosa cells. Other follicles with columnar cells, and atretic follicles, had the 'loopy' basal lamina phenotype. Thus, follicles have different basal laminae that relate to the morphology of the membrana granulosa.

  19. Interaction of relaxin-like gonad-stimulating substance with ovarian follicle cells of the starfish Asterina pectinifera.

    PubMed

    Mita, Masatoshi; Yamamoto, Kazutoshi; Nagahama, Yoshitaka

    2011-10-01

    Previously, gonad-stimulating substance (GSS), which acts as a gonadotropin, was purified from radial nerves of the starfish Asterina pectinifera and its structure was elucidated. Here, the interaction of GSS with receptors was examined in ovarian follicle cells, a target of GSS. In competitive experiments using radioiodinated and radioinert GSS, highly specific binding was observed in the microsomal/plasma membrane fraction of follicle cells. GSS scarcely bound in the cytosolic fraction. Scatchard plots showed the numbers of binding sites (NBS) in whole homogenate and the crude membrane to be 1.65 and 3.42 pmoles/mg protein, respectively. Dissociation constant (K (d)) values in these two preparations were almost the same at about 0.6-0.7 nM. Furthermore, it was shown that GSS stimulated adenylyl cyclase activity in follicle cell membranes in a dose-dependent manner that required GTP. Immunoblotting with specific antibodies for G-protein subunits after SDS-PAGE of the membrane preparation showed both stimulatory (Gs) and inhibitory (Gi) regulatory α-subunits for adenylyl cyclase and a β-subunit. The results strongly suggest that GSS interacts with G-protein-coupled receptors (GPCR) located in the follicle cell membrane to stimulate Gs-protein and adenylyl cyclase activity.

  20. Preservation of ovarian follicles reveals early evolution of avian reproductive behaviour.

    PubMed

    Zheng, Xiaoting; O'Connor, Jingmai; Huchzermeyer, Fritz; Wang, Xiaoli; Wang, Yan; Wang, Min; Zhou, Zhonghe

    2013-03-28

    The two groups of archosaurs, crocodilians and birds, form an extant phylogenetic bracket for understanding the reproductive behaviour of dinosaurs. This behaviour is inferred from preserved nests and eggs, and even gravid individuals. Data indicate that many 'avian' traits were already present in Paraves--the clade that includes birds and their close relatives--and that the early evolution of the modern avian form of reproduction was already well on its way. Like living neornithine birds, non-avian maniraptorans had daily oviposition and asymmetrical eggs with complex shell microstructure, and were known to protect their clutches. However, like crocodilians, non-avian maniraptorans had two active oviducts (one present in living birds), relatively smaller eggs, and may not have turned their eggs in the way that living birds do. Here we report on the first discovery of fossilized mature or nearly mature ovarian follicles, revealing a previously undocumented stage in dinosaur reproduction: reproductively active females near ovulation. Preserved in a specimen of the long bony-tailed Jeholornis and two enantiornithine birds from the Early Cretaceous period lacustrine Jehol Biota in northeastern China, these discoveries indicate that basal birds only had one functional ovary, but retained primitive morphologies as a result of their lower metabolic rate relative to living birds. They also indicate that basal birds reached sexual maturity before skeletal maturity, as in crocodiles and paravian dinosaurs. Differences in follicular morphology between Jeholornis and the enantiornithines are interpreted as forming an evolutionary gradient from the reproductive condition in paravian dinosaurs towards neornithine birds. Furthermore, differences between the two enantiornithines indicate that this lineage might also have evolved advanced reproductive traits in parallel to the neornithine lineage.

  1. Previtellogenic and vitellogenic oocytes in ovarian follicles of cultured siberian sturgeon Acipenser baerii (Chondrostei, Acipenseriformes).

    PubMed

    Żelazowska, Monika; Fopp-Bayat, Dorota

    2017-01-01

    Previtellogenic and vitellogenic oocytes in ovarian follicles from cultured Siberian sturgeon Acipenser baerii were examined. In previtellogenic oocytes, granular and homogeneous zones in the cytoplasm (the ooplasm) are distinguished. Material of nuclear origin, rough endoplasmic reticulum, Golgi complexes, complexes of mitochondria with cement and round bodies are numerous in the granular ooplasm. In vitellogenic oocytes, the ooplasm comprises three zones: perinuclear area, endoplasm and periplasm. The endoplasm contains yolk platelets, lipid droplets, and aggregations of mitochondria and granules immersed in amorphous material. In the nucleoplasm, lampbrush chromosomes, nucleoli, and two types of nuclear bodies are present. The first type of nuclear bodies is initially composed of fibrillar threads only. Their ultrastructure subsequently changes and they contain threads and medium electron dense material. The second type of nuclear bodies is only composed of electron dense particles. All nuclear bodies impregnate with silver, stain with propidium iodide, and are DAPI-negative. Their possible role is discussed. All oocytes are surrounded by follicular cells and a basal lamina which is covered by thecal cells. Egg envelopes are not present in previtellogenic oocytes. In vitellogenic oocytes, the plasma membrane (the oolemma) is covered by three envelopes: vitelline envelope, chorion, and extrachorion. Vitelline envelope comprises four sublayers: filamentous layer, trabecular layer 2 (t2), homogeneous layer, and trabecular layer 1 (t1). In the chorion, porous layer 1 and porous layer 2 are distinguished in most voluminous examined oocytes. Three micropylar cells that are necessary for the formation of micropyles are present between follicular cells at the animal hemisphere. J. Morphol. 278:50-61, 2017. ©© 2016 Wiley Periodicals,Inc.

  2. Decreased ovarian reserve in female Sprague-Dawley rats induced by isotretinoin (retinoic acid) exposure.

    PubMed

    Abali, Remzi; Yuksel, Mehmet Aytac; Aktas, Cevat; Celik, Cem; Guzel, Savas; Erfan, Gamze; Sahin, Onder

    2013-08-01

    Isotretinoin is a retinoid widely used for the treatment of severe nodulocystic acne. Although it has broad side effects, there is no well-designed study about its effects on the ovary. This study investigated possible toxic effects of isotretinoin on female gonads. A total of 30 female rats were randomly divided into three equal groups according to the dose of isotretinoin they were administered: 0 mg/kg/day (group 1), 7.5 mg/kg/day (group 2) or 15 mg/kg/day (group 3). Thirty days after the treatment, the effects of isotretinoin on the ovaries were evaluated with serum anti-Müllerian hormone (AMH) concentrations, apoptosis by TUNEL assay and immunohistochemical observations by proliferating cell nuclear antigen (PCNA). The percentage of atretic follicles was calculated for each stage of folliculogenesis. The serum AMH concentrations were found to be lower in both isotretinoin groups. The percentage of atretic follicles in both isotretinoin groups was higher than the control. The number of PCNA-positive granulosa cells was decreased in the isotretinoin groups. The number of ovarian follicles with apoptotic granulosa cells was increased in the experimental groups. These data are the first to identify that exposure of isotretinoin may be responsible for decreased ovarian reserve and toxic effects on rat ovaries.

  3. Antral follicle count as a marker of ovarian biological age to reflect the background risk of fetal aneuploidy.

    PubMed

    Grande, Maribel; Borobio, Virginia; Jimenez, Jose Miguel; Bennasar, Mar; Stergiotou, Iosifina; Peñarrubia, Joana; Borrell, Antoni

    2014-06-01

    Can antral follicle count (AFC) measured during pregnancy be used as a marker of ovarian age to assess the background risk of fetal aneuploidy? AFC was lower than expected according to maternal chronological age in trisomic pregnancies; therefore ovarian age could potentially reflect a more precise background risk of fetal aneuploidy screening. The decline in a woman's reproductive function is determined by a decline in the ovarian follicle pool and the quality of oocytes. The quantitative status of ovarian reserve can be indirectly assessed by AFC, but the role of AFC as an aneuploidy risk marker in pregnant women has not been assessed yet. Our study comprised a prospective cohort including 1239 singleton pregnancies scanned before 14 weeks in our center during a 14-month period. Reference ranges for AFC were constructed using 812 spontaneously conceived, chromosomally normal singleton ongoing pregnancies using the Lambda-Mu-Sigma method. The study population (n = 934) included 19 pregnancies with viable autosomal trisomies (trisomies 21, 18 and 13), 17 non-viable autosomal trisomies (other than 21, 18 or 13), 7 monosomies X, 1 sex trisomy and 3 triploidies (total n = 47 with chromosomal abnormalities). AFC in chromosomally abnormal pregnancies was plotted against the reference ranges. AFC multiple of the median was calculated according to the median AFC obtained by each year of age. Sixty-eight percent of women carrying a pregnancy with viable trisomies and 65% with non-viable trisomies presented an AFC below the 50th percentile. The median ovarian age in viable trisomies and non-viable trisomies was estimated to be 3 and 6 years above than median maternal age, respectively. However, the median ovarian age in monosomies X and triploidies was not higher than median maternal age. We did not assess the intra- and inter-observer reliability, or use specific three-dimensional analysis which may have advantages over our two-dimensional study. In clinical practice, a

  4. Ultrastructural observations of previtellogenic ovarian follicles of the caecilians Ichthyophis tricolor and Gegeneophis ramaswamii.

    PubMed

    Beyo, Reston S; Sreejith, Parameswaran; Divya, Lekha; Oommen, Oommen V; Akbarsha, Mohammad A

    2007-04-01

    The ultrastructural organization of the previtellogenic follicles of the caecilians Ichthyophis tricolor and Gegeneophis ramaswamii, of the Western Ghats of India, were observed. Both species follow a similar seasonal reproductive pattern. The ovaries contain primordial follicles throughout the year with previtellogenic, vitellogenic, or postvitellogenic follicles, depending upon the reproductive status. The just-recruited primordial follicle includes an oocyte surrounded by a single layer of follicle and thecal cells. The differentiation of the theca into externa and interna layers, the follicle cells into dark and light variants, and the appearance of primordial yolk platelets and mitochondrial clouds in the ooplasm mark the transition of the primordial follicle into a previtellogenic follicle. During further development of the previtellogenic follicle the following changes occur: i) the theca loses distinction as externa and interna; ii) all the follicle cells become the dark variant and increase in the complexity of ultrastructural organization; iii) the nucleus of the oocyte transforms into the germinal vesicle and there is amplification of the nucleoli; iv) the primordial yolk platelets of the cortical cytoplasm of the oocyte increase in abundance; v) the mitochondrial clouds fragment and the mitochondria move away from the clouds, leaving behind the cementing matrix, which contains membrane-bound vesicles of various sizes, either empty or filled with a lipid material; vi) the perivitelline space appears first as troughs at the junctional points between the follicle cells and oocyte, which subsequently spread all around to become continuous; vii) macrovilli and microvilli develop from the follicle cells and oocyte, respectively; and viii) the precursor material of the vitelline envelop arrives at the perivitelline space. The sequential changes in the previtellogenic follicles of two species of caecilians are described.

  5. Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles.

    PubMed

    Macchione, E; Epifano, O; Stefanini, M; Belin, D; Canipari, R

    2000-04-01

    Plasminogen activators (PAs) have been shown to be synthesized in ovarian follicles of several mammalian species, where they contribute to the ovulation process. The type of PA secreted by granulosa cells is species-specific. In fact, whereas in the rat, gonadotropins stimulate tissue-type PA (tPA) production, the same hormonal stimulation induces urokinase PA (uPA) secretion in mouse cells. To investigate in more detail the hormonal regulation of this system, we used the rat ovary as a model in which we analyzed the production of PAs by theca-interstitial (TI) and granulosa cells obtained from preovulatory follicles after gonadotropin stimulation. In untreated rats, uPA was the predominant enzyme in both TI and granulosa cells. After hormonal stimulation, an increase in uPA and tPA activity was observed in both cell types. Surprisingly, only tPA mRNA increased in a time-dependent manner in both cell types, while uPA mRNA increased only in TI cells and actually decreased in granulosa cells. These divergent results between uPA enzyme activity and mRNA levels in granulosa cells were explained by studying the localization of the enzyme. Analysis of granulosa cell lysates showed that after hormonal stimulation, 60-70% of the uPA behaved as a cell-associated protein, suggesting that uPA, already present in the follicle, accumulates on the granulosa cell surface through binding to specific uPA receptors. The redistribution of uPA in granulosa cells and the differing regulation of the two PAs by gonadotropins in the rat ovary suggest that the two enzymes might have different functions during the ovulation process. Moreover, the ability of antibodies anti-tPA and anti-uPA to significantly inhibit ovulation only when coinjected with hCG confirmed that the PA contribution to ovulation occurs at the initial steps.

  6. Plasma prorenin response to human chorionic gonadotropin in ovarian-hyperstimulated women: correlation with the number of ovarian follicles and steroid hormone concentrations.

    PubMed Central

    Itskovitz, J; Sealey, J E; Glorioso, N; Rosenwaks, Z

    1987-01-01

    Plasma prorenin and active renin were measured before and after human chorionic gonadotropin (hCG) administration in two groups of patients undergoing ovarian stimulation for 4-6 days with follicle-stimulating hormone alone or in combination with luteinizing hormone, for in vitro fertilization. Baseline total plasma renin (prorenin plus active renin; n = 12) averaged 25 +/- 8 ng/ml per hr (mean +/- SD). Total renin did not change during ovarian stimulation but it increased to 46 +/- 16 ng/ml per hr (P less than 0.05) 1 or 2 days later, just before hCG administration. Thirty-six hours after hCG administration, just before laparoscopy and egg retrieval, total renin was 123 +/- 97 ng/ml per hr; a peak of 182 +/- 143 ng/ml per hr occurred 2-6 days later--i.e., during the luteal phase of the menstrual cycle. In eight of the patients who did not conceive, total renin returned to baseline 14 days after hCG administration. In four who conceived, a nadir was reached (57 +/- 13 ng/ml per hr) 8-12 days after hCG administration and then total renin increased again as the plasma beta hCG measurement began to rise. By day 16 it averaged 225 +/- 157 ng/ml per hr. In a second group of five patients active renin and prorenin were measured separately. Active renin comprised less than 20% of the total renin at all times. It was unchanged until day 4 after hCG administration and then increased significantly only when plasma progesterone was high. Thus, the initial response to hCG was entirely due to an increase in prorenin. A highly significant correlation was observed between the number of follicles and the total renin increases on the day of aspiration (r = 0.93, P less than 0.001) and at the peak (r = 0.89, P less than 0.001). After hCG administration, a temporal relationship was observed between the rise in total renin and plasma estradiol and progesterone levels. These results demonstrate that plasma prorenin increases markedly after administration of hCG and that the rise is

  7. Global transcriptional expression in ovarian follicles from Tsaiya ducks (Anas platyrhynchos) with a high-fertilization rate.

    PubMed

    Wu, Shyh-Jong; Cheng, Yu-Shin; Liu, Hsiao-Lung; Wang, Hsing-He; Huang, Hsiu-Lin

    2016-05-01

    Novel candidates for biomarkers of a high-fertilization rate were identified here through global transcriptional profiling of ovarian follicles. Some other differentially expressed candidate genes were first noted to influence animal reproduction in our previous cDNA microarray analysis and are now recognized as markers for marker-assisted selection. In the present study, we compared gene expression in ovarian follicles from animals with high- and low-fertilization rates using an oligonucleotide array. On the basis of a fold change of greater than 1.2 and less than -1.2, a difference of >100 Affymetrix arbitrary units between the two groups, and a P value of less than 0.05, 47 genes were found to be associated with fertilization rate. GOEAST and MetaCore software were further used to identify the functional categories of genes that were differentially expressed. Then, we focused on three interesting genes associated with a high-fertilization rate: one of these genes was discovered to participate in signaling pathways of fertilization, and two genes take roles in lipid metabolism. An oligonucleotide array showed that the levels of orthodenticle homeobox 2 (OTX2) and lecithin:cholesterol acyltransferase (LCAT) gene expression were 1.62-fold and 1.95-fold higher in the high-fertilization rate group than in the low-fertilization rate group, respectively (P < 0.05). The level of apolipoprotein A-I (APOA1) gene expression was also higher in the high-fertilization rate group, with a difference of 2.31-fold (P < 0.05). The data were validated through quantitative polymerase chain reaction analysis. These results confirm the usefulness of the array technique and data mining methods in the discovery of new biomarkers and add knowledge to our understanding of the factors affecting fertilization rates in ovarian follicles. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Characterization of the mRNA expression of StAR and steroidogenic enzymes in zebrafish ovarian follicles.

    PubMed

    Ings, Jennifer S; Van Der Kraak, Glen J

    2006-08-01

    The objective of this study was to investigate the levels of expression of steroid biosynthetic enzymes and steroidogenic acute regulatory protein (StAR) at different stages of ovarian follicular development in zebrafish (Danio rerio), and to investigate the sites within the steroid biosynthetic pathway that may be regulated by gonadotropins. Ovarian follicles of sexually mature fish were separated into primary, previtellogenic, vitellogenic, and mature stages and the expression of StAR, P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 hydroxylase/lyase (P450c17), 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3), and P450 aromatase (P450aromA) was determined by Real time RT-PCR. The expression of all genes changed significantly as follicles grew, with a decrease in the expression of StAR, P450scc, 3beta-HSD and P450c17 with maturation, and an increase in the expression of 17beta-HSD3 during vitellogenesis and 17beta-HSD1 and P450aromA during previtellogenesis. In vitro incubation of vitellogenic follicles demonstrated that the expression of StAR, 17beta-HSD3, and P450aromA increased in response to hCG, and decreased in the absence of hCG. In contrast, the expression of P450scc, 3beta-HSD, P450c17, and 17beta-HSD1 remained constant between treatments and over time. Testosterone and estradiol production in the culture medium was stimulated by human chorionic gonadotropin (hCG). These experiments aid in the characterization of the roles and regulation of steroids throughout ovarian development, and suggest that gonadotropins play a key role in the regulation of StAR, 17beta-HSD3, and P450aromA in zebrafish.

  9. 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.

  10. Hormonal regulation of early follicle development in the rat ovary.

    PubMed

    Hsueh, A J; McGee, E A; Hayashi, M; Hsu, S Y

    2000-05-25

    Although earlier studies focused on the hormonal regulation of antral and preovulatory follicles, recent studies indicate the importance of the hormonal control mechanism for preantral follicles. The endocrine hormone FSH is not only a survival factor for early antral follicles but also a potent growth and differentiation factor for preantral follicles. In addition, KGF secreted by theca cells and c-kit ligand secreted by granulosa cells play paracrine roles in the regulation of preantral follicle growth and development. Furthermore oocyte-derived GDF-9 promotes the growth and differentiation of early follicles by acting on somatic cells in the follicle. It is likely that the genetic makeup of an oocyte could determine the secretion of oocyte hormones which would, in turn, regulate the growth and differentiation of the surrounding somatic cells of that follicle. A better understanding of the hormonal mechanisms underlying early follicle development could provide a refined culture system for the in vitro maturation of fertilizable oocytes and future design of fertility and contraceptive agents.

  11. 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.

  12. Significance of the ultrasonic morphology of preovulatory ovarian follicles prior to in vitro fertilization.

    PubMed

    Lenz, S; Lauritsen, G J; Lindenberg, S

    1983-01-01

    Forty-eight oocytes were collected from 47 follicles in 36 cycles from 24 patients with tubal infertility by ultrasonically guided percutaneous puncture under local anesthesia. The ultrasonic represented morphology of the aspirated follicles was compared to the presence of an egg-cumulus-mass in the follicular aspirate and the result of oocyte fertilization. There was a higher frequency of a cumulus-mass in the aspirate from follicles containing echoes (90 per cent) compared to ultrasonically empty follicles (37 per cent). Thirty-four oocytes were fertilized corresponding to a fertilization rate of 71 per cent. The mean diameter of follicles in the fertilized group was bigger (20.9 mm) than the mean diameter of follicles in the non-fertilized group (18.5 mm). The fertilization rate was higher in oocytes collected from follicles with internal echoes (87 per cent) compared to echo-free follicles (50 per cent). It seems that the ultrasonic demonstration of a fine cloud projecting into the follicle or a clouding of the cavity increases the possibility of collecting an oocyte suitable for in vitro fertilization.

  13. Effect of Rumex steudelii methanolic root extract on ovarian folliculogenesis and uterine histology in female albino rats.

    PubMed

    Solomon, T; Largesse, Z; Mekbeb, A; Eyasu, M; Asfaw, D

    2010-12-01

    A substantial number of the world population especially that of the developing countries rely on herbal products to control their fertility since ancient times. Rumex steudelii Hochst is one of the traditionally used antifertility plants in Ethiopia. Previous studies showed that the methanolic root extract of the plant had reversible antifertility effect in experimental animals. However, no study had hitherto been done on the antifertility activity of the methanolic root extract of Rumex steudelii on the ovary and uterus of female albino rats. To investigate the quantitative aspects of follicular development in the ovaries and uterine histology in cyclic female albino rats to get further information on the possible mechanism of antifertility effect of the methanolic extract of R. steudelii. The effect of the extract on uterine histology and ovarian follicular growth was determined after oral administration of the methanolic root extract of Rumex steudelii at 2.2, 2.5, 3.0 g/kg/day doses consecutively for 30 days. The extract significantly decreasing the number of healthy small antral, Graffian follicles and corpora lutea with concomitant significant increase in the number of atretic follicles of the same stage in dose dependent manner. Treatment at 3.0 g/Kg dose level in addition caused a significant decrease in the number of healthy primary, small preantral and large preantral follicles with concomitant significant increase in the number of atretic primary follicles. The ovarian and uterine wet weights are reduced significantly. The extracts also caused a significant decrease in the epithelial cell height, myometrial and stromal thickness in a dose dependent manner. The study demonstrated that the methanolic extract could cause atrophic changes in the uterus and disruption of ovarian folliculogenesis by inhibiting further development of the recruited ovarian follicles.

  14. 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.

  15. Follicle-stimulating hormone receptor (FSHR) alternative skipping of exon 2 or 3 affects ovarian response to FSH.

    PubMed

    Karakaya, Cengiz; Guzeloglu-Kayisli, Ozlem; Hobbs, Rebecca J; Gerasimova, Tsilya; Uyar, Asli; Erdem, Mehmet; Oktem, Mesut; Erdem, Ahmet; Gumuslu, Seyhan; Ercan, Deniz; Sakkas, Denny; Comizzoli, Pierre; Seli, Emre; Lalioti, Maria D

    2014-07-01

    Genes critical for fertility are highly conserved in mammals. Interspecies DNA sequence variation, resulting in amino acid substitutions and post-transcriptional modifications, including alternative splicing, are a result of evolution and speciation. The mammalian follicle-stimulating hormone receptor (FSHR) gene encodes distinct species-specific forms by alternative splicing. Skipping of exon 2 of the human FSHR was reported in women of North American origin and correlated with low response to ovarian stimulation with exogenous follicle-stimulating hormone (FSH). To determine whether this variant correlated with low response in women of different genetic backgrounds, we performed a blinded retrospective observational study in a Turkish cohort. Ovarian response was determined as low, intermediate or high according to retrieved oocyte numbers after classifying patients in four age groups (<35, 35-37, 38-40, >40). Cumulus cells collected from 96 women undergoing IVF/ICSI following controlled ovarian hyperstimulation revealed four alternatively spliced FSHR products in seven patients (8%): exon 2 deletion in four patients; exon 3 and exons 2 + 3 deletion in one patient each, and a retention of an intron 1 fragment in one patient. In all others (92%) splicing was intact. Alternative skipping of exons 2, 3 or 2 + 3 were exclusive to low responders and was independent of the use of agonist or antagonist. Interestingly, skipping of exon 3 occurs naturally in the ovaries of domestic cats--a good comparative model for human fertility. We tested the signaling potential of human and cat variants after transfection in HEK293 cells and FSH stimulation. None of the splicing variants initiated cAMP signaling despite high FSH doses, unlike full-length proteins. These data substantiate the occurrence of FSHR exon skipping in a subgroup of low responders and suggest that species-specific regulation of FSHR splicing plays diverse roles in mammalian ovarian function.

  16. Notch2 is required in somatic cells for breakdown of ovarian germ-cell nests and formation of primordial follicles.

    PubMed

    Xu, Jingxia; Gridley, Thomas

    2013-02-13

    In the mouse ovary, oocytes initially develop in clusters termed germ-cell nests. Shortly after birth, these germ-cell nests break apart, and the oocytes individually become surrounded by somatic granulosa cells to form primordial follicles. Notch signaling plays essential roles during oogenesis in Drosophila, and recent studies have suggested that Notch signaling also plays an essential role during oogenesis and ovary development in mammals. However, no in vivo loss-of-function studies have been performed to establish whether Notch family receptors have an essential physiological role during normal ovarian development in mutant mice. Female mice with conditional deletion of the Notch2 gene in somatic granulosa cells of the ovary exhibited reduced fertility, accompanied by the formation of multi-oocyte follicles, which became hemorrhagic by 7 weeks of age. Formation of multi-oocyte follicles resulted from defects in breakdown of the primordial germ-cell nests. The ovaries of the Notch2 conditional mutant mice had increased numbers of oocytes, but decreased numbers of primordial follicles. Oocyte numbers in the Notch2 conditional mutants were increased not by excess or extended cellular proliferation, but as a result of decreased oocyte apoptosis. Our work demonstrates that Notch2-mediated signaling in the somatic-cell lineage of the mouse ovary regulates oocyte apoptosis non-cell autonomously, and is essential for regulating breakdown of germ-cell nests and formation of primordial follicles. This model provides a new resource for studying the developmental and physiological roles of Notch signaling during mammalian reproductive biology.

  17. Notch2 is required in somatic cells for breakdown of ovarian germ-cell nests and formation of primordial follicles

    PubMed Central

    2013-01-01

    Background In the mouse ovary, oocytes initially develop in clusters termed germ-cell nests. Shortly after birth, these germ-cell nests break apart, and the oocytes individually become surrounded by somatic granulosa cells to form primordial follicles. Notch signaling plays essential roles during oogenesis in Drosophila, and recent studies have suggested that Notch signaling also plays an essential role during oogenesis and ovary development in mammals. However, no in vivo loss-of-function studies have been performed to establish whether Notch family receptors have an essential physiological role during normal ovarian development in mutant mice. Results Female mice with conditional deletion of the Notch2 gene in somatic granulosa cells of the ovary exhibited reduced fertility, accompanied by the formation of multi-oocyte follicles, which became hemorrhagic by 7 weeks of age. Formation of multi-oocyte follicles resulted from defects in breakdown of the primordial germ-cell nests. The ovaries of the Notch2 conditional mutant mice had increased numbers of oocytes, but decreased numbers of primordial follicles. Oocyte numbers in the Notch2 conditional mutants were increased not by excess or extended cellular proliferation, but as a result of decreased oocyte apoptosis. Conclusions Our work demonstrates that Notch2-mediated signaling in the somatic-cell lineage of the mouse ovary regulates oocyte apoptosis non-cell autonomously, and is essential for regulating breakdown of germ-cell nests and formation of primordial follicles. This model provides a new resource for studying the developmental and physiological roles of Notch signaling during mammalian reproductive biology. PMID:23406467

  18. Mate preference for dominant vs. subordinate males in young female Syrian hamsters (Mesocricetus auratus) following chemically-accelerated ovarian follicle depletion.

    PubMed

    Roosa, Kristen A; Place, Ned J

    2015-12-01

    Life history theory predicts that selectivity for mates generally declines as females age. We previously demonstrated this phenomenon in Syrian hamsters (Mesocricetus auratus), in that older females showed reduced preference for dominant over subordinate males. To test the hypothesis that decreased reproductive quality due to aging reduces mate preference, we decoupled reproductive and chronological age by treating young female hamsters with 4-vinylcyclohexene diepoxide (VCD), which destroys ovarian follicles and functionally accelerates ovarian follicle depletion without compromising the general health of rodents. In this study, VCD effectively reduced follicle numbers in young Syrian hamsters. VCD-treated and control females were allowed to choose between a dominant and a subordinate male in a Y-maze on the day of proestrus. Both VCD-treated and control females demonstrated preference for the dominant male by leaving a greater proportion of vaginal scent marks near him, which is a behavior that females display when soliciting prospective mates. However, there was no effect of treatment on the proportion of vaginal scent marks left for the dominant male. Furthermore, ovarian follicle numbers were not significantly correlated with any behaviors in either group. We conclude that accelerated ovarian follicle depletion does not reduce mate preference in young female hamsters.

  19. 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

  20. Ovarian development in athymic nude mice V. The effects of PMSG upon the numbers and growth of follicles in the early juvenile ovary.

    PubMed

    Lintern-Moore, S; Pantelouris, E M

    1976-01-01

    The composition and growth patterns of the ovarian follicle population have been determined in early juvenile athymic nude mice and their phenotypically normal littermates. Nude ovaries contained significantly more primordial follicles and fewer trilaminar follicles at 10 days of age. The rate of growth of the oocyte nucleolus was significantly less than in control ovaries. Treatment with PMSG from days 7 to 9 restored the rate of oocyte nucleolar growth in nudes to control levels. PMSG also increased the number of follicles commencing growth in both nudes and controls and the differences in the numbers of primordial and trilaminar follicles were no longer detectable. The data demonstrate that abnormalities in the ovarian follicle population of the nude are detectable as early as the 10th day of life and can be reversed by treatment with exogenous gonadotrophin. These results are discussed in relation to the role of the thymus gland in ovarian development and the competence of the early juvenile ovary to respond to gonadotrophin.

  1. Progressive Evaluation of Apoptosis, Proliferation, and Angiogenesis in Fresh Rat Ovarian Autografts Under Remote Ischemic Preconditioning.

    PubMed

    Damous, Luciana Lamarão; Silva, Sônia Maria da; Carbonel, Adriana Aparecida Ferraz; Simões, Manuel de Jesus; Baracat, Edmund Chada; Montero, Edna Frasson de Souza

    2016-06-01

    This study evaluated the remote ischemic preconditioning (R-IPC) early and late repercussion on fresh ovarian transplants, aiming to assess a probable protective effect in ovarian follicular pool. Sixty Wistar EPM-1 rats were used, divided in 2 study groups: ovarian transplantation (Tx) and Tx + R-IPC, submitted to ovary transplant with or without R-IPC, respectively. These groups were subdivided according to the date for euthanasia: 4th, 7th, 14th, 21st, and 30th days of the postoperatory period. Morphology, morphometry, neoangiogenesis (vascular endothelial growth factor [VEGF]), proliferative activity (Ki-67), and apoptosis (cleaved caspase-3) were evaluated. Remote ischemic preconditioning was performed in the common iliac artery. Fresh autologous ovarian tissue was implanted integrally in the retroperitoneum. All animals showed resumption of estrous phase after ovary transplantation. Remote ischemic preconditioning attenuated the lesions progressively from the 7th day, with greater number of the immature follicles (14 days, P < .05), but didn't affect mature follicles and corpora lutea (P > .05). Immunohistochemical analyzes, taken as a whole, show that R-IPC benefic effect is more evident in the later periods of evaluation, when a greater proliferative activity (14, 21, and 30 days, P < .05) and lesser cell apoptotic activity (21 and 30 days, P < .05). The VEGF expression was similar in all times (P > .05). Remote ischemic preconditioning could have a benefic effect in the progressive evaluation of freshly grafted ovarian, especially on the latest phases of the posttransplant period. The 14th day was a landmark in the recuperation of the graft. Further investigations are necessary to determine the role of R-IPC in this scenario and its effect in frozen-thawed ovarian tissue.

  2. SIRT1 activator (SRT1720) improves the follicle reserve and prolongs the ovarian lifespan of diet-induced obesity in female mice via activating SIRT1 and suppressing mTOR signaling.

    PubMed

    Zhou, Xiao-Ling; Xu, Jin-Jie; Ni, Yan-Hong; Chen, Xiao-Chun; Zhang, Hong-Xia; Zhang, Xing-Mei; Liu, Wei-Juan; Luo, Li-Li; Fu, Yu-Cai

    2014-10-21

    The prevalence of obesity is increasing worldwide and significantly affects fertility and reproduction in both men and women. Our recent study has shown that excess body fat accelerates ovarian follicle development and follicle loss in rats. The aim of the present study is to explore the effect of SIRT1 activator SRT1720 on the reserve of ovarian follicle pool and ovarian lifespan of obese mice and the underlying mechanism associated with SIRT1 and mTOR signaling. Adult female Kunming mice (n = 36) were randomly divided into three groups: the normal control (NC) group (n = 8), the caloric restriction (CR) group (fed 70% food of the NC group, n = 8) and the high-fat diet (HF) group (fed a rodent chow containing 20% fat, n = 20). After 4 months, the HF mice were further randomly divided into three groups: the control high-fat diet (CHF, n = 8) group (treated every day with an intraperitoneal injection of vehicle), the SRT1720 (SRT, n = 6) group (treated every other day with an intraperitoneal injection of SRT1720 (50 mg/kg)), the SRT1720 and nicotinamide (NAM, n = 6) group (treated every other day with an intraperitoneal injection of SRT1720 (50 mg/kg) and every day with an intraperitoneal injection of nicotinamide (100 mg/kg)). After 6 weeks of treatment, ovaries were harvested for histological and Western blotting analyses. The body weight, ovary weight and visceral fat in the SRT group were significantly lower than those in the CHF group at the end of treatment. Histological analysis showed that the SRT mice had significantly greater number and percentage of primordial follicles, but lower number and percentage of corpora lutea and atretic follicles than the CHF mice and NAM mice. Western blot analysis demonstrated that the levels of SIRT1, SIRT6, FOXO3a and NRF-1 protein expression significantly increased in the ovaries of SRT mice, whereas those of mTORC1, p-mTOR, p-p70S6K, NFκB and p53 decreased compared to the CHF and NAM mice. Our study suggests that

  3. Evaluation of the ovarian reserve in young low responders with normal basal levels of follicle-stimulating hormone using three-dimensional ultrasonography.

    PubMed

    Pellicer, A; Ardiles, G; Neuspiller, F; Remohí, J; Simón, C; Bonilla-Musoles, F

    1998-10-01

    To assess the ovarian content of selectable (2-5 mm) follicles using three-dimensional ultrasonography in low responders to ovarian stimulation for IVF. Prospective case-control study. IVF program at the Instituto Valenciano de Infertilidad. Ten low responders < or =35 years of age with normal basal serum FSH and eight control patients with normal response in a previous cycle. Blood was drawn under basal (day 3) conditions. Three-dimensional ultrasound was performed in both ovaries using a vaginal probe. Basal serum E2 and FSH measurements. The ovarian volume and the number of follicles > or =2 mm in each ovary were recorded and compared between the groups. Low-responder women had significantly higher serum FSH levels than controls despite having FSH values within the normal range. The number of selectable follicles and the total number of follicles with an antrum were significantly decreased in low responders as compared with normal responders. Ovarian volume did not differ between the groups. This study introduces three-dimensional ultrasound as a novel method for the evaluation of low responders. The results show that the most plausible explanation for low response in young women with normal serum FSH is diminished ovarian reserve.

  4. Sphingosine-1-phosphate and ceramide are associated with health and atresia of bovine ovarian antral follicles.

    PubMed

    Hernández-Coronado, C G; Guzmán, A; Espinosa-Cervantes, R; Romano, M C; Verde-Calvo, J R; Rosales-Torres, A M

    2015-02-01

    The follicle destiny towards ovulation or atresia is multi-factorial in nature and involves outcries, paracrine and endocrine factors that promote cell proliferation and survival (development) or unchain apoptosis as part of the atresia process. In several types of cells, sphingosine-1-phospate (S1P) promotes cellular proliferation and survival, whereas ceramide (CER) triggers cell death, and the S1P/CER ratio may determine the fate of the cell. The aim of present study was to quantify S1P and CER concentrations and their ratio in bovine antral follicles of 8 to 17 mm classified as healthy and atretic antral follicles. Follicles were dissected from cow ovaries collected from a local abattoir. The theca cell layer, the granulosa cells and follicular fluid were separated, and 17β-estradiol (E2) and progesterone (P4) concentrations were measured in the follicular fluid by radioimmunoassay. Based on the E2/P4 ratio, the follicles were classified as healthy (2.2±0.3) or atretic (0.2±0.3). In both follicular compartments (granulosa and theca cell layer), sphingolipids were extracted and S1P and CER concentrations were quantified by HPLC (XTerra RP18; 5 µm, 3.0×150 mm column). Results showed that in both follicular compartments, S1P concentrations were higher in healthy antral follicles than in atretic antral follicles (P<0.05). The concentration of CER in the granulosa cells was higher in atretic antral follicles than in healthy antral follicles, but no differences were observed in the theca cell layer. The S1P/CER ratio in both follicular compartments was also higher in healthy antral follicles. Interestingly, in these follicles, there was a 45-fold greater concentration of S1P than CER in the granulosa cells (P<0.05), whereas in the theca cell layer, S1P had only a 14-fold greater concentration than CER when compared with atretic antral follicles. These results suggest that S1P plays a role in follicle health, increasing cellular proliferation and survival. In

  5. Effect of Monochromatic Light on Expression of Estrogen Receptor (ER) and Progesterone Receptor (PR) in Ovarian Follicles of Chicken.

    PubMed

    Liu, Lingbin; Li, Diyan; Gilbert, Elizabeth R; Xiao, Qihai; Zhao, Xiaoling; Wang, Yan; Yin, Huadong; Zhu, Qing

    2015-01-01

    Artificial illumination is widely used in modern poultry houses and different wavelengths of light affect poultry production and behaviour. In this study, we measure mRNA and protein abundance of estrogen receptors (ERs) and progesterone receptors (PRs) in order to investigate the effect of monochromatic light on egg production traits and gonadal hormone function in chicken ovarian follicles. Five hundred and fifty-two 19-wk-old laying hens were exposed to three monochromatic lights: red (RL; 660 nm), green (GL; 560 nm), blue (BL; 480 nm) and control cool white (400-760 nm) light with an LED (light-emitting diode). There were 4 identical light-controlled rooms (n = 138) each containing 3 replicate pens (46 birds per pen). Water was supplied ad libitum and daily rations were determined according to the nutrient suggestions for poultry. Results showed that under BL conditions there was an increase in the total number of eggs at 300 days of age and egg-laying rate during the peak laying period. The BL and GL extended the duration of the peak laying period. Plasma melatonin was lowest in birds reared under BL. Plasma estradiol was elevated in the GL-exposed laying hens, and GL and BL increased progesterone at 28 wk of age. In the granulosa layers of the fifth largest preovulatory follicle (F5), the third largest preovulatory follicle (F3) and the largest preovulatory follicle (F1), ERα mRNA was increased by BL and GL. Treatment with BL increased ERβ mRNA in granulosa layers of F5, F3 and F1, while GL increased ERβ mRNA in F5 and F3. There was a corresponding increase in abundance of the proteins in the granulosa layers of F5, with an increase in PR-B, generated via an alternative splice site, relative to PR-A. Treatment with BL also increased expression of PR mRNA in all of the granulosa layers of follicles, while treatment with GL increased expression of PR mRNA in granulosa layers of SYF(small yellow follicle), F5 and F1. These results indicate that blue and green

  6. Growth differentiation factor-9 and anti-Müllerian hormone expression in cultured human follicles from frozen-thawed ovarian tissue.

    PubMed

    Sadeu, J C; Smitz, J

    2008-10-01

    In-vitro growth of frozen-thawed human follicles is perceived as a potential option for restoring women's fertility. The aims of this study were: (i) to test the usefulness of a defined serum-free medium for growth of frozen-thawed human follicles; and (ii) to evaluate the expression of growth differentiation factor-9 (GDF-9) and anti-Müllerian hormone (AMH) in cultured follicles. Frozen-thawed ovarian cortical pieces from 7-, 12-, 25- and 27-year-old women were cultured for 0, 7, 14, 21 and 28 days. Follicle developmental quality was evaluated and expression of proliferating cell nuclear antigen (PCNA) (day 21), GDF-9 (days 14 and 28) and AMH (day 21) was assessed by immunohistochemistry. Primary follicles and enclosed oocytes underwent significant growth at the end of culture (P < 0.05). Cultured follicles from all patients studied reached the early secondary stage and a few follicles from two patients developed up to the secondary stage. The rate of atresia was variable throughout the culture periods. PCNA was expressed in the granulosa cells at all the different follicular stages. AMH and GDF-9 immunostaining were found respectively in the granulosa cells and oocytes after several weeks of culture. The transition from resting to growing follicles leading to the development of secondary follicles showed the normal expression patterns of GDF-9 and AMH.

  7. 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

  8. Maintenance of Atlantic salmon (Salmo salar) at elevated temperature inhibits cytochrome P450 aromatase activity in isolated ovarian follicles.

    PubMed

    Watts, Marianne; Pankhurst, Ned W; King, Henry R

    2004-02-01

    Atlantic salmon (Salmo salar) broodstock were transferred from natural (12-16 degrees C) to controlled temperatures of 14, 18 or 22 degrees C for 3 months during vitellogenesis. Fertility and survival were significantly reduced in eggs from broodstock held at 22 degrees C relative to 14 or 18 degrees C. Endocrine mechanisms were disrupted after only one month at 22 degrees C, as evidenced by decreased plasma vitellogenin (Vtg) and increased plasma testosterone (T) levels and, at later stages, decreased levels of plasma 17beta-estradiol (E2). In vitro incubations of isolated ovarian follicles were carried out at monthly intervals, with follicles exposed to human chorionic gonadotropin, N-2-0-dibutyryladenosine 3,5-cyclic monophosphate, and the gonadal steroid precursors 17-hydroxyprogesterone, androstenedione, and T. After one month of exposure to controlled temperature, T synthesis was generally enhanced in response to all treatments at all temperatures, but E2 synthesis was inhibited at 22 degrees C, suggesting temperature impairment of cytochrome P450 aromatase (P450arom) synthesis or activity. The effect became less marked as follicles matured suggesting that temperature sensitivity is stage dependent. The results of this study suggest that the inhibitory effects of elevated temperature on E2 and Vtg synthesis, and subsequent egg development found in the present and earlier studies, arise at least partly, from temperature modulation of P450arom.

  9. Expression and regulation of SNAP-25 and synaptotagmin VII in developing mouse ovarian follicles via the FSH receptor.

    PubMed

    Choi, Sung Sik; Jung, Joo Young; Lee, Dong Ho; Kang, Ji Yoon; Lee, Sang Ho

    2013-02-01

    Soluble-NSF attachment protein receptor (SNARE) proteins play a role in vesicle fusion, exocytosis, and intracellular trafficking in neuronal cells as well as in fertilization and embryogenesis. We investigated the expression patterns of two SNARE proteins, SNAP-25 and synaptotagmin VII (SytVII), and their regulation by pregnant mare serum gonadotropin (PMSG) during mouse ovarian follicular development. Ovaries were obtained at 0, 12, 24, 36, and 48 h post-PMSG injection of immature mice. SNAP-25 and SytVII mRNA expression levels increased gradually in a time-dependant manner. However, protein levels revealed different patterns of expression, suggesting different translational regulation following PMSG stimulation. SNAP-25 and SytVII expression was closely associated with thickening of the granulosa cell (GC) layer and follicle morphological changes from a flattened to a cuboidal shape. To explore follicle stimulating hormone receptor (FSHR)-mediated regulation of their expression, GCs from preantral follicles were cultured to examine the effects of FSHR siRNA knockdown. FSHR siRNA abolished upregulation of the SNAREs in both PMSG and FSH-stimulated GCs. This abolished gene expression was rescued by adding dibutyryl cyclic AMP to the cultures. These results suggest that SNAP-25 and SytVII expression is regulated via the FSHR-cAMP pathway during follicular development.

  10. 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.

  11. Characterization, localization, and stage-dependent gene expression of gonadotropin receptors in chub mackerel (Scomber japonicus) ovarian follicles.

    PubMed

    Nyuji, Mitsuo; Kitano, Hajime; Shimizu, Akio; Lee, Jae Man; Kusakabe, Takahiro; Yamaguchi, Akihiko; Matsuyama, Michiya

    2013-06-01

    The pituitary gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are key regulators of gametogenesis in teleosts. However, little is known about the physiological mechanisms by which GtHs regulate asynchronous oocyte development in multiple-spawning marine fishes. We cloned cDNAs encoding GtH receptors (FSHR and LHR) from chub mackerel (Scomber japonicus). FSH and LH were purified by anion-exchange chromatography, gel filtration, and concanavalinA-agarose. When expressed in mammalian cells, FSHR and LHR responded strongly to their own ligands. By separating LH into two subunits by the use of reverse-phase chromatography, we found that the beta-subunit is responsible for signal transduction and the alpha-subunit may be important for holding hormone-receptor complex. In situ hybridization showed that only fshr was expressed in prefollicle and granulosa cells in oocytes at the perinucleolus and cortical alveolus stages, suggesting that FSH is involved in the primary and early secondary growth of oocytes. In ovarian follicles during vitellogenesis, both fshr and lhr were expressed in granulosa and thecal cells, and lhr was strongly expressed during germinal vesicle migration (GVM). Real-time PCR analysis of stage-dependent fshr and lhr expression showed that fshr expression was high in ovarian follicles throughout vitellogenesis and decreased during GVM, whereas lhr expression was low in early vitellogenesis, but increased markedly in the late phase of vitellogenesis, remaining high during GVM. These findings suggest that switching of the expression of FSHR to LHR controls the effects of FSH and/or LH on vitellogenesis and final oocyte maturation via steroid production in granulosa and thecal cells.

  12. 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.

  13. Evaluation of ovarian reserve using anti-müllerian hormone and antral follicle count in Sjögren's syndrome: Preliminary study.

    PubMed

    Karakus, Savas; Sahin, Ali; Durmaz, Yunus; Aydin, Huseyin; Yildiz, Caglar; Akkar, Ozlem; Dogan, Mansur; Cengiz, Ahmet; Cetin, Meral; Cetin, Ali

    2017-02-01

    The aim of this study was to determine ovarian reserve status using anti-müllerian hormone (AMH) level and antral follicle count (AFC) in patients with Sjögren's syndrome (SS). Twenty-four women with SS diagnosed according to the classification criteria proposed by the American-European Consensus Group and 25 healthy women as controls were enrolled in this study. Ovarian reserve was assessed on clinical findings, AFC, and serum AMH and reproductive hormone levels. Compared with the healthy controls, in the SS patients, the duration of menstrual cycle was significantly shorter (P = 0.043); serum AMH (P = 0.001) and AFC (P = 0.001) were significantly lower, and serum luteinizing hormone (LH) was significantly higher (P = 0.019). The right (P = 0.555) and left ovarian (P = 0.386) volumes were also lower but this did not reach statistical significance. Serum follicle-stimulating hormone (P = 0.327), estradiol (P = 0.241), and prolactin (P = 0.55) were similar between the two groups. Ovarian reserve may be reduced in SS patients. For the assessment of ovarian reserve, serum AMH and ovarian AFC with serum LH may be useful. Further studies with long-term follow-up are required to determine the course of ovarian reserve abnormalities and best possible biomarkers of reduced ovarian reserve in SS patients. © 2016 Japan Society of Obstetrics and Gynecology.

  14. Morphometry and immunohistochemistry of follicles growth and steroidogenesis in saharian wild sand rat, Psammomys obesus, ovary.

    PubMed

    Boubekri, Amina; Gernigon-Spychalowicz, Thérese; Khammar, Farida; Exbrayat, Jean-Marie

    2009-01-01

    The sand rat (Psammomys obesus) constitutes a model to study seasonal changes and several metabolic disorders. In order to perform breeding laboratory conditions, the reproductive function of this species living in North Occidental Algerian Sahara was studied. The aim of this work was to investigate the follicular growth changes and the steroidogenic associated aspects. The study was performed using morphometrical and immunohistochemical methods. From primordial to preantral states, the follicle diameter increased progressively from 17-20 mum to 192-225 mum. The preovulatory follicles reached about 500 mum in diameter. Immunoreactivity to progesterone, androstenediol and estradiol, varied in the different parts of the ovary and follicular cells. The progesterone antibody appeared clearly labelled in the theca interna of the growing follicle and increased in the granulosa; the androgen antibody was continuously weak and diffuses in all follicles; the estradiol labelling appeared weak and diffuse in preantral follicles then increased in antral follicles in both theca and granulosa or only in granulosa. In antral follicles, estradiol label was clearly localized in granulosa cells and totally devoid in theca cells. In Psammomys ovary, labels of hormone were diffuse or localized, weak or intense in the theca and or in the granulosa according to the follicle size.

  15. Urinary concentrations of ovarian steroid hormone metabolites and bioactive follicle-stimulating hormone in killer whales (Orcinus orchus) during ovarian cycles and pregnancy.

    PubMed

    Walker, L A; Cornell, L; Dahl, K D; Czekala, N M; Dargen, C M; Joseph, B; Hsueh, A J; Lasley, B L

    1988-12-01

    Reproductive hormone profiles of six captive killer whales (Orcinu orcus) from three Sea World aquaria were studied for intervals up to 2 yr. Daily urine samples and bimonthly blood samples were collected and analyzed for hormone concentration. Immunoreactive estrone conjugates, pregnanediol-3-glucoruonide, 20-alpha-hydroxyprogesterone as well as bioactive follicle-stimulating hormone (FSH) were measured in urine samples and indexed by creatinine concentrations of the same sample. In selected cases, serum progesterone concentrations were also measured. Three of the animals in the study became pregnant during the study period and two of these animals were evaluated during the time of conception and throughout most of gestation. From the data of the three animals that conceived, hormone profiles of the complete ovarian cycle, early pregnancy, and mid- to late gestation are described. The remaining three animals did not conceive and only one of these demonstrated hormone changes that indicated regular ovarian activity. The female reproductive pattern of the killer whale is characterized by a gestation of 17 mo and an ovarian cycle of 6-7 wk in duration. The hormone changes associated with the ovarian cycle of the killer whale are similar to those of most other mammalian species. A bimodal pattern of bioactive FSH with a pronounced rise of estrogen predominates the preovulatory hormone profile. After ovulation, increased progesterone production is observed for approximately 4 wk in the nonconceptive ovarian cycle. During the luteal phase and early pregnancy, when progesterone metabolites are elevated, estrogen metabolite excretion remains low. These data extend the application of urine collections for longitudinal studies involving hormone changes, particularly those involving nondomesticated species.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. 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.

  17. Developmental methoxychlor exposure affects multiple reproductive parameters and ovarian folliculogenesis and gene expression in adult rats

    SciTech Connect

    Armenti, AnnMarie E.; Zama, Aparna Mahakali; Passantino, Lisa; Uzumcu, Mehmet

    2008-12-01

    Methoxychlor (MXC) is an organochlorine pesticide with estrogenic, anti-estrogenic, and anti-androgenic properties. To investigate whether transient developmental exposure to MXC could cause adult ovarian dysfunction, we exposed Fischer rats to 20 {mu}g/kg/day (low dose; environmentally relevant dose) or 100 mg/kg/day (high dose) MXC between 19 days post coitum and postnatal day 7. Multiple reproductive parameters, serum hormone levels, and ovarian morphology and molecular markers were examined from prepubertal through adult stages. High dose MXC accelerated pubertal onset and first estrus, reduced litter size, and increased irregular cyclicity (P < 0.05). MXC reduced superovulatory response to exogenous gonadotropins in prepubertal females (P < 0.05). Rats exposed to high dose MXC had increasing irregular estrous cyclicity beginning at 4 months of age, with all animals showing abnormal cycles by 6 months. High dose MXC reduced serum progesterone, but increased luteinizing hormone (LH). Follicular composition analysis revealed an increase in the percentage of preantral and early antral follicles and a reduction in the percentage of corpora lutea in high dose MXC-treated ovaries (P < 0.05). Immunohistochemical staining and quantification of the staining intensity showed that estrogen receptor {beta} was reduced by high dose MXC while anti-Mullerian hormone was upregulated by both low- and high dose MXC in preantral and early antral follicles (P < 0.05). High dose MXC significantly reduced LH receptor expression in large antral follicles (P < 0.01), and down-regulated cytochrome P450 side-chain cleavage. These results demonstrated that developmental MXC exposure results in reduced ovulation and fertility and premature aging, possibly by altering ovarian gene expression and folliculogenesis.

  18. Detection of DNA damage in oocytes of small ovarian follicles following phosphoramide mustard exposures of cultured rodent ovaries in vitro

    SciTech Connect

    Petrillo, Stephanie K.; Desmeules, Patrice; Truong, To-Quyen; Devine, Patrick J.

    2011-06-01

    Healthy oocytes are critical for producing healthy children, but little is known about whether or not oocytes have the capacity to identify and recover from injury. Using a model ovotoxic alkylating drug, cyclophosphamide (CPA), and its active metabolite, phosphoramide mustard (PM), we previously showed that PM ({>=} 3 {mu}M) caused significant follicle loss in postnatal day 4 (PND4) mouse ovaries in vitro. We now investigate whether PM induces DNA damage in oocytes, examining histone H2AX phosphorylation ({gamma}H2AX), a marker of DNA double-strand breaks (DSBs). Exposure of cultured PND4 mouse ovaries to 3 and 0.1 {mu}M PM induced significant losses of primordial and small primary follicles, respectively. PM-induced {gamma}H2AX was observed predominantly in oocytes, in which foci of {gamma}H2AX staining increased in a concentration-dependent manner and peaked 18-24 h after exposure to 3-10 {mu}M PM. Numbers of oocytes with {>=} 5 {gamma}H2AX foci were significantly increased both 1 and 8 days after exposure to {>=} 1 {mu}M PM compared to controls. Inhibiting the kinases that phosphorylate H2AX significantly increased follicle loss relative to PM alone. In adult mice, CPA also induced follicle loss in vivo. PM also significantly decreased primordial follicle numbers ({>=} 30 {mu}M) and increased {gamma}H2AX foci ({>=} 3 {mu}M) in cultured PND4 Sprague-Dawley rat ovaries. Results suggest oocytes can detect PM-induced damage at or below concentrations which cause significant follicle loss, and there are quantitative species-specific differences in sensitivity. Surviving oocytes with DNA damage may represent an increased risk for fertility problems or unhealthy offspring.

  19. Regulatory Role of Gonadotropins and Local Factors Produced by Ovarian Follicles on In Vitro Resistin Expression and Action on Porcine Follicular Steroidogenesis.

    PubMed

    Rak, Agnieszka; Drwal, Eliza; Karpeta, Anna; Gregoraszczuk, Ewa Ł

    2015-06-01

    Resistin, a hormone secreted by adipocytes, is thought to be important in reproduction. Our previous study demonstrated resistin expression in porcine ovarian follicles and its direct effect on steroidogenesis. The aim of the current study was to evaluate the effect of gonadotropins and the local ovarian factors, such as insulin-like growth factor type 1 (IGF1) and steroids (progesterone, testosterone, and 17 beta-estradiol), on the expression and secretion of resistin, as well as its steroidogenic action. Porcine ovarian follicles were exposed to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) at 50-150 ng/ml, IGF1 (10-100 ng/ml), and steroids at 10(-8) to 10(-6) M for 24 h. Then, mRNA, protein expression, and medium concentration of resistin were determined using real-time PCR, Western blot analysis, and ELISA, respectively. In the subsequent experiments, ovarian follicles were exposed to resistin and/or FSH, LH, IGF1, and steroids, and ovarian steroidogenesis was analyzed. Additionally, we examined the direct effect of resistin on the protein expression of receptors for gonadotropins and investigated local factors. The results showed that gonadotropins and steroids have stimulatory effects but that IGF1 has an inhibitory effect on resistin expression and secretion. Resistin decreased gonadotropins and local hormone-induced steroid secretion and inhibited 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, and cytochrome P450 aromatase protein expression. Additionally, we demonstrated that resistin increased the expression of receptors for progesterone and testosterone. These findings all show that the expression and function of resistin are regulated by gonadotropins and local factors produced by ovarian follicles. © 2015 by the Society for the Study of Reproduction, Inc.

  20. Effects of soy phytoestrogens on pituitary-ovarian function in middle-aged female rats.

    PubMed

    Medigović, Ivana M; Živanović, Jasmina B; Ajdžanović, Vladimir Z; Nikolić-Kokić, Aleksandra L; Stanković, Sanja D; Trifunović, Svetlana L; Milošević, Verica Lj; Nestorović, Nataša M

    2015-12-01

    The aim of this study was to assess the effects of genistein (G) and daidzein (D) on the histological, hormonal, and functional parameters of the pituitary-ovarian axis in middle-aged female rats, and to compare these effects with the effects of estradiol (E), commonly used in the prevention and treatment of menopausal symptoms. Middle-aged (12 month old) Wistar female rats subcutaneously received 35 mg/kg of G, or 35 mg/kg of D, or 0.625 mg/kg of E every day for 4 weeks. Each of the treated groups had a corresponding control group. An intact control group was also established. G and D did not change the intracellular protein content within gonadotropic and lactotropic cells, but vacuolization was observed in all the cell types. In contrast, E caused an inhibition of gonadotropic and stimulation of lactotropic cells. Also, ovaries of middle-aged female rats exposed to G or D have more healthy primordial and primary follicles and less atretic follicles. E treatment in the ovaries had a mostly negative effect, which is reflected by the increased number of atretic follicles in all tested classes. G and D provoked decrease in CuZnSOD and CAT activity, while E treatment increased MnSOD and decreased CuZnSOD and GSHPx activity. All the treatments increased serum estradiol and decreased testosterone levels, while D and E increased the serum progesterone level. In conclusion, soy phytoestrogens exhibited beneficial effects on pituitary-ovarian function in middle-aged female rats, as compared to estradiol.

  1. Ganglionic adrenergic action modulates ovarian steroids and nitric oxide in prepubertal rat.

    PubMed

    Delgado, Silvia Marcela; Casais, Marilina; Sosa, Zulema; Rastrilla, Ana María

    2006-08-01

    Both peripheral innervation and nitric oxide (NO) participate in ovarian steroidogenesis. The purpose of this work was to analyse the ganglionic adrenergic influence on the ovarian release of steroids and NO and the possible steroids/NO relationship. The experiments were carried out in the ex vivo coeliac ganglion-superior ovarian nerve (SON)-ovary system of prepubertal rats. The coeliac ganglion-SON-ovary system was incubated in Krebs Ringer-bicarbonate buffer in presence of adrenergic agents in the ganglionic compartment. The accumulation of progesterone, androstenedione, oestradiol and NO in the ovarian incubation liquid was measured. Norepinephrine in coeliac ganglion inhibited the liberation of progesterone and increased androstenedione, oestradiol and NO in ovary. The addition of alpha and beta adrenergic antagonists also showed different responses in the liberation of the substances mentioned before, which, from a physiological point of view, reveals the presence of adrenergic receptors in coeliac ganglion. In relation to propranolol, it does not revert the effect of noradrenaline on the liberation of progesterone, which leads us to think that it might also have a "per se" effect on the ganglion, responsible for the ovarian response observed for progesterone. Finally, we can conclude that the ganglionic adrenergic action via SON participates on the regulation of the prepubertal ovary in one of two ways: either increasing the NO, a gaseous neurotransmitter with cytostatic characteristics, to favour the immature follicles to remain dormant or increasing the liberation of androstenedione and oestradiol, the steroids necessary for the beginning of the near first estral cycle.

  2. Nitric oxide and thyroid hormone receptor alpha 1 contribute to ovarian follicular development in immature hyper- and hypo-thyroid rats.

    PubMed

    Zheng, Kaizhi; Sulieman, Fedail Jaafar; Li, Junrong; Wei, Quanwei; Xu, Mulin; Shi, Fangxiong

    2015-03-01

    Thyroid dysfunction can cause ovarian cycle and ovulatory disturbances, however, the molecular link(s) between these two disorders remains largely unknown. In the current study, we examined the roles of nitric oxide synthase (NOS) and thyroid hormone receptor alpha 1 (TRα1) in these disorders using immature hyper-thyroid (hyper-T) and hypo-thyroid (hypo-T) rats. In comparison to controls, hyper-T rats had higher serum concentrations of triiodothyronine (T3) and thyroxine (T4), whereas hypo-T rats had lower serum T3 and T4. Serum estradiol (E2) level was decreased in both hyper-T and hypo-T animals and serum E2 in hyper-T rats were lower than in hypo-T rats. We found that neuronal NOS (nNOS) and TRα1 were present in oocytes, granulosa cells and theca cells of all examined rat groups. Ovarian nitric oxide (NO) content and the constitutive NOS (cNOS) activity in hyper-T rats were significantly decreased compared with control or hypo-T rats. Moreover, the number of large antral follicles was reduced in hyper-T rats, and number of primordial follicles was decreased in hypo-T rats compared with control rats. In conclusion, we observed an association between thyroid hormone and NO signaling pathways during the process of ovarian follicular development in immature rats. In hyperthyroidism, thyroid hormones induced an estrogen deficiency that inhibited the function of nNOS, resulting in the inhibition of NO synthesis and suppressed development of large antral follicles, while in hypothyroidism only development of primordial follicles was inhibited.

  3. Age-related changes in gene expression patterns of immature and aged rat primordial follicles.

    PubMed

    Govindaraj, Vijayakumar; Krishnagiri, Harshini; Chakraborty, Payal; Vasudevan, Madavan; Rao, A Jagannadha

    2017-02-01

    Women are born with millions of primordial follicles which gradually decrease with increasing age and this irreversible supply of follicles completely exhausts at menopause. The fertility capacity of women diminishes in parallel with aging. The mechanisms for reproductive aging are not fully understood. We have observed a decline in Brca1 mediated DNA repair in aging rat primordial follicles. To further understand the age-related molecular changes, we performed microarray gene expression analysis using total RNA extracted from immature (18 to 20 day old) and aged (400 to 450 day old) rat primordial follicles. The results of current microarray study revealed that there were 1,011 (>1.5 fold, p<0.05) genes differentially expressed between two groups in which 422 genes were up-regulated and 589 genes were down-regulated in aged rat primordial follicles compared to immature primordial follicles. The gene ontology and pathway analysis of differentially expressed genes revealed a critical biological function such as cell cycle, oocyte meiosis, chromosomal stability, transcriptional activity, DNA replication, and DNA repair were affected by age. This considerable difference in gene expression profiles may have an adverse influence on oocyte quality. Our data provide information on the processes that may contribute to aging and age-related decline in fertility.

  4. Concanavalin-A induces granulosa cell death and inhibits FSH-mediated follicular growth and ovarian maturation in female rats.

    PubMed

    Velasquez, Ethel V; Ríos, Mariana; Ortiz, María Elena; Lizama, Carlos; Nuñez, Elizabeth; Abramovich, Dalhia; Orge, Felipe; Oliva, Barbara; Orellana, Renán; Villalon, Manuel; Moreno, Ricardo D; Tesone, Marta; Rokka, Anne; Corthals, Garry; Croxatto, Horacio B; Parborell, Fernanda; Owen, Gareth I

    2013-05-01

    Reproductive success stems from a finely regulated balance between follicular maturation and atresia, in which the role of carbohydrate structure is poorly understood. Here, we describe for the first time a fraction of purified recombinant human FSH that is capable of bringing about the cell death of granulosa cells and preventing follicular maturation in a rat model. Further analysis by mass spectrometry revealed the presence of the lectin Concanavalin-A (Con-A) within this fraction of recombinant FSH. Using both the fractionated FSH and Con-A, the observed cell death was predominantly located to the granulosa cells. Ex vivo culture of rat follicles demonstrated that follicle degeneration occurred and resulted in the release of a denuded and deteriorated oocyte. Moreover, in vivo experiments confirmed an increase in atresia and a corresponding reduction confined to follicle in early antral stage. As a mechanism of action, Con-A reduces ovarian proliferation, Von Willebrand staining, and angiogenesis. Based on the observation that Con-A may induce granulosa cell death followed by follicle death, our results further demonstrate that follicular carbohydrate moiety is changing under the influence of FSH, which may allow a carbohydrate-binding lectin to increase granulosa cell death. The physiological consequences of circulating lectin-like molecules remain to be determined. However, our results suggest a potential exploitation of carbohydrate binding in fertility and ovarian cancer treatment. This work may shed light on a key role of carbohydrates in the still obscure physiological process of follicular selection and atresia.

  5. Cat and dog primordial follicles enclosed in ovarian cortex sustain viability after in vitro culture on agarose gel in a protein-free medium.

    PubMed

    Fujihara, M; Comizzoli, P; Wildt, D E; Songsasen, N

    2012-12-01

    Our objective was to examine the influences of differing media, protein supplementation and the microenvironment on cat vs dog primordial follicle viability in vitro. Ovarian cortical slices were cultured for 3, 9 or 15 days in α-minimum essential medium (α-MEM) or MEM supplemented with 10% fetal bovine serum (FBS), 10% knock-out serum replacement (KSR) or 0.1% polyvinyl alcohol (protein free). In a separate study, cat and dog ovarian tissues were cultured in protein-free α-MEM and MEM, respectively, in cell culture inserts, on 1.5% agarose gel or in 24-well cell culture plates (control). Follicle viability was assessed in both studies using calcein AM/ethidium homodimer and histological evaluation with haematoxylin/eosin staining. No cat follicle sustained viability beyond 9 days of in vitro culture in α-MEM compared to 37.5% of those incubated for 15 days in MEM in protein-free condition (p < 0.05). In contrast, α-MEM was superior (p < 0.05) to MEM in maintaining dog follicle viability (32.7% vs 8.1%) in protein-free condition at 15 days. Serum was detrimental (p < 0.05) to follicle survival in both species. Knock-out serum replacement supplementation and a protein-free condition supported cat follicle viability, whereas the latter was superior (p < 0.05) to the former for sustaining dog follicle survival. Likewise, dog follicle viability was enhanced (p < 0.05) by the agarose gel compared to the cell culture insert and control groups after 3 and 9 days of culture. For the cat, the agarose gel better (p < 0.05) supported follicle viability compared to the control, but was equivalent to the cell culture insert. Therefore, sustaining primordial follicle survival from intracortical ovarian slices requires a different in vitro microenvironment for the cat vs the dog. A key factor to enhancing survival of these early stage follicles in culture appears to be the use of agarose gel, which enhances follicle viability, perhaps by promoting gas exchange.

  6. Exosomal miR-10a derived from amniotic fluid stem cells preserves ovarian follicles after chemotherapy.

    PubMed

    Xiao, Guan-Yu; Cheng, Chun-Chun; Chiang, Yih-Shien; Cheng, Winston Teng-Kuei; Liu, I-Hsuan; Wu, Shinn-Chih

    2016-03-16

    Chemotherapy (CTx)-induced premature ovarian failure (POF) in woman remains clinically irreversible. Amniotic fluid stem cells (AFSCs) have shown the potential to treat CTx-induced POF; however, the underlying mechanism is unclear. Here we demonstrate that AFSC-derived exosomes recapitulate the anti-apoptotic effect of AFSCs on CTx-damaged granulosa cells (GCs), which are vital for the growth of ovarian follicles. AFSC-derived exosomes prevent ovarian follicular atresia in CTx-treated mice via the delivery of microRNAs in which both miR-146a and miR-10a are highly enriched and their potential target genes are critical to apoptosis. The down-regulation of these two miRNAs in AFSC-derived exosomes attenuates the anti-apoptotic effect on CTx-damaged GCs in vitro. Further, the administration of these miRNAs recapitulates the effects both in vitro and in vivo, in which miR-10a contributes a dominant influence. Our findings illustrate that miR-10a has potential as a novel therapeutic agent for the treatment of POF.

  7. Development of persistent ovarian follicles during synchronization of estrus influences the superovulatory response to FSH treatment in cattle.

    PubMed

    Wehrman, M E; Fike, K E; Kojima, F N; Bergfeld, E G; Cupp, A S; Mariscal, V; Sanchez, T; Kinder, J E

    1996-02-01

    The synchronization of estrus with synthetic progestins or progesterone (P(4)) results in the development of a large, persistent ovarian follicle. The objectives of the present study were to determine if development of a persistent ovarian follicle during synchronization of estrus suppresses recruitment of additional follicles during FSH treatment. On Day 5 of the estrous cycle (estrus = Day 0), beef cows were treated with 0.5 or 2.0 P(4) releasing intravaginal devices (PRIDs) for 8 d (Experiment 1, n = 20), 5 or 2 d (Experiment 2, n = 44) before initiation of FSH treatment. Prostaglandin F(2alpha) (25 mg) was administered on Days 5 and 6. Superovulation was induced with 24 mg of recombinant bovine FSH (rbFSH, Experiment 1) or 28 mg of FSH-P (Experiment 2) over a 3- or 4-d period, respectively. The PRIDs were removed concurrently with the 5th injection of rbFSH or FSH-P. There was a treatment-by-day interaction (P < 0.001) for the concentration of 17beta-estradiol in cows treated for 8, 5 or 2 d before FSH treatment. In Experiment 1, FSH treatment initiated 8 d after insertion of a 0.5 PRID did not affect the number of CL (6.9 +/- 1.4 vs 6.7 +/- 1.6), ova/embryos (3.7 +/-1.3 vs 3.0 +/- 1.3) and transferable embryos (2.4 +/- 0.9 vs 3.0 +/- 0.9) compared with that of the 2.0 PRIDs. In Experiment 2, FSH treatment initiated 5 d after insertion of a 0.5 PRID decreased the number of CL (4.0 +/- 0.5 vs 8.3 +/- 0.8; P < 0.001), ova/embryos (3.0 +/- 0.6 vs 5.9 +/- 1.2; P < 0.03) and transferable embryos (2.3 +/- 0.6 vs 5.1 +/- 1.0; P < 0.03) compared with that of a 2.0 PRID, respectively. Initiation of FSH treatment 2 d after insertion of a 0.5 PRID compared with a 2.0 PRID had no affect on the number of CL (8.0 +/- 2.1 vs 8.7 +/- 1.2), total ova (4.8 +/- 1.4 vs 6.9 +/- 1.4) and transferable embryos (2.9 +/- 1.2 vs 6.1 +/- 1.7). In conclusion, treatment with low doses of P(4) (0.5 PRID) for 5 d but not for 2 or 8 d before initiation of FSH treatment results in the

  8. Contribution of de novo synthesis of Gαs-proteins to 1-methyladenine production in starfish ovarian follicle cells stimulated by relaxin-like gonad-stimulating substance.

    PubMed

    Mita, Masatoshi; Haraguchi, Shogo; Uzawa, Haruka; Tsutsui, Kazuyoshi

    2013-11-01

    In starfish, the peptide hormone gonad-stimulating substance (GSS) secreted from nervous tissue stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) production by ovarian follicle cells. The hormonal action of GSS on follicle cells involves its receptor, G-proteins and adenylyl cyclase. However, GSS failed to induce 1-MeAde and cAMP production in follicle cells of ovaries during oogenesis. At the maturation stage, follicle cells acquired the potential to respond to GSS by producing 1-MeAde and cAMP. Adenylyl cyclase activity in follicle cells of fully grown stage ovaries was also stimulated by GSS in the presence of GTP. These activations depended on the size of oocytes in ovaries. The α subunit of Gs-proteins was not detected immunologically in follicle cells of oogenesis stage ovaries, although Gαi and Gαq were detectable. Using specific primers for Gαs and Gαi, expression levels of Gαs in follicle cells were found to increase significantly as the size of oocytes in ovaries increased, whereas the mRNA levels of Gαi were almost constant regardless of oocyte size. These findings strongly suggest the potential of follicle cells to respond to GSS by producing 1-MeAde and cAMP is brought by de novo synthesis of Gαs-proteins.

  9. Exposure to bisphenol A at physiological concentrations observed in Chinese children promotes primordial follicle growth through the PI3K/Akt pathway in an ovarian culture system.

    PubMed

    Zhao, Qian; Ma, Yan; Sun, Ning-Xia; Ye, Chen; Zhang, Qing; Sun, Shu-Han; Xu, Chen; Wang, Fang; Li, Wen

    2014-12-01

    The worldwide increase in the use of bisphenol A (BPA) has resulted in increased human exposure, which could affect human reproductive function. Few studies have investigated the effect of BPA exposure on the primordial follicle pool. In this study, we employed a neonatal ovarian culture system comprising organ obtained from female C57BL/6 pups on postnatal day 4 to assess the effect of BPA on the primordial follicle pool. Ovaries were cultured with BPA (0.1 μM, physiological concentration found in children's blood, and 1 μM, 10 μM) or vehicle for 10 days. Our study revealed that the primary follicle number increased during the early time points (⩽5 days), and we observed a reduction in the primordial follicle pool at a later time point (day 10). This reduction at day 10 was due to increased follicle activation and reduced follicle atresia, as determined by immunohistochemistry for Ki-67 and active caspase-3. Then we examined the phosphatidylinositol-3-kinase (PI3K)/Akt pathway, which is known to be important for early follicle growth. BPA exposure induced the upregulation of the PI3K/Akt pathway, which was reversed by concomitant treatment with PI3K inhibitor. Our results reveal a novel mechanism for BPA-induced primordial follicle activation that involves the PI3K/Akt pathway. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Phosphodiesterases in the rat ovary: effect of cAMP in primordial follicles.

    PubMed

    Petersen, Tonny Studsgaard; Stahlhut, Martin; Andersen, Claus Yding

    2015-07-01

    Phosphodiesterases (PDEs) are important regulators of the intracellular cAMP concentration, which is a central second messenger that affects a multitude of intracellular functions. In the ovaries, cAMP exerts diverse functions, including regulation of ovulation and it has been suggested that augmented cAMP levels stimulate primordial follicle growth. The present study examined the gene expression, enzyme activity and immunolocalization of the different cAMP hydrolysing PDEs families in the rat ovary. Further, the effect of PDE4 inhibition on primordial follicle activation in cultured neonatal rat ovaries was also evaluated. We found varied expression of all eight families in the ovary with Pde7b and Pde8a having the highest expression each accounting for more than 20% of the total PDE mRNA. PDE4 accounted for 15-26% of the total PDE activity. Immunoreactive PDE11A was found in the oocytes and PDE2A in the corpora lutea. Incubating neonatal rat ovaries with PDE4 inhibitors did not increase primordial follicle activation or change the expression of the developing follicle markers Gdf9, Amh, Inha, the proliferation marker Mki67 or the primordial follicle marker Tmeff2. In addition, the cAMP analogue 8-bromo-cAMP did not increase AKT1 or FOXO3A phosphorylation associated with follicle activation or increase the expression of Kitlg known to be associated with follicle differentiation but did increase the Tmeff2, Mki67 and Inha expression in a dose-dependent manner. In conclusion, this study shows that both Pde7b and Pde8a are highly expressed in the rodent ovary and that PDE4 inhibition does not cause an increase in primordial follicle activation. © 2015 Society for Reproduction and Fertility.

  11. Inhibition of ovarian cancer cell proliferation in vivo and incorporation of /sup 3/H-thymidine in vitro after follicle regulatory protein administration

    SciTech Connect

    Rodgers, K.E.; Montz, F.J.; Scott, L.; Condon, S.; Fujimori, K.; diZerega, G.S.

    1989-01-01

    Follicle regulatory protein immunoreactivity and biologic activity were measured in ascites from a patient with juvenile granulosa cell tumor. Microscopic examination of immunohistochemical staining of a juvenile granulosa cell tumor with anti-follicle regulatory protein antisera showed homogeneous cytosolic expression of follicle regulatory protein throughout the tumor. Tumor cells were injected subcutaneously into nude mice. Partially purified follicle regulatory protein (50 micrograms/day) was then injected daily for 10 days, or for 25 days once the tumor became palpable. Treatment with follicle regulatory protein significantly slowed the rate of tumor growth with both treatments. To test the tissue specificity of the effect, a metastatic, well-differentiated endometrial adenocarcinoma was also grown in nude mice. Follicle regulatory protein treatment did not alter the rate of tumor growth. An in vitro clonigenic assay confirmed these in vivo results. Partially purified follicle regulatory protein had a biphasic effect on the proliferation of juvenile granulosa tumor cell but did not affect the proliferation of endometrial adenocarcinoma cells. Clonigenic assays were performed on five ovarian adenocarcinomas passaged in vitro, and these tumor cells exhibited a biphasic response to follicle regulatory protein. Immunoneutralization studies showed that this biphasic response was due to impurities in the follicle regulatory protein preparations. The longer the exposure of the tumor cells to follicle regulatory protein, the greater the degree of inhibition of proliferation. In summary, administration of follicle regulatory protein slowed tumor growth through a direct effect on the tumor cell rather than an indirect effect on the hormonal or immune status of the host.

  12. Di-n-Butyl Phthalate Disrupts the Expression of Genes Involved in Cell Cycle and Apoptotic Pathways in Mouse Ovarian Antral Follicles1

    PubMed Central

    Craig, Zelieann R.; Hannon, Patrick R.; Wang, Wei; Ziv-Gal, Ayelet; Flaws, Jodi A.

    2012-01-01

    ABSTRACT Di-n-butyl phthalate (DBP) is present in many consumer products, such as infant, beauty, and medical products. Several studies have shown that DBP causes reproductive toxicity in rodents, but no studies have evaluated its effects on ovarian follicles. Therefore, we used a follicle culture system to evaluate the effects of DBP on antral follicle growth, cell cycle and apoptosis gene expression, cell cycle staging, atresia, and 17β-estradiol (E2) production. Antral follicles were isolated from adult CD-1 mice and exposed to DBP at 1, 10, 100, and 1000 μg/ml for 24 or 168 h. Follicles treated with vehicle or DBP at 1–100 μg/ml grew over time, but DBP at 1000 μg/ml significantly suppressed follicle growth. Regardless of effect on follicle growth, DBP-treated follicles had decreased mRNA for cyclins D2, E1, A2, and B1 and increased p21. Levels of the proapoptotic genes Bax, Bad, and Bok were not altered by DBP treatment, but DBP 1000 μg/ml increased levels of Bid and decreased levels of the antiapoptotic gene Bcl2. DBP-treated follicles contained significantly more cells in G1 phase, significantly less cells in S, and exhibited a trend for fewer cells in G2. Although DBP did not affect E2 production and atresia at 24 h, follicles treated with DBP had reduced levels of E2 at 96 h and underwent atresia at 168 h. These data suggest that DBP targets antral follicles and alters the expression of cell cycle and apoptosis factors, causes cell cycle arrest, decreases E2, and triggers atresia, depending on dose. PMID:23242528

  13. Quantitative expression of antiapoptotic and proapoptotic genes in sheep ovarian follicles grown in vivo or cultured in vitro.

    PubMed

    Praveen Chakravarthi, V; Kona, S S R; Siva Kumar, A V N; Bhaskar, M; Rao, V H

    2015-03-01

    To test the hypothesis that the poor development of the oocytes in cultured ovarian follicles of mammals is due to aberrant expression of developmentally important genes, quantitative expression patterns of Bcl2 (B-cell leukemia/lymphoma 2; antiapoptotic) and Bax (Bcl2-associated X protein; proapoptotic) genes in preantral, early antral, antral, large antral follicles, and cumulus-oocyte complexes (COCs) grown in vivo or cultured in vitro were studied. The level and pattern of expression of Bcl2 in the cumulus cells isolated from different development stages of in vivo- and in vitro-grown ovarian follicles were similar suggesting that in vitro culture did not alter the expression of this antiapoptotic gene in the cumulus cells. However, between the in vivo- and in vitro-grown ovarian follicles (1) Bcl2 expression levels in the oocytes from antral follicles (2.21 ± 0.14 vs. 0.87 ± 0.19), large antral follicles (0 ± 0.35 vs. 1.56 ± 0.13), and COCs (0.45 ± 0.31 vs. 2.69 ± 0.15), Bax expression levels in the (2) cumulus cells from early antral (2.09 ± 0.11 vs. 0.98 ± 0.13) and large antral follicle (0.63 ± 0.44 vs. 0 ± 0.21), and (3) oocytes from antral follicles (1.65 ± 0.20 vs. 0.97 ± 0.15), large antral follicles (0.93 ± 0.18 vs. 2.08 ± 0.11), and COCs (1.03 ± 0.17 vs. 2.09 ± 0.11) were significantly different (P ≤ 0.05). Similarly, Bcl2 to Bax ratios were also significantly different between some but not all stages of in vivo and in vitro development. From the present results, it is concluded that imbalance in the expression of proapoptotic and antiapoptotic genes may be an important cause for the compromised development potential of the oocytes in cultured ovarian follicles of sheep. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. 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

  15. Effects of frutalin on early follicle morphology, ultrastructure and gene expression in cultured goat ovarian cortical tissue.

    PubMed

    Soares, Maria A A; Costa, José J N; Vasconcelos, Gisvani L; Ribeiro, Regislane P; Souza, José C; Silva, André L C; Van den Hurk, Robert; Silva, José R V

    2017-02-15

    Frutalin is a galactose-binding lectin that has an irreversible cytotoxic effect on HeLa cervical cancer cells, by inducing apoptosis and inhibiting cell proliferation. It was previously shown that after in vitro incubation, frutalin is internalized into HeLa cells nucleus, which indicates that frutalin apoptosis-inducing activity might be linked with its nuclear localization. Considering that drugs commonly used for cancer treatment have a deleterious effect on germ cells, the aim of this study was to evaluate the effect of frutalin on the activation, survival, ultrastructure and gene expression in follicles cultured within ovarian tissue. Goat ovarian fragments were cultured for 6 days in α-MEM+ alone or supplemented with frutalin (1, 10, 50, 100 or 200 µg/ml). Non-culturad and cultured tissues were processed for histological and ultrastructural analysis and they were also stored to evaluate the expression of anti- and pro-apoptotic genes by quantitative polymerase chain reaction (qPCR). The results showed that the frutalin, at all concentrations tested, reduced follicular survival when compared with control medium. Higher concentrations of frutalin (50, 100 or 200 µg/ml) also reduced follicular survival when compared with those tissues cultured with 1 or 10 µg/ml of frutalin. The ultrastructural analysis showed that atretic cultured follicles had retracted oocytes and a large number of vacuoles spread throughout the cytoplasm. In addition, signs of damage of mitochondrial membranes and cristae were observed. Moreover, although a dose-response effect on gene expression has not been observed, when compared with tissues culture in control medium, the presence of frutalin increased in mRNA expression pro-apoptotic genes. In conclusion, frutalin reduces follicular survival at all concentrations tested, its effects being more pronounced when high concentrations of this lectin (50, 100 and 200 µg/ml) are used. Gene expression profile and ultrastrutural features of

  16. Assessment of fertility protection and ovarian reserve with GnRH antagonist in rats undergoing chemotherapy with cyclophosphamide.

    PubMed

    Lemos, Claudia N C D; Reis, Fernando M; Pena, Guilherme N; Silveira, Laila C; Camargos, Aroldo F

    2010-05-18

    Reproductive function following chemotherapy is of increasing importance given that survival rates are improving. We assessed whether a gonadotropin-releasing hormone antagonist (GnRHant; cetrorelix) could promote ovarian protection against damage due to chemotherapy. Forty-two female Wistar rats were used in this study. Animals were divided into four groups: group I (n=9) received placebo twice; group II (n=12) received placebo+cyclophosphamide (CPA); group III (n=12) received GnRHant+CPA; and group IV (n=9) received GnRHant+placebo. After medication, the estrous cycle was studied through vaginal smears. Rats were mated, pregnancy was documented and the number of live pups evaluated. Afterwards, rat ovaries were removed and prepared for histological studies. The ovarian cross-sectional area was measured and follicles were counted. Cyclic changes in vaginal smears were observed in all but one animal after treatment, but group II had a significantly lower rate of animals with proestrus or estrus (p<0.01). The offspring was markedly reduced by CPA treatment (group II, 3.00+/-1.33 pups vs. group I, 11.44+/-0.78 pups, p<0.01) and this effect was partly reversed by pre-treatment with GnRHant (group III, 7.00+/-1.31 pups). The ovarian cross-sectional area was not significantly different between groups, neither was the number of individual follicle types. However, rats in Group IV had a higher total number of ovarian follicles than those in the control group (17.1+/-1.22 vs. 10.9+/-0.70, p<0.05). The use of a GnRHant before CPA chemotherapy provided protection of fertility.

  17. Cellular heterogeneity in the membrana granulosa of developing rat follicles: assessment by flow cytometry and lectin binding.

    PubMed

    Kerketze, K; Blaschuk, O W; Farookhi, R

    1996-07-01

    The hormone-mediated maturation of ovarian follicles is apparently accompanied by position-specific differentiation of cells of the membrana granulosa. We have assessed the extent of this cellular heterogeneity by flow cytometry using a variety of fluorescein isothiocyanate-labeled lectins as probes. Follicular development was stimulated in immature rats by treatment with either diethylstilbestrol (DES) or equine CG (eCG). Lectin binding to monodispersed rat granulosa cells was then analyzed by flow cytometry. Our results demonstrate that there are two distinct populations of small (4-7 microM) and large (9-12 microM) granulosa cells in follicles from DES- and eCG-treated animals. Both populations appear to be mitotically active and show specific lectin-binding characteristics. Six lectins (canavalia ensiforms, triticum vulgaris, maclura pomifera, erythrina cristagalli, jacalin, and vicia villosa) bind equally to both small and large granulosa cells from the DES- and eCG-treated rats. In contrast, no binding to either cell population was detected with six other lectins (dolichos biflorus, griffonia simplicifolia-II, lycopersicon esculentum, datura stramonium, solanum tuberosum, and ulex europaeus). Furthermore, four galactose-binding lectins (bauhinia purpurea, glysine maximus, griffonia simplicifolia-I, and arachis hypogaea) were found to identify specific subsets of granulosa cells. Three of these lectins (bauhinia purpurea, glysine maximus, and griffonia simplicifolia-I) bind to only small granulosa cells from either DES- or eCG- treated immature rats. The fourth lectin (arachis hypogaea) identifies subpopulations of both small and large granulosa cells. Application of the four galactose-specific lectins to fixed sections of frozen ovaries demonstrated binding to the perioocyte and cumulus granulosa cells. We conclude that cellular heterogeneity exists within the follicular epithelium at various stages-specific lectin-binding sites.

  18. Expression of amelogenin and effects of cyclosporin A in developing hair follicles in rats.

    PubMed

    Yoo, Hong-Il; Lee, Gye-Hyeok; Lee, Su-Young; Kang, Jee-Hae; Moon, Jung-Sun; Kim, Min-Seok; Kim, Sun-Hun

    2016-01-01

    Amelogenin, an enamel matrix protein has been considered to be exclusively expressed by ameloblasts during odontogenesis. However, burgeoning evidence indicates that amelogenin is also expressed in non-mineralizing tissues. Under the hypothesis that amelogenin may be a functional molecule in developing hair follicles which share developmental features with odontogenesis, this study for the first time elucidated the presence and functional changes of amelogenin and its receptors during rat hair follicle development. Amelogenin was specifically localized in the outer epithelial root sheath of hair follicles. Its expression appeared in the deeper portion of hair follicles, i.e. the bulbar and suprabulbar regions rather than the superficial region. Lamp-1, an amelogenin receptor, was localized in either follicular cells or outer epithelial sheath cells, reflecting functional changes during development. The expression of amelogenin splicing variants increased in a time-dependent manner during postnatal development of hair follicles. Amelogenin expression was increased by treatment with cyclosporin A, which is an inducer of anagen in the hair follicle, whereas the level of Lamp-1 and -2 was decreased by cyclosporin A treatment. These results suggest that amelogenin may be a functional molecule involved in the development of the hair follicle rather than an inert hair shaft matrix protein. © 2015 Anatomical Society.

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

    PubMed

    Aad, Pauline Y; Echternkamp, Sherrill E; Sypherd, David D; Schreiber, Nicole B; Spicer, Leon J

    2012-10-01

    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 theca (TC) cells of small (1-5 mm) antral follicles by in situ hybridization and of larger (5-17 mm) antral follicles by real-time RT-PCR from ovaries of cyclic cows genetically selected (Twinner) or not selected (control) for twin ovulations. Expression of IHH mRNA was localized to GC and cumulus cells, whereas PTCH1 mRNA was greater in TC than in GC. Estrogen-active (E-A; follicular fluid concentration of estradiol > progesterone) versus estrogen-inactive follicles had a greater abundance of mRNA for IHH in GC and PTCH1 in TC. Abundance of IHH mRNA in GC was not affected by cow genotype, whereas TC PTCH1 mRNA was less in large E-A follicles of Twinners than in controls. In vitro, estradiol and wingless-type (WNT) 3A increased IHH mRNA in IGF1-treated GC. IGF1 and BMP4 treatments decreased PTCH1 mRNA in small TC. Estradiol and LH increased PTCH1 mRNA in IGF1-treated TC from large and small follicles, respectively. In summary, functional status of ovarian follicles was associated with differences in hedgehog signaling in GC and TC. We hypothesize that as follicles grow and develop, increased free IGF1 may suppress expression of IHH mRNA by GC and PTCH1 mRNA by TC, and these effects are regulated in a paracrine way by estradiol and other intra- and extragonadal factors.

  20. 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

  1. 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

    SciTech Connect

    Craig, Zelieann R. Hannon, Patrick R. Flaws, Jodi A.

    2013-11-01

    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 prevent 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

  2. Characterization and significance of adhesion and junction-related proteins in mouse ovarian follicles.

    PubMed

    Mora, Jocelyn M; Fenwick, Mark A; Castle, Laura; Baithun, Marianne; Ryder, Timothy A; Mobberley, Margaret; Carzaniga, Raffaella; Franks, Stephen; Hardy, Kate

    2012-05-01

    In the ovary, initiation of follicle growth is marked by cuboidalization of flattened granulosa cells (GCs). The regulation and cell biology of this shape change remains poorly understood. We propose that characterization of intercellular junctions and associated proteins is key to identifying as yet unknown regulators of this important transition. As GCs are conventionally described as epithelial cells, this study used mouse ovaries and isolated follicles to investigate epithelial junctional complexes (tight junctions [TJ], adherens junctions [AJ], and desmosomes) and associated molecules, as well as classic epithelial markers, by quantitative PCR and immunofluorescence. These junctions were further characterized using ultrastructural, calcium depletion and biotin tracer studies. Junctions observed by transmission electron microscopy between GCs and between GCs and oocyte were identified as AJs by expression of N-cadherin and nectin 2 and by the lack of TJ and desmosome-associated proteins. Follicles were also permeable to biotin, confirming a lack of functional TJs. Surprisingly, GCs lacked all epithelial markers analyzed, including E-cadherin, cytokeratin 8, and zonula occludens (ZO)-1alpha+. Furthermore, vimentin was expressed by GCs, suggesting a more mesenchymal phenotype. Under calcium-free conditions, small follicles maintained oocyte-GC contact, confirming the importance of calcium-independent nectin at this stage. However, in primary and multilayered follicles, lack of calcium resulted in loss of contact between GCs and oocyte, showing that nectin alone cannot maintain attachment between these two cell types. Lack of classic markers suggests that GCs are not epithelial. Identification of AJs during GC cuboidalization highlights the importance of AJs in regulating initiation of follicle growth.

  3. Embryos of the viviparous dermapteran, Arixenia esau develop sequentially in two compartments: terminal ovarian follicles and the uterus.

    PubMed

    Tworzydlo, Waclaw; Kisiel, Elzbieta; Bilinski, Szczepan M

    2013-01-01

    Three main reproductive strategies have been described among insects: most common oviparity, ovoviviparity and viviparity. In the latter strategy, the embryonic development takes place within the body of the mother which provides gas exchange and nutrients for embryos. Here we present the results of histological and EM analyses of the female reproductive system of the viviparous earwig, Arixenia esau, focusing on all the modifications related to the viviparity. We show that in the studied species the embryonic development consists of two "physiological phases" that take place in two clearly disparate compartments, i.e. the terminal ovarian follicle and the uterus. In both compartments the embryos are associated with synthetically active epithelial cells. We suggest that these cells are involved in the nourishment of the embryo. Our results indicate that viviparity in arixeniids is more complex than previously considered. We propose the new term "pseudoplacento-uterotrophic viviparity" for this unique two-phase reproductive strategy.

  4. Transcriptome profiling of granulosa cells from bovine ovarian follicles during atresia

    PubMed Central

    2014-01-01

    Background The major function of the ovary is to produce oocytes for fertilisation. Oocytes mature in follicles surrounded by nurturing granulosa cells and all are enclosed by a basal lamina. During growth, granulosa cells replicate and a large fluid-filled cavity (the antrum) develops in the centre. Only follicles that have enlarged to over 10 mm can ovulate in cows. In mammals, the number of primordial follicles far exceeds the numbers that ever ovulate and atresia or regression of follicles is a mechanism to regulate the number of oocytes ovulated and to contribute to the timing of ovulation. To better understand the molecular basis of follicular atresia, we undertook transcriptome profiling of granulosa cells from healthy (n = 10) and atretic (n = 5) bovine follicles at early antral stages (< 5 mm). Results Principal Component Analysis (PCA) and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. These analyses and size-frequency plots of coefficients of variation of signal intensities revealed that the healthy follicles were more heterogeneous. Examining the differentially-expressed genes the most significantly affected functions in atretic follicles were cell death, organ development, tissue development and embryonic development. The overall processes influenced by transcription factor gene TP53 were predicted to be activated, whereas those of MYC were inhibited on the basis of known interactions with the genes in our dataset. The top ranked canonical pathway contained signalling molecules common to various inflammatory/fibrotic pathways such as the transforming growth factor-β and tumour necrosis factor-α pathways. The two most significant networks also reflect this pattern of tissue remodelling/fibrosis gene expression. These networks also contain molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming

  5. 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

  6. 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

  7. Fluoride Exposure, Follicle Stimulating Hormone Receptor Gene Polymorphism and Hypothalamus-pituitary-ovarian Axis Hormones in Chinese Women.

    PubMed

    Zhao, Ming Xu; Zhou, Guo Yu; Zhu, Jing Yuan; Gong, Biao; Hou, Jia Xiang; Zhou, Tong; Duan, Li Ju; Ding, Zhong; Cui, Liu Xin; Ba, Yue

    2015-09-01

    The effects of fluoride exposure on the functions of reproductive and endocrine systems have attracted widespread attention in academic circle nowadays. However, it is unclear whether the gene-environment interaction may modify the secretion and activity of hypothalamus-pituitary- ovarian (HPO) axis hormones. Thus, the aim of this study was to explore the influence of fluoride exposure and follicle stimulating hormone receptor (FSHR) gene polymorphism on reproductive hormones in Chinese women. A cross sectional study was conducted in seven villages of Henan Province, China during 2010-2011. A total of 679 women aged 18-48 years were recruited through cluster sampling and divided into three groups, i.e. endemic fluorosis group (EFG), defluoridation project group (DFPG), and control group (CG) based on the local fluoride concentration in drinking water. The serum levels of gonadotropin releasing hormone (GnRH), follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) were determined respectively and the FSHR polymorphism was detected by real time PCR assay. The results provided the preliminary evidence indicating the gene-environment interaction on HPO axis hormones in women.

  8. The histone acetyltransferases CBP and Chameau integrate developmental and DNA replication programs in Drosophila ovarian follicle cells.

    PubMed

    McConnell, Kristopher H; Dixon, Michael; Calvi, Brian R

    2012-10-01

    DNA replication origin activity changes during development. Chromatin modifications are known to influence the genomic location of origins and the time during S phase that they initiate replication in different cells. However, how chromatin regulates origins in concert with cell differentiation remains poorly understood. Here, we use developmental gene amplification in Drosophila ovarian follicle cells as a model to investigate how chromatin modifiers regulate origins in a developmental context. We find that the histone acetyltransferase (HAT) Chameau (Chm) binds to amplicon origins and is partially required for their function. Depletion of Chm had relatively mild effects on origins during gene amplification and genomic replication compared with previous knockdown of its ortholog HBO1 in human cells, which has severe effects on origin function. We show that another HAT, CBP (Nejire), also binds amplicon origins and is partially required for amplification. Knockdown of Chm and CBP together had a more severe effect on nucleosome acetylation and amplicon origin activity than knockdown of either HAT alone, suggesting that these HATs collaborate in origin regulation. In addition to their local function at the origin, we show that Chm and CBP also globally regulate the developmental transition of follicle cells into the amplification stages of oogenesis. Our results reveal a complexity of origin epigenetic regulation by multiple HATs during development and suggest that chromatin modifiers are a nexus that integrates differentiation and DNA replication programs.

  9. Stress promotes development of ovarian cysts in rats: the possible role of sympathetic nerve activation.

    PubMed

    Paredes, A; Gálvez, A; Leyton, V; Aravena, G; Fiedler, J L; Bustamante, D; Lara, H E

    1998-06-01

    Activation of the sympathetic innervation precedes the induction of polycystic ovaries in rats given estradiol valerate (EV). The mechanism of induction by EV may thus involve both direct and neurogenic components. We tested this hypothesis using a combined cold and restraint stress to induce an increase in sympathetic tone, including that of the ovarian sympathetic nerves. Three weeks after the start of stress we found: 1. An increase in the content of norepinephrine (NE) in the celiac ganglion. 2. An increase in the release of NE from the ovary. 3. An unchanged NE uptake by the ovary. 4. An unchanged content of NE in the ovary. The ovarian content of neuropeptide Y (NPY) (colocalized with NE) was significantly decreased. These results suggest that NE synthesis and its secretion are increased during this period and correlate with the increase in secretion of androgens and estradiol, the development of precystic follicles, and a decrease in the ovulatory rate. After 11 wk, NE release had returned to control values, whereas the ovarian NE content had risen significantly, suggesting a maintained high rate of NE synthesis. In the ovary, NPY contents, steroid secretion, morphology, and ovulation had returned to the control state. These results suggest the participation of an extraovarian factor that might act locally to control the release of NE from the ovary, and further support the hypothesis that increased sympathetic activity plays a role in the development and maintenance of ovarian cysts.

  10. Impaired ovarian response to exogenous gonadotropins in female rat offspring born to mothers perinatally exposed to Bisphenol A.

    PubMed

    Santamaría, C G; Rodriguez, H A; Abud, J E; Rivera, O E; Muñoz-de-Toro, M; Luque, E H

    2017-06-23

    The ovary is sensitive to disruption by the environmental estrogen Bisphenol A (BPA). Our aim was to investigate whether perinatal exposure to BPA (50μg/kgday), orally administered, affects ovarian response to exogenous gonadotrophins (PMSG or PMSG+hCG) in prepubertal female offspring. An altered response to gonadotrophins was observed in BPA-exposed rats. Increased proportion of antral follicles, altered levels of ovarian steroidogenic enzymes, gonadotropin receptors, AR and ERβ were observed in PMSG group. Besides that, in response to PMSG+hCG, a persistent high Fshr mRNA expression and a decreased number of follicles with high expression of PR before ovulation were observed. After ovulation, there was an increase in antral atretic follicles, reduced Lhcgr mRNA expression and high serum levels of E2. Therefore, an early exposure to a low dose of BPA during perinatal period induces ovarian changes leading to an altered response to exogenous gonadotropin treatment later in life. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Molecular cloning and mRNA expression analysis of ornithine decarboxylase antizyme 2 in ovarian follicles of the Sichuan white goose (Anser cygnoides).

    PubMed

    He, Hui; Kang, Bo; Jiang, Dongmei; Ma, Rong; Bai, Lin

    2014-07-25

    The ornithine decarboxylase antizyme 2 (OAZ2) gene is a member of the antizyme gene family. Antizymes play pivotal roles in various cellular pathways, including polyamine anabolism and apoptosis. The molecular structure and expression profile of the OAZ2 in goose ovarian follicles have not been reported. In this study, the OAZ2 cDNA sequence of the Sichuan white goose was cloned (Anser cygnoides), and phylogenetic and structural analyses of the OAZ2 were performed. The expression profiling of OAZ2 mRNA in goose ovarian follicles was examined using quantitative real-time PCR. The sequence analysis showed that the 756 bp OAZ2 sequence contained two overlapping open reading frames (ORF). ORF1 was 99 bp in length, and encoded a 32 aa polypeptide. ORF2 was 477 bp in length, and encoded a 158 aa polypeptide. The frameshift site that initiates the translation of ORF2 was located at nucleotide position 97 in the OAZ2. The analysis of OAZ2 mRNA expression in hierarchical follicles showed that the level of OAZ2 mRNA was higher in the SWF and F2 follicular stages than that in the ovarian stroma (P<0.05). The lowest level of OAZ2 expression was detected in the ovarian stroma. These results suggest that the highly conserved frameshift region plays an important role in sustaining the function of OAZs. Furthermore, the significantly higher level of OAZ2 mRNA in the SWF stage indicates that OAZ2 may be involved in recruiting hierarchical follicles. Our results also suggest that OAZ2 may augment the effects of OAZ1 in follicle development.

  12. Ovarian aging and the activation of the primordial follicle reserve in the long-lived Ames dwarf and the short-lived bGH transgenic mice.

    PubMed

    Saccon, Tatiana D; Moreira, Fabiana; Cruz, Luis A; Mondadori, Rafael G; Fang, Yimin; Barros, Carlos C; Spinel, L; Bartke, A; Masternak, Michal M; Schneider, A

    2017-11-05

    The aim of this study was to evaluate the effect of growth hormone (GH) in the maintenance of the ovarian primordial follicle reserve. Ovaries from 16 mo old GH-deficient Ames Dwarf (df/df) and Normal (N/df) mice were used. A subgroup of df/df and N mice received GH or saline injections for six weeks starting at 14 mo of age. In addition, ovaries from 12 mo old mice overexpressing bovine GH (bGH) and controls were used. df/df mice had higher number of primordial and total follicles than N/df mice (p < 0.05), while GH treatment decreased follicle counts in both genotypes (p < 0.05). In addition, bGH mice had lower number of primordial and total follicles than the controls (p < 0.05). pFoxO3a levels were higher in mice treated with GH and in bGH mice (p < 0.05) when comparing with age match controls. These results indicate that increased circulating GH is associated with a reduced ovarian primordial follicle reserve and increased pFoxO3a content in oocytes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Molecular cloning and expression of prostaglandin F2α receptor isoforms during ovulation in the ovarian follicles of Xenopus laevis.

    PubMed

    Liu, Zhiming; Su, Xiurong; Li, Taiwu; Pan, Daodong; Sena, Johnny; Dhillon, Jasvinder

    2010-11-01

    Prostaglandins F2α levels increase during ovulatory period in Xenopus laevis in response to stimulation by gonadotropins and progesterone. PGF2α exerts its effects on ovulation through interaction with its receptor (FP) in ovaries. Little is known about the characteristics of the FP receptor and its regulation during the ovulatory period in non-mammalian species. In the present study, two isoforms of prostaglandin F receptor (FP A and B) cDNAs were isolated from Xenopus laevis ovarian tissues using reverse transcription-polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). The cDNAs of FP A and FP B were sequenced. In Xenopus laevis ovary, FP A and B mRNA levels were up-regulated during gonadotropin- and progresterone-induced ovulation in vitro. The mRNA level of FP B was higher than that of FP A. Moreover, FP A and FP B mRNA levels were measured in various tissues including eye, liver, lungs, heart, muscle, ovary, and skin. Overall, FP B mRNA level was approximately 10- to 100-fold higher than that of FP A, except in the muscle and skin where FP A mRNA level was comparable to that of FP B. The results suggest that in Xenopus ovarian follicles FP receptors play an important role during gonadotropin- and progesterone-induced ovulation.

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

    PubMed

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

    2012-08-01

    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 [E(2)], 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 E(2) 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. Published by Elsevier Inc.

  15. Cisplatin Induces Overactivation of the Dormant Primordial Follicle through PTEN/AKT/FOXO3a Pathway which Leads to Loss of Ovarian Reserve in Mice

    PubMed Central

    Chang, Eun Mi; Lim, Eunjin; Yoon, Sookyoung; Jeong, Kyungah; Bae, Sijeong; Lee, Dong Ryul; Yoon, Tae Ki

    2015-01-01

    Cisplatin is a first-line chemotherapeutic agent for ovarian cancer that acts by promoting DNA cross links and adduct. However drug resistance and considerable side effects including reproductive toxicity remain a significant challenge. PTEN is well known as a tumor suppressor function which plays a fundamental role in the regulation of the cell cycle, apoptosis and development of cancer. At the same time PTEN has been revealed to be critically important for the maintenance of the primordial follicle pool. In this study, we investigated the role of PTEN/Akt/FOXO3 pathway in cisplatin-induced primordial follicle depletion. Cisplatin induced ovarian failure mouse model was used to evaluate how this pathway involves. In vitro maturation was used for oocyte rescue after cisplatin damage. We found that cisplatin treatment decreased PTEN levels, leading to a subsequent increase in the phosphorylation of key molecules in the pathway. The activation of the PTEN/Akt/FOXO3 pathway cascade increased cytoplasmic translocation of FOXO3a in cisplatin-treated follicles, which in turn increased the pool size of growing follicles, and rapidly depleted the number of dormant follicles. Once activated, the follicles were more prone to apoptosis, and their cumulus cells showed a loss of luteinizing hormone (LH) receptor expression, which leads to failure during final maturation and ovulation. In vitro maturation to rescue oocytes in a cisplatin-treated mouse model resulted in successful maturation and fertilization. This study is the first to show the involvement of the PTEN/Akt/FOXO3 pathway in premature ovarian failure after cisplatin treatment and the possibility of rescue through in vitro maturation. PMID:26656301

  16. Adrenomedullin in rat follicles and corpora lutea: expression, functions and interaction with endothelin-1

    PubMed Central

    2011-01-01

    Background Adrenomedullin (ADM), a novel vasorelaxant peptide, was found in human/rat ovaries. The present study investigated the interaction of ADM and endothelin-1 (ET-1) in follicles and newly formed corpora lutea (CL) and the actions of ADM on progesterone production in CL during pregnancy. Methods The peptide and gene expression level of adrenomedullin in small antral follicles, large antral follicles and CL was studied by real-time RT-PCR and EIA. The effect of ADM treatment on oestradiol production in 5-day follicular culture and on progesterone production from CL of different pregnant stages was measured by EIA. The interaction of ADM and ET-1 in follicles and CL at their gene expression level was studied by real-time RT-PCR. Results In the rat ovary, the gene expression of Adm increased during development from small antral follicles to large antral follicles and CL. In vitro treatment of preantral follicular culture for 5 days with ADM increased oestradiol production but did not affect follicular growth or ovulation rate. The regulation of progesterone production by ADM in CL in culture was pregnancy-stage dependent, inhibitory at early and late pregnancy but stimulatory at mid-pregnancy, which might contribute to the high progesterone production rate of the CL at mid-pregnancy. Moreover, the interaction between ADM and ET-1 at both the production and functional levels indicates that these two vasoactive peptides may form an important local, fine-tuning regulatory system together with LH and prolactin for progesterone production in rat CL. Conclusions As the CL is the major source of progesterone production even after the formation of placenta in rats, ADM may be an important regulator in progesterone production to meet the requirement of pregnancy. PMID:21824440

  17. Ammonia concentrations in different size classes of ovarian follicles of sheep (Ovis aries): Possible mechanisms of accumulation and its effect on oocyte and granulosa cell growth in vitro.

    PubMed

    Nandi, S; Gupta, P S P; Mondal, S

    2016-03-01

    The present study investigated the concentrations and the mechanisms of accumulation of ammonia in different sizes of ovarian follicles and the effect of ammonia on oocyte and granulosa cell growth and functions in vitro with sheep (Ovis aries) as an animal model. The effects of cyclicity, seasonality, phases of the estrous cycle, and seasons (environmental) on ammonia concentrations in follicular fluid were also investigated. The effect of ammonia on in vitro development of oocytes (maturation rate, viability rate, cleavage rate, morulae/blastocysts yield) recovered from different sizes of follicles was examined at the levels of 0, 50, 100, 150, 250, 300, and 500 μM. Same concentrations of ammonia were examined on growth parameters (metabolic activity, viability, cell number increment, monolayer formation, apoptosis rate) and hormone (progesterone, estrogen) secretion activity of granulosa cells in vitro. Results suggested as the follicle size increased, ammonia concentrations decreased. The ammonia concentrations in ovine follicular fluid were found to be 261.5 ± 32.4, 157.7 ± 19.2, and 42.9 ± 8.3 μM, respectively, for small, medium, and large follicles. The corresponding ranges were 290 to 238 μM, 184 to 142 μM, and 70 to 22 μM. The differences were due to more accumulation of fluid, less metabolic activity of granulosa cells, and elevation of protein, potassium, and chloride as the follicle size increased. The seasonality and phases of the estrous cycle did not have any effect on ammonia level in ovarian follicles. Ammonia concentrations in all size classes of follicles examined were significantly reduced in ewes during hot seasons compared to cold seasons and in acyclic animals compared to cyclic ones. Ammonia impaired oocyte development at 300 μM when the oocytes were isolated from small follicles and at 250 μM when the oocytes were isolated from medium and large follicles. In contrast, ammonia caused the negative impact on granulosa cells growth

  18. Effect of Nrf2 on rat ovarian tissues against atrazine-induced anti-oxidative response

    PubMed Central

    Zhao, Fan; Li, Kun; Zhao, Lijing; Liu, Jian; Suo, Qi; Zhao, Jing; Wang, Hebin; Zhao, Shuhua

    2014-01-01

    The environmental persistence and bioaccumulation of herbicide atrazine may pose a significant threat to human health. In this experiment, Wistar rats were treated by 5, 25 and 125 mg·kg-1 atrazine respectively for 28 days, and the oxidative stress responses as well as the activations of Nrf2 signaling pathway in ovarian tissues induced by atrazine were observed. The results showed that after be treated by atrazine, the proportion of atretic follicles in the rat ovary were increased, the contents of NO and MDA in the tissue homogenates were increased, the over-expressed Nrf2 transferred into the nuclei and played an antioxidant role by up-regulated the expression of II phase detoxifying enzymes such as HO1 and NQO1 and the expression of antioxidant enzymes such as CAT, SOD and GSH-PX. PMID:25031697

  19. Involvement of Gαs-proteins in the action of relaxin-like gonad-stimulating substance on starfish ovarian follicle cells.

    PubMed

    Mita, Masatoshi; Haraguchi, Shogo; Watanabe, Miho; Takeshige, Yuki; Yamamoto, Kazutoshi; Tsutsui, Kazuyoshi

    2014-09-01

    Gonad-stimulating substance (GSS) in starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. In breeding season (stage V), GSS stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) by ovarian follicle cells. The hormonal action of GSS is mediated through the activation of its receptor, G-proteins and adenylyl cyclase. It has been reported that GSS fails to induce 1-MeAde and cyclic AMP (cAMP) production in follicle cells of ovaries during oogenesis (stage IV). This study examined the regulatory mechanism how ovarian follicle cells acquire the potential to respond to GSS by producing 1-MeAde and cAMP. Because the failure of GSS action was due to G-proteins of follicle cells, the molecular structures of Gαs, Gαi, Gαq and Gβ were identified in follicle cells of starfish Asterina pectinifera. The cDNA sequences of Gαs, Gαi, Gαq and Gβ consisted of ORFs encoding 379, 354, 353 and 353 amino acids. The expression levels of Gαs were extremely low in follicle cells at stage IV, whereas the mRNA levels increased markedly in stage V. On contrary, the mRNA levels of Gαi were almost constant regardless of stage IV and V. These findings strongly suggest that de novo synthesis of Gαs-proteins is contributed to the action of GSS on follicle cells to produce 1-MeAde and cAMP.

  20. Transcriptomic diversification of developing cumulus and mural granulosa cells in mouse ovarian follicles.

    PubMed

    Wigglesworth, Karen; Lee, Kyung-Bon; Emori, Chihiro; Sugiura, Koji; Eppig, John J

    2015-01-01

    Cumulus cells and mural granulosa cells (MGCs) have functionally distinct roles in antral follicles, and comparison of their transcriptomes at a global and systems level can propel future studies on mechanisms underlying their functional diversity. These cells were isolated from small and large antral follicles before and after stimulation of immature mice with gonadotropins, respectively. Both cell types underwent dramatic transcriptomic changes, and differences between them increased with follicular growth. Although cumulus cells of both stages of follicular development are competent to undergo expansion in vitro, they were otherwise remarkably dissimilar with transcriptomic changes quantitatively equivalent to those of MGCs. Gene ontology analysis revealed that cumulus cells of small follicles were enriched in transcripts generally associated with catalytic components of metabolic processes, while those from large follicles were involved in regulation of metabolism, cell differentiation, and adhesion. Contrast of cumulus cells versus MGCs revealed that cumulus cells were enriched in transcripts associated with metabolism and cell proliferation while MGCs were enriched for transcripts involved in cell signaling and differentiation. In vitro and in vivo models were used to test the hypothesis that higher levels of transcripts in cumulus cells versus MGCs is the result of stimulation by oocyte-derived paracrine factors (ODPFs). Surprisingly ∼48% of transcripts higher in cumulus cells than MGCs were not stimulated by ODPFs. Those stimulated by ODPFs were mainly associated with cell division, mRNA processing, or the catalytic pathways of metabolism, while those not stimulated by ODPFs were associated with regulatory processes such as signaling, transcription, phosphorylation, or the regulation of metabolism.

  1. Unilateral or bilateral vagotomy induces ovulation in both ovaries of rats with polycystic ovarian syndrome

    PubMed Central

    2013-01-01

    Background Injecting estradiol valerate (EV) to pre-pubertal or adult female rat results in effects similar to those observed in women with polycystic ovarian syndrome (PCOS). One of the mechanisms involved in PCOS development is the hyperactivity of the sympathetic nervous system. In EV-induced PCOS rats, the unilateral sectioning of the superior ovarian nerve (SON) restores ovulation of the innervated ovary. This suggests that, in addition to the sympathetic innervation, other neural mechanisms are involved in the development/maintenance of PCOS. The aims of present study were analyze if the vagus nerve is one of the neural pathways participating in PCOS development. Methods Ten-day old rats were injected with EV dissolved in corn oil. At 24-days of age sham-surgery, unilateral, or bilateral sectioning of the vagus nerve (vagotomy) was performed on these rats. The animals were sacrificed at 90–92 days of age, when they presented vaginal estrous preceded by a pro-estrus smear. Results In EV-induced PCOS rats, unilateral or bilateral vagotomy restored ovulation in both ovaries. Follicle-stimulating hormone (FSH) levels in PCOS rats with unilateral or bilateral vagotomy were lower than in control rats. Conclusions This result suggests that in EV-induced PCOS rats the vagus nerve is a neural pathway participating in maintaining PCOS. The vagus nerve innervates the ovaries directly and indirectly through its synapsis in the celiac-superior-mesenteric ganglion, where the somas of neurons originating in the SON are located. Then, it is possible that vagotomy effects in EV-induced PCOS rats may be explained as a lack of communication between the central nervous system and the ovaries. PMID:23866168

  2. 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.

  3. Levels of BMP-6 mRNA in goat ovarian follicles and in vitro effects of BMP-6 on secondary follicle development.

    PubMed

    Frota, Isana M A; Leitão, Cintia C F; Costa, José J N; van den Hurk, Robert; Saraiva, Márcia V A; Figueiredo, José R; Silva, José R V

    2013-08-01

    Expression of BMP-6 mRNA was quantified by real-time polymerase chain reaction (PCR) and the BMP-6 protein was demonstrated by immunohistochemistry in the primordial, primary, secondary, small and large antral follicles of goat. Furthermore, the influence of BMP-6 on increase in diameter, antrum formation and expression of BMP-6 and FSH-R in in vitro cultured secondary follicles was studied. Therefore, goat primordial, primary and secondary follicles, as well as small and large antral follicles were obtained and the mRNA levels of BMP-6 were quantified by PCR in real time. Expression of BMP-6 protein in goat follicles was demonstrated by immunohistochemistry. The influence of BMP-6 in the presence or absence of follicle-stimulating hormone (FSH) on both the development of secondary follicles and the expression of mRNA for BMP-6 and FSH-R was evaluated after 6 days of culture. Furthermore, the follicular diameter and the formation of the antrum were evaluated before and after 6 days of culture and compared by Kruskal-Wallis and chi-squared tests (P < 0.05), respectively. The results show that the level of mRNA for BMP-6 in primary and secondary follicles was significantly higher than in the primordial follicles (P < 0.05). Similar levels of BMP-6 mRNA were observed in cumulus-oocyte complexes and mural granulosa/theca cells from small and large antral follicles, respectively. BMP-6 protein was expressed in oocytes of all categories of follicles and in granulosa cells from secondary follicles onwards. Addition of BMP-6 to the culture medium increased the diameter of secondary follicles mainly by antrum formation after 6 days' culture, in the presence or absence of FSH (P < 0.05). Furthermore, addition of FSH resulted in increased levels of BMP-6 mRNA in these follicles (P < 0.05). Simultaneous administration of FSH and BMP-6 enhanced the levels of FSH receptor (FSH-R) mRNA (P < 0.05). It is concluded that BMP-6 mRNA is increased during transition from primordial to

  4. Desmocollin 3 mediates follicle stimulating hormone-induced ovarian epithelial cancer cell proliferation by activating the EGFR/Akt signaling pathway.

    PubMed

    Yang, Xiao; Wang, Jing; Li, Wen-Ping; Jin, Zhi-Jun; Liu, Xiao-Jun

    2015-01-01

    Follicle-stimulating hormone (FSH) is associated with the pathogenesis of ovarian cancer. We sought to explore whether desmocollin 3 (Dsc3) mediates FSH-induced ovarian epithelial cancer cell proliferation and whether the EGFR/Akt signaling pathway may be involved in this process. Dsc3 positivity in ovarian tissue specimens from 72 patients was assessed by immunohistochemistry. The positive expression rates of Dsc3 were similar in ovarian cancer tissues (24/31:77.4%) and borderline ovarian tumor tissues (18/22:81.8%) (P>0.05), but were significantly higher in these cancerous tissues than in benign ovarian cyst tissues (3/19:15.8%) (P<0.05). Consistently, the expression of Dsc3 in four out of five ovarian cancer cells (HO8910, Skov3ip, Skov and Hey cells, but not ES-2 and in borderline ovarian MCV152 tumor cells was higher than in the immortalized ovarian epithelial cell line, Moody. FSH up-regulated the expression of Dsc3 and EGFR in a dose- and time-dependent manner. Furthermore, a converse relationship between the expression of Dsc3, EFGR and PI3K/Akt signaling was elucidated using RNA interference and PI3K/Akt inhibitor in the absence and presence of FSH. A role for these proteins in FSH-induced cell proliferation was verified, highlighting their interdependence in mediating ovarian cancer cell function. These results suggest that Dsc3 can mediate FSH-induced ovarian cancer cell proliferation by activating the EGFR/Akt signaling pathway.

  5. Pregnancy after ovarian superovulation by transdermal delivery of follicle-stimulating hormone.

    PubMed

    Zech, Nicolas H; Murtinger, Maximilian; Uher, Petr

    2011-06-30

    Because of its size of 32 kDa and physicochemical properties, urinary FSH cannot permeate intact skin. We report the first pregnancy after laser microporation and transdermal delivery of FSH for ovarian superovulation as a substitute for SC or IM injections. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. Molecular cloning of the SMAD4 gene and its mRNA expression analysis in ovarian follicles of the Yangzhou goose (Anser cygnoides).

    PubMed

    Huang, Z; Yuan, X; Wang, M; Wu, N; Song, Y; Chen, Y; Zhang, Y; Xu, Q; Chen, G; Zhao, W

    2016-08-01

    Mothers against decapentaplegic homolog 4 (SMAD4) is an important protein in animal reproduction. It plays pivotal roles in cellular pathways, including apoptosis. The expression profile of the SMAD4 gene in goose ovarian follicles has not been reported. In this study, the SMAD4 coding sequence was cloned from the Yangzhou goose. A phylogenetic analysis was performed and mRNA expression was examined in various tissues using quantitative real-time PCR. An alternative splice form of SMAD4, SMAD4-b having 1656 bp, was identified. SMAD4-a mRNA was widely expressed in various healthy tissues, whereas SMAD4-b was very weakly expressed. SMAD4 mRNA in the ovary and oviduct was significantly higher than that in the pituitary and hypothalamus. SMAD4 mRNA expression analysis in hierarchical follicles showed that the level of SMAD4 mRNA was higher in large white follicles and post-ovulatory follicles than in the other follicles. The results indicate that SMAD4 might be involved in the recruitment of hierarchical follicles.

  7. Molecular cloning and mRNA expression analysis of antizyme inhibitor 1 in the ovarian follicles of the Sichuan white goose.

    PubMed

    Ma, Rong; Jiang, Dongmei; Kang, Bo; Bai, Lin; He, Hui; Chen, Ziyu; Yi, Zhixin

    2015-08-15

    Antizyme inhibitor 1 (Azin1) plays critical roles in various cellular pathways, including ornithine decarboxylase regulation, polyamine anabolism and uptake and cell proliferation. However, the molecular characteristics of the AZIN1 gene and its expression profile in goose tissues and ovarian follicles have not been reported. In this study, the AZIN1 cDNA of the Sichuan white goose (Anser cygnoides) was cloned, and analyzed for its phylogenetic and physiochemical properties. The expression profile of AZIN1 mRNA in geese tissues and ovarian follicles were examined using quantitative real-time PCR. The results showed that the open reading frame of the AZIN1 cDNA is 1,353 bp in length, encoding a 450 amino acid protein with a molecular weight of 50 kDa. Out of all tissues examined, AZIN1 expression was highest in the adrenal gland and lowest in breast muscle. There was also a high expression of AZIN1 in the cerebellum and isthmus of oviduct. With follicular development, AZIN1 gene expression gradually increased, and its expression in F1 was significantly higher than in F5 (P<0.05). AZIN1 expression was also significantly higher in the POF1 than in the other follicles (P<0.05), and there was a low mRNA expression of AZIN1 in atretic follicles. The results of AZIN1 expression profiling in ovarian follicles suggest that AZIN1 may play an important role in the progression of follicular development, potentially through regulating polyamine levels.

  8. HemoHIM improves ovarian morphology and decreases expression of nerve growth factor in rats with steroid-induced polycystic ovaries.

    PubMed

    Kim, Sung Ho; Lee, Hae June; Kim, Joong Sun; Moon, Changjong; Kim, Jong Choon; Bae, Chun Sik; Park, Hae Ran; Jung, Uhee; Jo, Sung Kee

    2009-12-01

    Estradiol valerate (EV)-induced polycystic ovaries (PCOs) in rats cause the anovulation and cystic ovarian morphology. We investigated whether treatment with HemoHIM influences the ovarian morphology and the expression of nerve growth factor (NGF) in an EV-induced PCO rat model. PCO was induced by a single intramuscular injection of EV (4 mg, dissolved in sesame oil) in adult cycling rats. HemoHIM was either administered orally (100 mg/kg of body weight/day) for 35 consecutive days or injected intraperitoneally (50 mg/kg of body weight) every other day after EV injection. Ovarian morphology was almost normalized, and NGF was normalized in the PCO + HemoHIM group. HemoHIM lowered the high numbers of antral follicles and increased the number of corpora lutea in PCOs. The results are consistent with a beneficial effect of HemoHIM in the prevention and treatment of PCO syndrome.

  9. 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.

  10. Comparison of transabdominal and transvaginal pelvic ultrasonography for ovarian follicle assessment in in vitro fertilisation.

    PubMed

    O'Shea, R T; Forbes, K L; Scopacasa, L; Jones, W R

    1988-01-01

    The emergence of transvaginal ultrasound-guided oocyte retrieval provided an opportunity to review and improve the traditional transabdominal ultrasonic approach for follicular tracking in in vitro fertilisation (IVF). This technique requires a full bladder, which may cause extreme discomfort. Hence, to provide both effective and comfortable monitoring, we instituted a study comparing transabdominal and transvaginal ultrasonography, with regard to patient preference and follicular number, size and dominance, in patients undergoing IVF and gamete intrafallopian transfer (GIFT). Commencing usually on day 9 of the treatment cycle, 45 patients were scanned on 55 occasions, initially abdominally (with a full bladder) and subsequently vaginally, using a transvaginal 7.5 MHz sector transducer. Follicular number was identical in 78% of cases, with the majority of the remaining patients showing an extra follicle on vaginal assessment. There were no significant differences in overall or dominant follicular diameters with either technique. Overall, 85% unashamedly preferred the vaginal approach. We believe that tracking follicular development in IVF treatment cycles is efficient and popular using the vaginal sector transducer and accordingly have ceased all assessments using the abdominal probe. Subsequently, 450 follicle scans have been performed with virtually the unanimous approval of our patients.

  11. Dose dependent effect of resveratrol in preventing cisplatin-induced ovarian damage in rats: An experimental study.

    PubMed

    Atli, Mine; Engin-Ustun, Yaprak; Tokmak, Aytekin; Caydere, Muzaffer; Hucumenoglu, Sema; Topcuoglu, Canan

    2017-09-01

    This study aimed to evaluate the effect of resveratrol in preventing cisplatin (CP) induced ovarian damage in rats. Twenty-eight female Wistar albino rats were separated into four groups. No medication was given to group 1. Over the 21-day study period, low-dose resveratrol was given to group 2, high-dose resveratrol was given to group 3, and saline was administered to group 4. On the 15th day of medication, all groups except for group 1 were treated with a single dose of CP. Serum levels of anti-Mullerian hormone (AMH) were tested at baseline and on the 15th and 21st days. All rats underwent oophorectomy one week after CP application. Primordial, primary, secondary, and tertiary follicles were counted microscopically. No significant difference was observed among the groups in mean AMH levels according to follow-up time. The numbers of primary and primordial follicles were statistically significantly higher in group 2 than in group 4 (p<0.05). The number of tertiary follicles was statistically significantly higher in group 1 than in groups 3 and 4 (p<0.05), but it was not statistically significantly different than in group 2. Resveratrol, particularly at low-doses, can prevent CP induced ovarian damage by maintaining the numbers of primordial and primary follicles. Further studies are needed to study the effect of resveratrol on human ovaries. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  12. Concentration of progesterone during the development of the ovulatory follicle: II. Ovarian and uterine responses.

    PubMed

    Cerri, R L A; Chebel, R C; Rivera, F; Narciso, C D; Oliveira, R A; Amstalden, M; Baez-Sandoval, G M; Oliveira, L J; Thatcher, W W; Santos, J E P

    2011-07-01

    Two experiments evaluated the influence of altering the concentrations of progesterone during the development of the ovulatory follicle on the composition of the follicular fluid, circulating LH and PGF(2α) metabolite (PGFM), and expression of endometrial progesterone receptor and estrogen receptor-α. In both experiments, the estrous cycles were presynchronized (GnRH and progesterone insert followed by insert removal and PGF(2α) 7 d later, and GnRH after 48 h) and cows were then enrolled in 1 of 2 treatments 7 d later (study d -16): high progesterone (HP) or low progesterone (LP). In experiment 1 (n=19), cows had their estrous cycle synchronized starting on study d -9 (GnRH and progesterone insert on d -9, and insert removal and PGF(2α) on d -2). In experiment 2 (n=25), cows were submitted to the same synchronization protocol as in experiment 1, but had ovulation induced with GnRH on study d 0. In experiment 1, plasma was sampled on d -4 and analyzed for concentrations of LH; the dominant follicle was aspirated on d 0 and the fluid analyzed for concentrations of progesterone, estradiol, and free and total IGF-1. In experiment 2, follicular development and concentrations of progesterone and estradiol in plasma were evaluated until study d 16. Uterine biopsies were collected on d 12 and 16 for progesterone receptor and estrogen receptor-α protein abundance. An estradiol/oxytocin challenge for PGFM measurements in plasma was performed on d 16. In experiments 1 and 2, LP cows had lower plasma concentrations of progesterone and greater concentrations of estradiol, and had larger ovulatory follicle diameter (20.4 vs. 17.2mm) at the end of the synchronization protocol than HP cows. Concentration of LH tended to be greater for LP than HP cows (0.98 vs. 0.84 ng/mL). The dominant follicle of LP cows had greater concentration of estradiol (387.5 vs. 330.9 ng/mL) and a lower concentration of total IGF-1 (40.9 vs. 51.7 ng/mL) than that of HP cows. In experiment 2

  13. 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.

    PubMed

    Echternkamp, S E; Aad, P Y; Eborn, D R; Spicer, L J

    2012-07-01

    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 were to 1) assess relationships among aromatase (CYP19A1), IGF-1 (IGF1), IGF-2 receptor (IGF2R), and FSH receptor (FSHR) mRNA expression in small (≤5 mm) antral follicles and 2) determine their association with increased numbers of developing follicles in ovaries of Twinner females. Ovaries were collected from mature, cyclic (d 3 to 6) Twinner (n = 11), and Control (n = 12) cows at slaughter and pieces of cortical tissue were fixed and embedded in paraffin. Expression of mRNA was evaluated by in situ hybridization using (35)S-UTP-labeled antisense and sense probes for CYP19A1, FSHR, IGF1, and IGF2R mRNA. Silver grain density was quantified within the granulosa and theca cells of individual follicles (2 to 7 follicles/cow) by Bioquant image analysis. Follicles of Twinners tended to be smaller in diameter than Controls (1.9 ± 0.1 vs. 2.3 ± 0.1 mm; P = 0.08), but thickness of granulosa layer did not differ (P > 0.1) by genotype. Relative abundance of CYP19A1 (P < 0.01) and FSHR (P < 0.05) mRNA was greater in granulosa cells of Twinners vs. Controls, respectively, whereas IGF2R mRNA expression was less in both granulosa (P < 0.01) and theca (P < 0.05) cells in follicles of Twinners vs. Controls, respectively. Abundance of CYP19A1 mRNA in granulosa cells was correlated negatively with IGF2R mRNA expression in both granulosa (r = -0.33; P < 0.01) and theca (r = -0.21; P = 0.05) cells. Expression of IGF1 mRNA was primarily in granulosa cells, including cumulus cells, and its expression did not differ between Twinners vs. Controls (P > 0.10). Detected increases in CYP19A1 and FSHR, but not IGF1, mRNA expression along with decreases in IGF2R mRNA expression in individual

  14. A regulatory role of androgen in ovarian steroidogenesis by rat granulosa cells.

    PubMed

    Hasegawa, Toru; Kamada, Yasuhiko; Hosoya, Takeshi; Fujita, Shiho; Nishiyama, Yuki; Iwata, Nahoko; Hiramatsu, Yuji; Otsuka, Fumio

    2017-09-01

    Excess androgen and insulin-like growth factor (IGF)-I in the ovarian follicle has been suggested to be involved in the pathophysiology of polycystic ovary syndrome (PCOS). Here we investigated the impact of androgen and IGF-I on the regulatory mechanism of ovarian steroidogenesis using rat primary granulosa cells. It was revealed that androgen treatment with dihydrotestosterone (DHT) amplified progesterone synthesis in the presence of FSH and IGF-I, whereas it had no significant effect on estrogen synthesis by rat granulosa cells. In accordance with the effects of androgen on steroidogenesis, DHT enhanced the expression of progesterogenic factors and enzymes, including StAR, P450scc and 3βHSD, and cellular cAMP synthesis induced by FSH and IGF-I. Of note, treatment with DHT and IGF-I suppressed Smad1/5/8 phosphorylation and transcription of the BMP target gene Id-1, suggesting that androgen and IGF-I counteract BMP signaling that inhibits FSH-induced progesterone synthesis in rat granulosa cells. DHT was revealed to suppress the expression of BMP-6 receptors, consisting of ALK-2, ALK-6 and ActRII, while it increased the expression of inhibitory Smads in rat granulosa cells. In addition, IGF-I treatment upregulated androgen receptor (AR) expression and DHT treatment suppressed IGF-I receptor expression on rat granulosa cells. Collectively, the results indicate that androgen and IGF-I mutually interact and accelerate progesterone production, at least in part, by regulating endogenous BMP signaling in rat granulosa cells. Cooperative effects of androgen and IGF-I counteract endogenous BMP-6 activity in rat granulosa cells, which is likely to be functionally linked to the steroidogenic property shown in the PCOS ovary. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Morphological evidence of apoptosis and the prevalence of apoptotic versus mitotic cells in the membrana granulosa of ovarian follicles during spontaneous and induced atresia in ewes.

    PubMed

    Jolly, P D; Smith, P R; Heath, D A; Hudson, N L; Lun, S; Still, L A; Watts, C H; McNatty, K P

    1997-04-01

    Apoptosis is a process by which granulosa cells are thought to be deleted during ovarian follicular atresia. The aims of the present studies, using sheep as the experimental model, were to determine 1) whether morphological changes in cells composing the membrana granulosa during the process of atresia conformed with the general criteria of apoptotic cell death as assessed using tissue sections stained with hematoxylin and eosin; 2) whether cells classified as apoptotic on the basis of their morphology contained fragmented DNA using an in situ 3' end-labeling technique; and 3) the degree of apoptosis and mitosis within the granulosa cell populations of large antral follicles (> or = 3 mm in diameter) during both spontaneous and experimentally induced atresia using stereological methods. The results showed that most degenerate granulosa cells in follicles undergoing atresia display the morphological characteristics of apoptosis, suggesting that this is the most common pathway of cell deletion. Typical features were cells containing nuclei with marginated chromatin; cells with a single small densely staining nucleus (pyknotic appearance); cells with multiple smaller, densely staining nuclear fragments; and densely staining membrane-bound bodies (apoptotic bodies) either singly or in clusters. Cells with morphological features more typical of oncosis or necrosis were sometimes observed, but mainly during the later stages of atresia. All cells classified as apoptotic on the basis of morphological criteria contained fragmented DNA as measured by 3' end-labeling. Apoptotic bodies and/or cells were found in all follicles examined, including those classified as healthy. The overall prevalence of apoptotic cells plus apoptotic bodies expressed as a percentage of the total granulosa cell number per follicle varied from 0.02% to 0.20% in healthy follicles, varied from 0.21% to 2.00% in follicles in early (primary) atresia, and was > 2.0% in follicles in later (secondary

  16. Ovarian Toxicity in Female Rats after Oral Administration of Melamine or Melamine and Cyanuric Acid.

    PubMed

    Sun, Jiarui; Zhang, Xinchen; Cao, Yinan; Zhao, Qiling; Bao, Endong; Lv, Yingjun

    2016-01-01

    Although the toxicity of melamine to the kidneys and testes is well known, few studies have investigated the effects of melamine on female reproductive organs. Therefore, this study explores the effects of oral administration melamine or melamine and cyanuric acid for 28 days on the ovaries of female rats. Rats that were exposed to the mixture exhibited reduced ovarian and uterine weights, a shorter estrous cycle, and reduced serum estrogen and progesterone levels compared to rats that were exposed to melamine and control rats. Furthermore, morphological analysis revealed pathological changes in the ovaries of rats exposed to melamine or the mixture, such as more atretic follicles and necrosis of oocytes and granulosa cells. TUNEL staining revealed that the exposed groups had a higher proportion of TUNEL-positive granulosa cells than the control group, and the mRNA expressions of SOD1, GPX1, GPX2, P450scc, 17β-HSD I, and 17β-HSD II were reduced in the exposure groups compared with the control group. These results indicated that exposure to melamine alone or to the melamine-cyanuric acid mixture could damage the ovaries in rats.

  17. Ovarian Toxicity in Female Rats after Oral Administration of Melamine or Melamine and Cyanuric Acid

    PubMed Central

    Sun, Jiarui; Zhang, Xinchen; Cao, Yinan; Zhao, Qiling; Bao, Endong; Lv, Yingjun

    2016-01-01

    Although the toxicity of melamine to the kidneys and testes is well known, few studies have investigated the effects of melamine on female reproductive organs. Therefore, this study explores the effects of oral administration melamine or melamine and cyanuric acid for 28 days on the ovaries of female rats. Rats that were exposed to the mixture exhibited reduced ovarian and uterine weights, a shorter estrous cycle, and reduced serum estrogen and progesterone levels compared to rats that were exposed to melamine and control rats. Furthermore, morphological analysis revealed pathological changes in the ovaries of rats exposed to melamine or the mixture, such as more atretic follicles and necrosis of oocytes and granulosa cells. TUNEL staining revealed that the exposed groups had a higher proportion of TUNEL-positive granulosa cells than the control group, and the mRNA expressions of SOD1, GPX1, GPX2, P450scc, 17β-HSD I, and 17β-HSD II were reduced in the exposure groups compared with the control group. These results indicated that exposure to melamine alone or to the melamine-cyanuric acid mixture could damage the ovaries in rats. PMID:26866683

  18. Serum levels of reproductive hormones and ultrasonographic monitoring of ovarian follicles in female cloned dogs.

    PubMed

    Hong, So Gun; Oh, Hyun Ju; Park, Jung Eun; Kang, Jung Taek; Kim, Min Jung; Yoon, Jung Hee; Chang, Jin Hwa; Kim, Min Kyu; Jang, Goo; Lee, Byeong Chun

    2010-01-01

    This study investigated the reproductive characteristics in 2 cloned female beagle dogs (Clones A and B) during proestrus and early estrus. The pre-ovulatory estradiol peak occurred 2 days before the pre-ovulatory luteinizing hormone (LH) surge, while the follicle stimulating hormone surge started concomitantly with the LH surge in both cloned dogs. Serum progesterone levels increased during proestrus and estrus and its concentration on the day of the LH surge was 3.59 and 2.71 ng/ml in Clones A and B, respectively. Gradual follicular growth was observed by ultrasonography during proestrus. Although the numbers are limited, these cloned female dogs showed normal ranges of reproductive hormone levels and follicular changes during proestrous and early estrous stages of the cycle.

  19. Quality of Oocytes Derived from Vitrified Ovarian Follicles Cultured in Two- and Three-Dimensional Culture System in the Presence and Absence of Kit Ligand.

    PubMed

    Abdi, Shabnam; Salehnia, Mojdeh; Hosseinkhani, Saman

    2016-08-01

    The aim of this study was to evaluate the effects of Kit Ligand (KL) on the growth of vitrified follicle, oocyte quality, and embryo development in two- and three-dimensional culture systems. Vitrified and nonvitrified mouse whole ovaries were cultured for 1 week, then their isolated preantral follicles were cultured for 12 days in two- or three-dimensional culture systems in the presence and absence of KL. The growth and diameter of follicles, maturation of oocytes, and hormonal level were assessed. Finally, embryo developmental rate and oocytes reactive oxygen species (ROS) levels, adenosine triphosphate (ATP) content, and distribution of mitochondria were examined. The results showed the maturation and survival rates and steroidogenesis of follicles were significantly higher not only in the three-dimensional culture system but also in the presence of KL (p < 0.001). The embryo development, ATP content, and ROS level of collected oocytes were not significantly different in all groups. Mitochondrial distribution within the oocyte was similar in all groups of the study. Our results demonstrated that KL supplementation during follicular culture in two- and three-dimensional culture systems appeared to significantly increase follicular function and development. The ovarian vitrification had no harmful effect on the steroidogenesis, growth, and maturation of follicles.

  20. [Effects of acupuncture on PI3K/Akt/mTOR signaling pathway in rats with premature ovarian failure].

    PubMed

    Zhang, Yimin; Yu, Bin; Chen, Jia; Zhao, Zhisheng; Wang Jiali; Huang, Fasen; Lin, Yuee; Wang, Mengwei; Zhang, Yupei; Wei, Bo

    2015-01-01

    To explore the effects of acupuncture and medication on PI3K/Akt/mTOR signaling pathway in rats with premature ovarian failure. Ten of fifty SPF-grade female SD rats were randomly selected into a normal group, and the remaining 40 rats were treated with intraperitoneal injection of cyclophospha mide (30 mg/kg) for consecutive 5 days to establish rat model of premature ovarian failure. Thirty five successful rat models were randomly divided into a model group (9 cases), a medication group (9 cases), an acupuncture group A (9 cases) and an acupuncture group B (8 cases). The rats in the model group and normal group did not receive any treatment. The rats in the medication group were treated with intragastric administration of diethylstil bestrol, once a day. The rats in the acupuncture group A and acupuncture group B were respectively treated with acupuncture at different acupoints, twice a day. All the treatment was given for 4 weeks. After the treatment, enzyme-linked immunosorbent assay (ELISA) was applied to test the levels of estradiol (E2), progesterone (P), follicle stimulating hormone (FSH) and luteotropic hormone (LH). The ovarian tissue sample was processed with hematoxylin eosin (HE) staining as well as RNA and protein extraction to test the mRNA expression of estrogen receptor alpha (ERalpha), estrogen receptor beta (ERP), phosphatidylinositol 3-kinase/serine/threonine kinase (PI3K), protein kinase B (Akt) and mammalian target of rapamycin (mTOR). High-dose short-term in- tervention of cyclophosphamide could establish rat model of premature ovarian failure with a successful rate of 87.5%. Compared with the normal group, the vaginal smear in the model group was featured with signs of estro gen deficiency, early-follicle reduction, structural damage to the follicle, and reducing number of mature follicles; the level of E2 was significantly reduced (P<0.05), levels of P, FSH and ILH were increased (all P<0.05), and mRNA expression of estrogen-related ERP3, PI

  1. [Effects of follicle stimulating hormone on proliferation, apoptosis, migration and invasion of ovarian carcinoma cells: an in vitro experiment].

    PubMed

    Huang, Yan; Zhao, Yu-Qing; Su, Min; Gao, Shu-Jun; Jin, Hong-Yan; Feng, You-Ji

    2007-09-18

    To investigate the effects of follicle stimulating hormone (FSH) on the proliferation, apoptosis, migration and invasion of ovarian cancer cells. Ovarian cancer cells of the lines SKOV-3 and ES-2 were cultured, and treated by FSH of the concentrations of 10, 20, 40, 80, and 160 mU/ml for 48 h or 24 h respectively. The cells without FSH treatment were used as control cells. The proliferative effects of the cells were detected by MTT colorimetry. The apoptosis and cell cycle were examined by flow cytometry. The matrix metalloproteinases-2 (MMP-2) protein levels in the supernatant were determined by zymography. The cytoplasm levels of MMP-2 protein in cells were tested by Western blotting. RT-PCR was used to detect the expression of MMP-2 mRNA in cells. The migration and invasion of the cells were examined. The a values of the SKOV-3 treated with FSH of the concentrations of 10 - 160 mU/ml were all significantly higher than those without FSH treatment (all P < 0.01). The apoptosis rates of the SKOV-3 treated with FSH of the concentrations 10 - 160 mU/ml were (0.94 +/- 0.06)%, (0.71 +/- 0.03)%, (0.22 +/- 0.02)%, (0.32 +/- 0.02)%, and (0.55 +/- 0.05)% respectively, all significantly lower than those without FSH treatment [(1.30 +/- 0.10)%, all P < 0.01]. After treatment with FSH of the concentrations 40 to 160 mU/ml the percentages of the SKOV-3 at the stage G(0)/G(1) gradually decreased and the cells at the stage S gradually increased compared with the control groups (all P < 0.05). The MMP-2 mRNA and protein expression levels of the SKOV-3 increased with the concentration increase of FSH (P < 0.05 or P < 0.01). Boyden chamber invasive assay showed that the numbers of the SKOV-3 that penetrated the basement membrane were (157 +/- 20)/hp (x200), significantly higher than those of the control groups [(27 +/- 9)/hp, P < 0.01]. Scarification test showed that the distance between scratches of the FSH-treated SKOV-3 cells was significantly shorter than that of the control

  2. Ovarian hyperstimulation syndrome due to follicle-stimulating hormone-secreting pituitary adenomas.

    PubMed

    Caretto, Amelia; Lanzi, Roberto; Piani, Cecilia; Molgora, Michela; Mortini, Pietro; Losa, Marco

    2017-07-04

    Gonadotroph adenomas are pituitary adenomas with inefficient and variable secretory characteristics, that is why they are usually considered as a subgroup of nonfunctioning pituitary adenomas (NFPA) and are recognized only at immunohistochemistry. When gonadotroph adenomas secrete active hormones, they may cause spontaneous ovarian hyperstimulation syndrome (OHSS) in premenopausal women. Aim of our study is to describe three women with OHSS diagnosed before the removal of the adenoma and to calculate the prevalence of OHSS in premenopausal women with a clinical diagnosis of NFPA. We reviewed clinical records of premenopausal women that underwent neurosurgery for NFPA at our centre between 1993 and 2014. OHSS was diagnosed in patients with high levels of FSH, suppressed LH, hyperestrogenism, abdominal symptoms, polymenorrhea, enlarged ovaries with cysts or previous surgery for ovarian cysts. 171 women were included into the study; 62 (36.6%) had a gonadotroph adenoma diagnosed at immunohistochemistry. Two patients were retrospectively diagnosed as having OHSS due to gonadotroph adenoma and three had OHSS diagnosed before neurosurgery. The prevalence of OHSS was 2.9% in the overall group of patients with NFPA and 8.1% among patients with a gonadotroph adenoma detected at immunohistochemistry. Frequency of OHSS due to a gonadotroph adenoma is not negligible. Increased awareness of the characteristic clinical and hormonal picture should permit an early detection of this condition in premenopausal women with a pituitary adenoma.

  3. Expression of factors involved in apoptosis and cell survival is correlated with enzymes synthesizing lysophosphatidic acid and its receptors in granulosa cells originating from different types of bovine ovarian follicles.

    PubMed

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

    2017-09-06

    Lysophosphatidic acid (LPA) regulates reproductive processes in the cow. Ovarian granulosa cells play a pivotal role in follicle growth and development. Nevertheless, the role of LPA in the local regulation of granulosa cell function in different follicle categories in the bovine ovary has not been investigated. Ovarian follicles were divided into healthy, transitional and atretic categories. The expression levels of AX, PLA2, LPARs and factors involved in apoptosis and cell survival processes in granulosa cells in different types of follicles were measured by real-time PCR. The correlations between the expression levels of AX, PLA2, LPARs and the examined factors were measured. The immunolocalization of AX, PLA2 and LPARs in different ovarian follicles was examined by immunohistochemistry. Statistical analyses were conducted in GraphPad using a one-way ANOVA followed by the Kruskal-Wallis multiple comparison test or a correlation analysis followed by Pearson's test. The expression levels of AX, PLA2 and LPARs, with the major role of LPAR2 and PLA2, were found in the granulosa cells originating from different follicle types. The expression levels of the factors involved in cell apoptosis (TNFα and its receptors, FAS, FASL, CASP3, CASP8, β-glycan, and DRAK2) were significantly higher in the granulosa cells of the atretic follicles compared to the healthy follicles. A number of correlations between LPARs, AX, PLA2 and factors associated with apoptosis were observed in the atretic but not in the healthy follicles. A greater expression of the factors involved in differentiation and proliferation in the granulosa cells (DICE1 and SOX2) was found in the healthy follicles in comparison with the atretic. A number of correlations between LPARs, AX, PLA2 and the factors associated with cell survival were observed in the healthy but not in the atretic follicles. Granulosa cells are the target of LPA action and the source of LPA synthesis in the bovine ovarian follicle. We

  4. Inhibition of follicle-stimulating hormone-induced preovulatory follicles in rats treated with a nonsteroidal negative allosteric modulator of follicle-stimulating hormone receptor.

    PubMed

    Dias, James A; Campo, Brice; Weaver, Barbara A; Watts, Julie; Kluetzman, Kerri; Thomas, Richard M; Bonnet, Béatrice; Mutel, Vincent; Poli, Sonia M

    2014-01-01

    We previously described a negative allosteric modulator (NAM) of FSHR (ADX61623) that blocked FSH-induced cAMP and progesterone production but did not block estradiol production. That FSHR NAM did not affect FSH-induced preovulatory follicle development as evidenced by the lack of an effect on the number of FSH-dependent oocytes found in the ampullae following ovulation with hCG. A goal is the development of a nonsteroidal contraceptive. Toward this end, a high-throughput screen using human FSHR identified an additional nonsteroidal small molecule (ADX68692). Although ADX68692 behaved like ADX61623 in inhibiting production of cAMP and progesterone, it also inhibited FSH-induced estradiol in an in vitro rat granulosa primary cell culture bioassay. When immature, noncycling female rats were injected subcutaneously or by oral dosing prior to exogenous FSH administration, it was found that ADX68692 decreased the number of oocytes recovered from the ampullae. The estrous cycles of mature female rats were disrupted by administration by oral gavage of 25 mg/kg and 10 mg/kg ADX68692. In the highest dose tested (25 mg/kg), 55% of animals cohabited with mature males had implantation sites compared to 33% in the 10 mg/kg group and 77% in the control group. A surprising finding was that a structural analog ADX68693, while effectively blocking progesterone production with similar efficacy as ADX68692, did not block estrogen production and despite better oral availability did not decrease the number of oocytes found in the ampullae even when used at 100 mg/kg. These data demonstrate that because of biased antagonism of the FSHR, nonsteroidal contraception requires that both arms of the FSHR steroidogenic pathway must be effectively blocked, particularly estrogen biosynthesis. Thus, a corollary to these findings is that it seems reasonable to propose that the estrogen-dependent diseases such as endometriosis may benefit from inhibition of FSH action at the ovary using the FSHR

  5. Monitoring preantral follicle survival and growth in bovine ovarian biopsies by repeated use of neutral red and cultured in vitro under low and high oxygen tension.

    PubMed

    Jorssen, Ellen P A; Langbeen, An; Fransen, Erik; Martinez, Emilia L; Leroy, Jo L M R; Bols, Peter E J

    2014-08-01

    The development and optimization of preantral follicle culture methods are crucial in fertility preservation strategies. As preantral follicle dynamics are usually assessed by various invasive techniques, the need for alternative noninvasive evaluation tools exists. Recently, neutral red (NR) was put forward to visualize preantral follicles in situ within ovarian cortical fragments. However, intense light exposure of NR-stained tissues can lead to cell death because of increased reactive oxygen species production, which is also associated with elevated oxygen tension. Therefore, we hypothesize that after repeated NR staining, follicle viability and dynamics can be altered by changes in oxygen tension. In the present study, we aim (1) to determine whether NR can be used to repeatedly assess follicular growth, activation, and viability and (2) to assess the effect of a low (5% O2) or high (20% O2) oxygen tension on the viability, growth, and stage transition of preantral follicles cultured in vitro by means of repeated NR staining. Cortical slices (n = 132; six replicates) from bovine ovaries were incubated for 3 hours at 37 °C in a Leibovitz medium with 50 μg/mL NR. NR-stained follicles were evaluated in situ for follicle diameter and morphology. Next, cortical fragments were individually cultured in McCoy's 5A medium for 6 days at 37 °C, 5% CO2, and 5% or 20% O2. On Days 4 and 6, the fragments were restained by adding NR to the McCoy's medium and follicles were reassessed. In both low and high oxygen tension treatment groups, approximately 70% of the initial follicles survived a 6-day in vitro culture, but no significant difference in follicle survival on Day 4 or 6 could be observed compared with Day 0 (P > 0.05). A significant decrease in the number of primordial and increase in primary and secondary follicles was observed within 4 days of culture (P < 0.001). In addition, a significant increase of the mean follicle diameter in NR-stained follicles was

  6. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling

    PubMed Central

    Abedini, Atefeh; Zamberlam, Gustavo; Lapointe, Evelyne; Tourigny, Catherine; Boyer, Alexandre; Paquet, Marilène; Hayashi, Kanako; Honda, Hiroaki; Kikuchi, Akira; Price, Christopher; Boerboom, Derek

    2015-01-01

    Whereas the roles of the canonical wingless-type MMTV (mouse mammary tumor virus) integration site family (WNT) signaling pathway in the regulation of ovarian follicle growth and steroidogenesis are now established, noncanonical WNT signaling in the ovary has been largely overlooked. Noncanonical WNTs, including WNT5a and WNT11, are expressed in granulosa cells (GCs) and are differentially regulated throughout follicle development, but their physiologic roles remain unknown. Using conditional gene targeting, we found that GC-specific inactivation of Wnt5a (but not Wnt11) results in the female subfertility associated with increased follicular atresia and decreased rates of ovulation. Microarray analyses have revealed that WNT5a acts to down-regulate the expression of FSH-responsive genes in vitro, and corresponding increases in the expression of these genes have been found in the GCs of conditional knockout mice. Unexpectedly, we found that WNT5a regulates its target genes not by signaling via the WNT/Ca2+ or planar cell polarity pathways, but rather by inhibiting the canonical pathway, causing both β-catenin (CTNNB1) and cAMP responsive element binding (CREB) protein levels to decrease via a glycogen synthase kinase-3β-dependent mechanism. We further found that WNT5a prevents follicle-stimulating hormone and luteinizing protein from up-regulating the CTNNB1 and CREB proteins and their target genes, indicating that WNT5a functions as a physiologic inhibitor of gonadotropin signaling. Together, these findings identify WNT5a as a key regulator of follicle development and gonadotropin responsiveness.—Abedini, A., Zamberlam, G., Lapointe, E., Tourigny, C., Boyer, A., Paquet, M., Hayashi, K., Honda, H., Kikuchi, A., Price, C., Boerboom, D. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling. PMID:26667040

  7. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling.

    PubMed

    Abedini, Atefeh; Zamberlam, Gustavo; Lapointe, Evelyne; Tourigny, Catherine; Boyer, Alexandre; Paquet, Marilène; Hayashi, Kanako; Honda, Hiroaki; Kikuchi, Akira; Price, Christopher; Boerboom, Derek

    2016-04-01

    Whereas the roles of the canonical wingless-type MMTV (mouse mammary tumor virus) integration site family (WNT) signaling pathway in the regulation of ovarian follicle growth and steroidogenesis are now established, noncanonical WNT signaling in the ovary has been largely overlooked. Noncanonical WNTs, including WNT5a and WNT11, are expressed in granulosa cells (GCs) and are differentially regulated throughout follicle development, but their physiologic roles remain unknown. Using conditional gene targeting, we found that GC-specific inactivation ofWnt5a(but notWnt11) results in the female subfertility associated with increased follicular atresia and decreased rates of ovulation. Microarray analyses have revealed that WNT5a acts to down-regulate the expression of FSH-responsive genesin vitro, and corresponding increases in the expression of these genes have been found in the GCs of conditional knockout mice. Unexpectedly, we found that WNT5a regulates its target genes not by signalingviathe WNT/Ca(2+)or planar cell polarity pathways, but rather by inhibiting the canonical pathway, causing both β-catenin (CTNNB1) and cAMP responsive element binding (CREB) protein levels to decreaseviaa glycogen synthase kinase-3β-dependent mechanism. We further found that WNT5a prevents follicle-stimulating hormone and luteinizing protein from up-regulating the CTNNB1 and CREB proteins and their target genes, indicating that WNT5a functions as a physiologic inhibitor of gonadotropin signaling. Together, these findings identify WNT5a as a key regulator of follicle development and gonadotropin responsiveness.-Abedini, A., Zamberlam, G., Lapointe, E., Tourigny, C., Boyer, A., Paquet, M., Hayashi, K., Honda, H., Kikuchi, A., Price, C., Boerboom, D. WNT5a is required for normal ovarian follicle development and antagonizes gonadotropin responsiveness in granulosa cells by suppressing canonical WNT signaling.

  8. Expressed microRNA associated with high rate of egg production in chicken ovarian follicles.

    PubMed

    Wu, N; Gaur, U; Zhu, Q; Chen, B; Xu, Z; Zhao, X; Yang, M; Li, D

    2016-10-26

    MicroRNA (miRNA) is a highly conserved class of small noncoding RNA about 19-24 nucleotides in length that function in a specific manner to post-transcriptionally regulate gene expression in organisms. Tissue miRNA expression studies have discovered a myriad of functions for miRNAs in various aspects, but a role for miRNAs in chicken ovarian tissue at 300 days of age has not hitherto been reported. In this study, we performed the first miRNA analysis of ovarian tissues in chickens with low and high rates of egg production using high-throughput sequencing. By comparing low rate of egg production chickens with high rate of egg production chickens, 17 significantly differentially expressed miRNAs were found (P < 0.05), including 11 known and six novel miRNAs. We found that all 11 known miRNAs were involved mainly in pathways of reproduction regulation, such as steroid hormone biosynthesis and dopaminergic synapse. Additionally, expression profiling of six randomly selected differentially regulated miRNAs were validated by quantitative real-time polymerase chain reaction (RT-qPCR). Some miRNAs, such as gga-miR-34b, gga-miR-34c and gga-miR-216b, were reported to regulate processes such as proliferation, cell cycle, apoptosis and metastasis and were expressed differentially in ovaries of chickens with high rates of egg production, suggesting that these miRNAs have an important role in ovary development and reproductive management of chicken. Furthermore, we uncovered that a significantly up-regulated miRNA-gga-miR-200a-3p-is ubiquitous in reproduction-regulation-related pathways. This miRNA may play a special central role in the reproductive management of chicken, and needs to be further studied for confirmation.

  9. Structural Changes in Large Intestinal Aggregated Lymphoid Follicles in Wistar Rats During Postnatal Ontogeny.

    PubMed

    Tikhonov, E A

    2015-09-01

    Quantitative evaluation of aggregated lymphoid follicles in various compartments of the large intestine was carried out in Wistar rats of different age: newborn (3-4 days), prepubertal (20-30 days), adult (2-3 months), and old (16-18 months). No aggregated lymphoid tissue was detected in the large intestinal mucosa of newborn animals. The cecum of prepubertal, adult, and old animals contained solitary patches with 7-9 follicles. Higher percentage of aggregated lymphoid tissue, associated with colonic mucosa, was explained by enlargement of the lymphoid patch area and of the number of solitary lymphoid follicles during the postnatal ontogenesis. The mean area of a patch in the distal part of the colon in prepubertal, adult, and old animals was 3.2, 2.5, and 2.2 times larger than in the medial part of the intestine, the number of follicles per patch was 2.8, 2.8, and 2.5 times higher, respectively. The differences were significant only for the two younger groups.

  10. Effects of flavonoids from semen cuscutae on the hippocampal-hypothalamic-pituitary-ovarian sex hormone receptors in female rats exposed to psychological stress.

    PubMed

    Ke, J; Duan, R

    2013-01-01

    To investigate the effects of flavonoids from semen cuscutae (FSCs) on the hippocampal-hypothalamic-pituitary-ovarian sex hormone receptors in female rats exposed to psychological stress and to explore the related mechanism. Flavonoids were obtained from semen cuscutae using solvent extraction and polyamide column chromatography. Sound, light, and electricity were combined into psychological stress for endocrine dysfunction model establishment in female rats. The effects of FSCs on estrogen receptor (ER) in the hippocampus, hypothalamus, and pituitaries, as well as on follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in the ovaries of the psychologically stressed rats were quantitatively analyzed using immunohistochemistry and image analysis. FSCs increased ER expression in the hippocampus, hypothalamus, and pituitaries, as well as LHR expression in the ovaries, but had no effect on FSHR expression in the ovaries. FSCs are an effective medicine in the treatment of ovarian endocrine dysfunction in psychologically stressed rats.

  11. A quantitative electron microscopic analysis of the membrana granulosa of rat preovulatory follicles.

    PubMed

    Zoller, L C

    1984-01-01

    The ultrastructure of the membrana granulosa (MG) of rat preovulatory follicles was examined using stereological techniques. Organelles studied were nuclei, mitochondria, lipid droplets (LD), lysosomes, and smooth and rough endoplasmic reticulum (SER, RER). The peripheral region of the MG contained the greatest volume of mitochondria, LD and SER, organelles associated with steroidogenesis. The volume of RER, an organelle associated with protein production, was greatest in the cumulus oophorus. These results corroborate previous analyses and demonstrate that the rat MG is composed of discrete subregions.

  12. CONFOCAL LASER SCANNING MICROSCOPY OF RAT FOLLICLE DEVELOPMENT

    EPA Science Inventory

    This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pr...

  13. CONFOCAL LASER SCANNING MICROSCOPY OF RAT FOLLICLE DEVELOPMENT

    EPA Science Inventory

    This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pr...

  14. Gonadotropin stimulation of steroid synthesis and metabolism in the Rana pipiens ovarian follicle: sequential changes in endogenous steroids during ovulation, fertilization and cleavage stages.

    PubMed

    Morrill, Gene A; Schatz, Frederick; Kostellow, Adele; Bloch, Eric

    2006-05-01

    Steroid synthesis and metabolism have been followed in Rana pipiens ovarian follicles, denuded oocytes and eggs during ovulation, fertilization and cleavage stages (blastula formation). Under physiological conditions, gonadotropin stimulation of the fully grown follicle leads to progesterone synthesis from [(3)H]acetate as well as formation of much smaller amounts of 17alpha-hydroxyprogesterone, androstenedione, pregnanedione and pregnanediol. Progesterone levels increase during completion of the first meiotic division, but by ovulation progesterone disappears from the egg. Plasma membrane-bound progesterone is taken up into the oocyte cortical granules and is largely metabolized to 5alpha-pregnane-3alphaol,20-one and 5beta-pregnane-3alpha,17alpha,20beta-triol coincident with internalization of 60% of the oocyte surface (and >90% of bound progesterone) by the end of the hormone-dependent period. The principal steroid in the ovulated egg is 5beta-pregnane-3alpha,17alpha,20beta-triol. There is a rapid efflux of 5beta-pregnane-3alpha,17alpha,20beta-triol into the medium immediately following fertilization and residual steroid levels remain low in the developing blastula. Dissociated blastulae cells prepared from stage 9 1/2 embryos concentrate both pregnenolone and progesterone from the medium with minimal metabolism. The results indicate that the ovarian follicle has the ability to synthesize and metabolize progesterone but that this ability disappears in the ovulated egg. The progesterone metabolites formed during meiosis are largely released at fertilization.

  15. Screening for DNA adducts in ovarian follicles exposed to benzo[a]pyrene and cigarette smoke condensate using liquid chromatography-tandem mass spectrometry.

    PubMed

    Yao, Chunhe; Foster, Warren G; Sadeu, Jean C; Siddique, Shabana; Zhu, Jiping; Feng, Yong-Lai

    2017-01-01

    A rapid mass spectrometric method was applied to non-targeted screening of DNA adducts in follicular cells (granulosa cells and theca cells) from isolated ovarian follicles that were exposed in-vitro to benzo[a]pyrene (B[a]P) and cigarette smoke condensate (CSC) for 13days of culture. The method employed a constant neutral loss (CNL) scan to identify chromatographic peaks associated to a neutral loss of deoxyribose moiety of DNA nucleosides. These peaks were subsequently analyzed by a product ion scan in tandem mass spectrometry to elucidate structures of DNA adducts. The identification was further confirmed through synthesis of proposed DNA adducts where possible. Three DNA adducts, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-dG (BPDE-dG), phenanthrene 1,2-quinone-dG (PheQ-dG) and B[a]P-7,8-quinone-dG (BPQ-dG) were identified in the follicular cells from isolated ovarian follicles exposed to B[a]P. Along with these three, an additional DNA adduct, 4-aminobiphenyl-dG, was identified in the follicular cells from isolated ovarian follicles exposed to CSC. The amounts of the identified DNA adducts in follicular cells increased in a dose-dependent manner for both B[a]P (0, 1.5, 5, 15 and 45ng/mL) and CSC (0, 30, 60, 90 and 130μg/mL). The results revealed that B[a]P-related DNA adducts were the major adducts in the ovarian follicular cells exposed to CSC. The results also revealed that two oxidative biomarkers, 8-hydroxy-2-deoxy guanosine (8-OH-dG) and 8-isoprostane (8-IsoP), in both B[a]P-exposed and CSC-exposed ovarian follicles had strong correlations with the three DNA adducts, BPDE-dG, BPQ-dG and PheQ-dG. A pathway to describe formation of DNA adducts was proposed based on the DNA adducts observed.

  16. Nutrient limitation results in juvenile hormone-mediated resorption of previtellogenic ovarian follicles in mosquitoes.

    PubMed

    Clifton, Mark E; Noriega, Fernando G

    2011-09-01

    Juvenile hormone (JH) is a central hormonal regulator of previtellogenic development in female Aedes aegypti mosquitoes. JH levels are low at eclosion and increase during the first day after adult emergence. This initial rise in JH is essential for female reproductive maturation. After previtellogenic maturation is complete, the mosquito enters a 'state-of-arrest' during which JH synthesis continues at a slower pace and further ovary development is repressed until a blood meal is taken. By examining the relationships between juvenile hormone, follicular resorption and nutrition in A. aegypti, we were able to define a critical role of JH during the previtellogenic resting stage. The rate of follicular resorption in resting stage mosquitoes is dependent on nutritional quality. Feeding water alone caused the rate of follicular resorption to reach over 20% by day 7 after emergence. Conversely, feeding a 20% sucrose solution caused resorption to remain below 5% during the entire experimental period. Mosquitoes fed 3% sucrose show rates of resorption intermediate between water and 20% sucrose and only reached 10% by day 7 after emergence. Follicular resorption is related to JH levels. Ligated abdomens separated from a source of JH (the corpora allata) showed an increase in resorption comparable to similarly aged starved mosquitoes (16%). Resorption in ligated abdomens was reduced to 6% by application of methoprene. The application of methoprene was also sufficient to prevent resorption in intact mosquitoes starved for 48 h (14% starved vs. 4% starved with methoprene). Additionally, active caspases were localized to resorbing follicles indicating that an apoptotic cell-death mechanism is responsible for follicular resorption during the previtellogenic resting stage. Taken together, these results indicate that JH mediates reproductive trade-offs in resting stage mosquitoes in response to nutrition.

  17. Nutrient limitation results in juvenile hormone-mediated resorption of previtellogenic ovarian follicles in mosquitoes

    PubMed Central

    Clifton, Mark E.; Noriega, Fernando G.

    2011-01-01

    Juvenile hormone (JH) is a central hormonal regulator of previtellogenic development in female Aedes aegypti mosquitoes. JH levels are low at eclosion and increase during the first day after adult emergence. This initial rise in JH is essential for female reproductive maturation. After previtellogenic maturation is complete, the mosquito enters a ‘state-of-arrest’ during which JH synthesis continues at a slower pace and further ovary development is repressed until a blood meal is taken. By examining the relationships between juvenile hormone, follicular resorption and nutrition in A. aegypti, we were able to define a critical role of JH during the previtellogenic resting stage. The rate of follicular resorption in resting stage mosquitoes is dependent on nutritional quality. Feeding water alone caused the rate of follicular resorption to reach over 20% by day 7 after emergence. Conversely, feeding a 20% sucrose solution caused resorption to remain below 5% during the entire experimental period. Mosquitoes fed 3% sucrose show rates of resorption intermediate between water and 20% sucrose and only reached 10% by day 7 after emergence. Follicular resorption is related to JH levels. Ligated abdomens separated from a source of JH (the corpora allata) showed an increase in resorption comparable to similarly aged starved mosquitoes (16%). Resorption in ligated abdomens was reduced to 6% by application of methoprene. The application of methoprene was also sufficient to prevent resorption in intact mosquitoes starved for 48 hours (14% starved vs. 4% starved with methoprene). Additionally, active caspases were localized to resorbing follicles indicating that an apoptotic cell-death mechanism is responsible for follicular resorption during the previtellogenic resting stage. Taken together, these results indicate that JH mediates reproductive trade-offs in resting stage mosquitoes in response to nutrition. PMID:21708165

  18. 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.

  19. Effects of dominant follicle aspiration and treatment with recombinant bovine somatotropin (BST) on ovarian follicular development in nelore (Bos indicus) heifers.

    PubMed

    Buratini, J; Price, C A; Visintin, J A; Bó, G A

    2000-08-01

    Follicle ablation has been recognized as an efficient method of follicular wave synchronization. Treatment with recombinant bovine somatotropin (BST) has been shown to enhance follicular development in Bos taurus. This experiment assessed the effects of these treatments in Nelore (B. indicus) heifers. Eight cycling Nelore heifers were randomly assigned to 3 different treatments. On Day 2 of a synchronized cycle (Day 0 = day of ovulation), heifers assigned to Treatments 1 and 2 received 2 mL of saline, whereas heifers assigned to Treatment 3 received 320 mg of BST. On Day 5, the first-wave dominant follicle was ablated by ultrasound-guided transvaginal aspiration in heifers in Treatments 2 and 3, and all heifers received an injection of prostaglandin on Day 11. Aspiration of the dominant follicle advanced and synchronized (P < 0.05) the day of second-wave emergence (6.9 +/- 0.1 vs. 8.4 +/- 0.4) and the day of the pre-wave FSH peak (6.0 +/- 0.0 vs. 6.9 +/- 0.4), and increased FSH peak concentrations (381 +/- 21 vs. 292 +/- 30; pg/mL; P < 0.01). Recombinant bovine somatotropin treatment caused a two-fold increase in plasma insulin-like growth factor-I (IGF-I) concentrations (P < 0.001) and resulted in a 36% increase in the number of small follicles (<5 mm; P < 0.001) compared with saline-treated heifers. In summary, in agreement with previous reports on B. taurus, dominant follicle aspiration synchronized ovarian follicular development, and BST treatment increased peripheral concentrations of IGF-I in Nelore heifers. Recombinant bovine somatotropin also increased the number of small follicles, but this response appeared to be inferior to that reported for B. taurus.

  20. Participation of Gs-proteins in the action of relaxin-like gonad-stimulating substance (GSS) for 1-methyladenine production in starfish ovarian follicle cells.

    PubMed

    Mita, Masatoshi; Yamamoto, Kazutoshi; Nakamura, Masaru; Takeshige, Yuki; Watanabe, Miho; Nagahama, Yoshitaka

    2012-05-01

    Gonad-stimulating substance (GSS) in starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. Recently, we purified GSS from radial nerves in the starfish Asterina pectinifera and identified the chemical structure as a heterodimer composed of two different peptides (A- and B-chain) with disulfide cross-linkages. This study examined the hormonal action of GSS on ovarian follicle cells obtained from ovaries in growing (stage IV) and fully grown (stage V) stages, and particularly the mode of signal transduction. The action of GSS on 1-MeAde production by follicle cells in stage V was mediated through the production of cAMP. In contrast, GSS failed to induce 1-MeAde and cAMP production by follicle cells in stage IV. According to competitive experiments using radioiodinated and radioinert GSS, highly specific binding was observed in follicle cells, though their affinities and numbers in stage IV were inferior to those in stage V. Interestingly, Gsα was not detected immunologically in follicle cell membranes of stage IV. Gβ was also faint in stage IV. Although adenylyl cyclase activity in stage V was dose-dependently activated by GSS in the presence of GTP, neither GSS in the presence of GTP nor nonhydrolyzable GTP analogs were effective on the activity in stage IV. These findings strongly suggest that the failure of GSS to produce 1-MeAde is because of a lack of Gs-proteins in follicle cells at stage IV.

  1. A novel follicle-stimulating hormone receptor mutation causing primary ovarian failure: a fertility application of whole exome sequencing.

    PubMed

    Bramble, Matthew S; Goldstein, Ellen H; Lipson, Allen; Ngun, Tuck; Eskin, Ascia; Gosschalk, Jason E; Roach, Lara; Vashist, Neerja; Barseghyan, Hayk; Lee, Eric; Arboleda, Valerie A; Vaiman, Daniel; Yuksel, Zafer; Fellous, Marc; Vilain, Eric

    2016-04-01

    Can whole exome sequencing (WES) and in vitro validation studies be used to find the causative genetic etiology in a patient with primary ovarian failure and infertility? A novel follicle-stimulating hormone receptor (FSHR) mutation was found by WES and shown, via in vitro flow cytometry studies, to affect membrane trafficking. WES may diagnose up to 25-35% of patients with suspected disorders of sex development (DSD). FSHR mutations are an extremely rare cause of 46, XX gonadal dysgenesis with primary amenorrhea due to hypergonadotropic ovarian failure. A WES study was followed by flow cytometry studies of mutant protein function. The study subjects were two Turkish sisters with hypergonadotropic primary amenorrhea, their parents and two unaffected sisters. The affected siblings and both parents were sequenced (trio-WES). Transient transfection of HEK 293T cells was performed with a vector containing wild-type FSHR as well as the novel FSHR variant that was discovered by WES. Cellular localization of FSHR protein as well as FSH-stimulated cyclic AMP (cAMP) production was evaluated using flow cytometry. Both affected sisters were homozygous for a previously unreported missense mutation (c.1222G>T, p.Asp408Tyr) in the second transmembrane domain of FSHR. Modeling predicted disrupted secondary structure. Flow cytometry demonstrated an average of 48% reduction in cell-surface signal detection (P < 0.01). The mean fluorescent signal for cAMP (second messenger of FSHR), stimulated by FSH, was reduced by 50% in the mutant-transfected cells (P < 0.01). This is an in vitro validation. All novel purported genetic variants can be clinically reported only as 'variants of uncertain significance' until more patients with a similar phenotype are discovered with the same variant. We report the first WES-discovered FSHR mutation, validated by quantitative flow cytometry. WES is a valuable tool for diagnosis of rare genetic diseases, and flow cytometry allows for quantitative

  2. A homologous radioreceptor assay for rat follicle-stimulating hormone

    SciTech Connect

    Findley, W.E.; Steinberger, A.

    1983-09-01

    A homologous radioreceptor assay (RRA) for rat FSH (rFSH), which is both sensitive and easy to perform, is described. The receptor preparation is isolated from a 12,000 X G pellet of rat testes homogenate prepared with a high speed tissue grinder using Tris buffer. The sensitivity of the assay extends below 10 ng rFSH RP-1 (approximately 0.13 ng purified I-3 rat FSH), and the range of the assay spans 2 orders of magnitude. The specific binding of the (/sup 125/I)rFSH tracer is 22-28% of the total tracer added. Such binding, which exceeds previously published values by 2- to 4-fold, allows the addition of relatively small amounts of total tracer radioactivity, which, in turn, contributes to a low nonspecific binding and highly reproducible values for replicates (coefficient of variation, approximately 3.0%). This represents the first homologous RRA for rFSH using testicular receptors. Likewise, the sensitivity and reproducibility exceed those of previous RRAs for rFSH.

  3. 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

  4. 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.

  5. EFFECT OF ATRAZINE ON OVARIAN FUNCTION IN THE RAT

    EPA Science Inventory

    The effect of the chlorotriazine herbicide, atrazine, on ovarian function was studied in Long-Evans hooded (LE-hooded) and SpragucDawley (SD) rats. Atrazine was administered by gavage for 21 d to females displaying regular 4-d estrous cycles. In both sfrains, 75 mg/kg/d disrupted...

  6. EFFECT OF ATRAZINE ON OVARIAN FUNCTION IN THE RAT

    EPA Science Inventory

    The effect of the chlorotriazine herbicide, atrazine, on ovarian function was studied in Long-Evans hooded (LE-hooded) and SpragucDawley (SD) rats. Atrazine was administered by gavage for 21 d to females displaying regular 4-d estrous cycles. In both sfrains, 75 mg/kg/d disrupted...

  7. Characterization of rat hair follicle stem cells selected by vario magnetic activated cell sorting system.

    PubMed

    Huang, Enyi; Lian, Xiaohua; Chen, Wei; Yang, Tian; Yang, Li

    2009-10-30

    Hair follicle stem cells (HfSCs) play crucial roles in hair follicle morphogenesis and hair cycling. These stem cells are self-renewable and have the multi-lineage potential to generate epidermis, sebaceous glands, and hair follicle. The separation and identification of hair follicle stem cells are important for further research in stem cell biology. In this study, we report on the successful enrichment of rat hair follicle stem cells through vario magnetic activated cell sorting (Vario MACS) and the biological characteristics of the stem cells. We chose the HfSCs positive surface markers CD34, alpha 6-integrin and the negative marker CD71 to design four isolation strategies: positive selection with single marker of CD34, positive selection with single marker of alpha 6-integrin, CD71 depletion followed by CD34 positive selection, and CD71 depletion followed by alpha 6-integrin positive selection. The results of flow cytometry analysis showed that all four strategies had ideal effects. Specifically, we conducted a series of researches on HfSCs characterized by their high level of CD34, termed CD34(bri) cells, and low to undetectable expression of CD34, termed CD34(dim) cells. CD34(bri) cells had greater proliferative potential and higher colony-forming ability than CD34(dim) cells. Furthermore, CD34(bri) cells had some typical characteristics as progenitor cells, such as large nucleus, obvious nucleolus, large nuclear:cytoplasmic ratio and few cytoplasmic organelles. Our findings clearly demonstrated that HfSCs with high purity and viability could be successfully enriched with Vario MACS.

  8. Effects of sub-lethal levels of dichlorodiphenyltrichloroethane and dichlorodiphenyldichloroethylene on in vitro steroid biosynthesis by ovarian follicles or steroid metabolism by embryos of rainbow trout (Oncorhynchus mykiss).

    PubMed

    Petkam, Rakpong; Renaud, Rick; Lin, Lucy; Boermans, Herman; Leatherland, John

    2005-07-01

    This study examined the possibility that DDT and DDE, at sub-lethal exposure levels, exert direct effects on the biotransformation of gonadal steroids by rainbow trout (Oncorhynchus mykiss) ovarian follicles and embryos. Ovarian follicles were co-incubated with DDT or DDE at 0.01 or 1 mg l-1 to examine effects of the pesticides on basal or cAMP-activated steroidogenesis. Ovarian preparations were incubated with radiolabelled [3H]pregnenolone ([3H]P5), and the tritiated metabolites of [3H]P5 metabolism were separated using high-performance liquid chromatography (HPLC). Testosterone (T) and 17beta-estradiol (E2) production were also measured using radioimmunoassay (RIA). Embryos were either exposed to the pesticides in ovo, or co-incubated in vitro with the pesticides. The effect of the pesticides on embryo steroid biotransformation was examined using a range of radioactively labelled substrates, including [3H]P5, [3H]progesterone ([3H]P4), [3H]T and [3H]E2. At the concentrations used, the pesticides had no significant effect on the relative amounts of unconjugated radiolabelled steroids formed by the biotransformation of [3H]P5 under conditions of basal or cAMP-stimulated ovarian steroidogenesis. However, DDT and DDE appeared to reduce the basal accumulation of androgen as a product of P5 biotransformation by ovarian follicles. Basal or cAMP-stimulated total estrogen production was not affected. In addition, DDT at 1 mg l-1 and DDE at 0.01 mg l-1 significantly increased and decreased cAMP-stimulated T accumulation, respectively. Also DDT at 0.01 mg l-1 and DDE at 1 mg l-1 significantly increased and decreased basal E2 accumulation, respectively. The steroid metabolites synthesized from the different substrates by embryos were essentially similar in both controls and pesticide-exposed groups, and the survival of embryos to hatch was not significantly affected by pesticide exposure, in ovo, with an approximately 90% hatchability in all treatment groups. This study

  9. Functional histology of the ovarian follicles as determined by follicular fluid concentrations of steroids and IGF-1 in Camelus dromedarius.

    PubMed

    Kafi, M; Maleki, M; Davoodian, N

    2015-04-01

    Ovaries were collected from sexually mature non-pregnant dromedary she-camels. Follicles 6 to 19 mm in diameter per pair of ovaries were randomly selected and classified into clear (n = 30), or opaque (n = 14) based on macroscopic examination of the follicle surface, and then were divided into four classes: clear follicles with 6- 9.9 and 10-19 mm diameter; opaque follicles with 6- 9.9 and 10-19 mm diameter. Follicular fluid (FF) was aspirated for measurement of estradiol-17β, progesterone and IGF-I concentrations, and then a section of tissue through the exposed surface of the follicle wall was removed and fixed in and processed for histological examination. Mean (±SE) number of clear follicles observed on the ovaries that contained a large dominant follicle was less than that on the ovaries which contained a large atretic follicle (p < 0.05; 2.6 ± 1 vs 8.6 ± 0.6). In conclusion, the estrogenic large follicles have suppressive effects on the growth of other follicles. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Prostaglandin E2 (EP) Receptors Mediate PGE2-Specific Events in Ovulation and Luteinization Within Primate Ovarian Follicles

    PubMed Central

    Kim, Soon Ok; Harris, Siabhon M.

    2014-01-01

    Prostaglandin E2 (PGE2) is a key mediator of ovulation. All 4 PGE2 receptors (EP receptors) are expressed in the primate follicle, but the specific role of each EP receptor in ovulatory events is poorly understood. To examine the ovulatory events mediated via these EP receptors, preovulatory monkey follicles were injected with vehicle, the PG synthesis inhibitor indomethacin, or indomethacin plus PGE2. An ovulatory dose of human chorionic gonadotropin was administered; the injected ovary was collected 48 hours later and serially sectioned. Vehicle-injected follicles showed normal ovulatory events, including follicle rupture, absence of an oocyte, and thickening of the granulosa cell layer. Indomethacin-injected follicles did not rupture and contained oocytes surrounded by unexpanded cumulus; granulosa cell hypertrophy did not occur. Follicles injected with indomethacin plus PGE2 were similar to vehicle-injected ovaries, indicating that PGE2 restored the ovulatory changes inhibited by indomethacin. Additional follicles were injected with indomethacin plus an agonist for each EP receptor. EP1, EP2, and EP4 agonists each promoted aspects of follicle rupture, but no single EP agonist recapitulated normal follicle rupture as seen in follicles injected with either vehicle or indomethacin plus PGE2. Although EP4 agonist-injected follicles contained oocytes in unexpanded cumulus, the absence of oocytes in EP1 agonist- and EP2 agonist-injected follicles suggests that these EP receptors promote cumulus expansion. Surprisingly, the EP3 agonist did not stimulate any of these ovulatory changes, despite the high level of EP3 receptor expression in the monkey follicle. Therefore, agonists and antagonists selective for EP1 and EP2 receptors hold the most promise for control of ovulatory events in women. PMID:24506073

  11. Prostaglandin E2 (EP) receptors mediate PGE2-specific events in ovulation and luteinization within primate ovarian follicles.

    PubMed

    Kim, Soon Ok; Harris, Siabhon M; Duffy, Diane M

    2014-04-01

    Prostaglandin E2 (PGE2) is a key mediator of ovulation. All 4 PGE2 receptors (EP receptors) are expressed in the primate follicle, but the specific role of each EP receptor in ovulatory events is poorly understood. To examine the ovulatory events mediated via these EP receptors, preovulatory monkey follicles were injected with vehicle, the PG synthesis inhibitor indomethacin, or indomethacin plus PGE2. An ovulatory dose of human chorionic gonadotropin was administered; the injected ovary was collected 48 hours later and serially sectioned. Vehicle-injected follicles showed normal ovulatory events, including follicle rupture, absence of an oocyte, and thickening of the granulosa cell layer. Indomethacin-injected follicles did not rupture and contained oocytes surrounded by unexpanded cumulus; granulosa cell hypertrophy did not occur. Follicles injected with indomethacin plus PGE2 were similar to vehicle-injected ovaries, indicating that PGE2 restored the ovulatory changes inhibited by indomethacin. Additional follicles were injected with indomethacin plus an agonist for each EP receptor. EP1, EP2, and EP4 agonists each promoted aspects of follicle rupture, but no single EP agonist recapitulated normal follicle rupture as seen in follicles injected with either vehicle or indomethacin plus PGE2. Although EP4 agonist-injected follicles contained oocytes in unexpanded cumulus, the absence of oocytes in EP1 agonist- and EP2 agonist-injected follicles suggests that these EP receptors promote cumulus expansion. Surprisingly, the EP3 agonist did not stimulate any of these ovulatory changes, despite the high level of EP3 receptor expression in the monkey follicle. Therefore, agonists and antagonists selective for EP1 and EP2 receptors hold the most promise for control of ovulatory events in women.

  12. Effects of Spirulina on Cyclophosphamide-Induced Ovarian Toxicity in Rats: Biochemical and Histomorphometric Evaluation of the Ovary

    PubMed Central

    Yener, Nese Arzu; Sinanoglu, Orhun; Ilter, Erdin; Celik, Aygen; Sezgin, Gulbuz; Midi, Ahmet; Aksungar, Fehime

    2013-01-01

    Cyclophosphamide (Cyc) is known to cause ovotoxicity and infertility in women. Our aim is to investigate the possible ovotoxic effects of Cyc and possible antioxidant and protective effects of blue-green algae, Spirulina (Sp), in rat ovaries. Eighteen rats were given: group I (n = 6, control); group II (n = 6, CP), a single dose Cyc; group III (n = 6, Sp+Cyc), 7 days Sp+single dose Cyc. Tissue malondialdehyde (MDA) levels, superoxide dismutase (SOD), and catalase (CAT) activities are assessed biochemically. Normal and atretic primordial and primary follicle counts for all sections obtained for each ovary are calculated. Mean number of follicle counts for each group are compared. In Sp+Cyc group, tissue MDA levels were significantly lower than those in the CP and higher than those in the C group (CP > Sp+Cyc > C). Tissue SOD activity was significantly higher in Sp+Cyc group than that in the CP group and lower than that in the C group (C > Sp+Cyc > C). No statistically significant difference was found between the ovarian CAT activities in any group. Histomorphometrically, there was also no significant difference between the mean numbers of normal and atretic small follicle counts. Our results suggest that single dose Cyc has adverse effects on oxidant status of the ovaries and Sp has protective effects in Cyc-induced ovotoxicity. PMID:23762559

  13. Characterization of persistent follicles induced by prolonged treatment with progesterone in dairy cows: an experimental model for the study of ovarian follicular cysts.

    PubMed

    Díaz, Pablo U; Stangaferro, Matías L; Gareis, Natalia C; Silvia, William J; Matiller, Valentina; Salvetti, Natalia R; Rey, Florencia; Barberis, Fabián; Cattaneo, Luciano; Ortega, Hugo H

    2015-10-15

    Cystic ovarian disease (COD) is a major factor contributing to poor reproductive efficiency of lactating dairy cows. The objective of the present study was to analyze the endocrine profile, growth dynamics, and histologic characteristics of persistent ovarian follicles-cysts developing in response to long-term administration of intermediate levels of progesterone. To this end, after synchronization of cows, a low dose of progesterone was administered for 5, 10, and 15 days after the expected day of ovulation in treated cows (groups P5, P10, and P15, respectively), using an intravaginal progesterone-releasing device. A significant increase in diameter was detected on Day 11 of progesterone treatment and thereafter (P < 0.05), and at Day 15 of persistence, the diameter of the persistent follicle reached a mean of 23 ± 0.6 mm. Microscopically, the persistent follicles had a complete granulosa, an intensely vascularized theca interna, and a collagenous theca externa layer. Temporal changes in the serum concentrations of estradiol, progesterone, and FSH were detected (effects of time, P < 0.01). Progesterone treatment completely inhibited the LH preovulatory surge in treated cows and affected the basal concentration of LH. The pulse frequency remained high at 5 and 10 days of persistence and declined (P < 0.05) after 15 days of persistence. The LH pulse concentration and pulse amplitude had a significant reduction (P < 0.05) during follicular persistence. Changes in the serum levels of estradiol, progesterone, 17-hydroxyprogesterone, and testosterone in serum and follicular fluid were also observed. In serum, estradiol increased gradually from proestrus to Day 10 of follicular persistence (P < 0.05), progesterone showed an increase (P < 0.05) at Day 5 of follicular persistence, 17-hydroxyprogesterone showed a significant decrease at 5 days of follicular persistence in relation to proestrus, and testosterone showed a significant increase (P < 0.05) from proestrus and

  14. Maternal High-Fat Diet-Induced Loss of Fetal Oocytes Is Associated with Compromised Follicle Growth in Adult Rat Offspring1

    PubMed Central

    Tsoulis, Michael W.; Chang, Pauline E.; Moore, Caroline J.; Chan, Kaitlyn A.; Gohir, Wajiha; Petrik, James J.; Vickers, Mark H.; Connor, Kristin L.; Sloboda, Deborah M.

    2016-01-01

    Maternal obesity predisposes offspring to metabolic and reproductive dysfunction. We have shown previously that female rat offspring born to mothers fed a high-fat (HF) diet throughout pregnancy and lactation enter puberty early and display aberrant reproductive cyclicity. The mechanisms driving this reproductive phenotype are currently unknown thus we investigated whether changes in ovarian function were involved. Wistar rats were mated and randomized to: dams fed a control diet (CON) or dams fed a HF diet from conception until the end of lactation (HF). Ovaries were collected from fetuses at Embryonic Day (E) 20, and neonatal ovaries at Day 4 (P4), prepubertal ovaries at P27 and adult ovaries at P120. In a subset of offspring, the effects of a HF diet fed postweaning were evaluated. The present study shows that fetuses of mothers fed a HF diet had significantly fewer oocytes at E20, and in neonates, have reduced AMH signaling that may facilitate an increased number of assembled primordial follicles. Both prepubertally and in adulthood, ovaries show increased follicular atresia. As adults, offspring have reduced FSH responsiveness, low expression levels of estrogen receptor alpha (Eralpha), the oocyte-secreted factor, Gdf9, oocyte-specific RNA binding protein, Dazl, and high expression levels of the granulosa-cell derived factor, AMH, in antral follicles. Together, these data suggest that ovarian compromise in offspring born to HF-fed mothers may arise from changes already observable in the fetus and neonate and in the long term, associated with increased follicular atresia through adulthood. PMID:26962114

  15. Development and application of a rat ovarian gene expression database.

    PubMed

    Jo, Misung; Gieske, Mary C; Payne, Charles E; Wheeler-Price, Sarah E; Gieske, Joseph B; Ignatius, Ignatius V; Curry, Thomas E; Ko, Chemyong

    2004-11-01

    The pituitary gonadotropins play a key role in follicular development and ovulation through the induction of specific genes. To identify these genes, we have constructed a genome-wide rat ovarian gene expression database (rOGED). The database was constructed from total RNA isolated from intact ovaries, granulosa cells, or residual ovarian tissues collected from immature pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin-treated rats at 0 h (no PMSG), 12 h, and 48 h post PMSG, as well as 6 and 12 h post human chorionic gonadotropin. The total RNA was used for DNA microarray analysis using Affymetrix Rat Expression Arrays 230A and 230B (Affymetrix, Santa Clara, CA). The microarray data were compiled and used for display of individual gene expression profiles through specially developed software. The final rOGED provides immediate analysis of temporal gene expression profiles for over 28,000 genes in intact ovaries, granulosa cells, and residual ovarian tissue during follicular growth and the preovulatory period. The accuracy of the rOGED was validated against the gene profiles for over 20 known genes. The utility of the rOGED was demonstrated by identifying six genes that have not been described in the rat periovulatory ovary. The mRNA expression patterns and cellular localization for each of these six genes (estrogen sulfotransferase, synaptosomal-associated protein 25 kDa, runt-related transcription factor, calgranulin B, alpha1-macroglobulin, and MAPK phosphotase-3) were confirmed by Northern blot analyses and in situ hybridization, respectively. The current findings demonstrate that the rOGED can be used as an instant reference for ovarian gene expression profiles, as well as a reliable resource for identifying important yet, to date, unknown ovarian genes.

  16. 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....

  17. DETERMINATION OF IODINE CONCENTRATION AND DISTRIBUTION IN RAT THYROID FOLLICLES BY ELECTRON-PROBE MICROANALYSIS

    PubMed Central

    Robison, William L.; Davis, David

    1969-01-01

    The concentration and the distribution of iodine in various sized follicles of rat thyroid glands have been analyzed by electron-probe microanalysis. The results of the iodine analysis were grouped according to uncorrected lumen diameter size. No significant differences in iodine concentration were observed among the various size categories. When the results for all follicles from a given sample were pooled, the standard error of the mean was approximately 4%. Usually 40–50 follicles per animal were analyzed. The concentration of iodine ranged from 0.9 to 2.1% by weight among individual animals. Scanning pictures and step-scan analysis showed the iodine distribution to be quite uniform across the colloid area. Several techniques of sample preparation were used; they produced no significant differences in the iodine concentrations observed. Sodium concentration, also determined in all samples, was found to vary from 2 to 9% by weight. Because of the mobility of the sodium ion, its distribution was greatly affected by the method of sample preparation. The technique that best preserved the natural chemistry of the sample was that of freezing the tissue, sectioning, and then freeze-drying. PMID:5824060

  18. Effect of medroxyprogesterone acetate (Provera) on ovarian radiosensitivity

    SciTech Connect

    Jarrell, J.; YoungLai, E.V.; McMahon, A.; Barr, R.; O'Connell, G.; Belbec, L.

    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. 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.

  19. The effects of insulin and follicle-simulating hormone (FSH) during in vitro development of ovarian goat preantral follicles and the relative mRNA expression for insulin and FSH receptors and cytochrome P450 aromatase in cultured follicles.

    PubMed

    Chaves, Roberta N; Duarte, Ana Beatriz G; Rodrigues, Giovanna Q; Celestino, Juliana J H; Silva, Gerlane M; Lopes, Claudio Afonso P; Almeida, Anderson P; Donato, Mariana A M; Peixoto, C A; Moura, Arlindo A A; Lobo, Carlos H; Locatelli, Yann; Mermillod, Pascalle; Campello, Claudio C; Figueiredo, Jose Ricardo

    2012-09-01

    The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 μg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles.

  20. N-hexane inhalation during pregnancy alters DNA promoter methylation in the ovarian granulosa cells of rat offspring.

    PubMed

    Li, Hong; Liu, Jin; Sun, Yan; Wang, Wenxiang; Weng, Shaozheng; Xiao, Shihua; Huang, Huiling; Zhang, Wenchang

    2014-08-01

    The N-hexane-induced impact on the reproductive system of the offspring of animals exposed to n-hexane has caused great concern. Pregnant Wistar rats inhaled 500, 2 500 or 12 500 ppm n-hexane during gestational days 1-20. Clinical characteristics and developmental indices were observed. Ovarian granulosa cells were extracted from F1 rats, the number of follicles was determined in ovarian slices and promoter methylation was assessed using MeDIP-Chip. Several methods were used to analyze the scanned genes, including the Gene Ontology Consortium tools, the DAVID Functional Annotation Clustering Tool, hierarchical clustering and KEGG pathway analysis. The results indicated that the live pups/litter ratio was significantly lowest in the 12 500 ppm group. A significant decrease in secondary follicles and an increase in atresic follicles were observed in the 12 500 ppm group. The number of shared demethylated genes was higher than that of the methylated genes, and the differentially methylated genes were enriched in cell death and apoptosis, cell growth and hormone regulation. The methylation profiles of the offspring from the 500 ppm and control groups were different from those of the 2500 and 12 500 ppm groups. Furthermore, the methylation status of genes in the PI3K-Akt and NF-kappa B signaling pathways was changed after n-hexane exposure. The Cyp11a1, Cyp17a1, Hsd3b1, Cyp1a1 and Srd5a1 promoters were hypermethylated in the n-hexane-exposed groups. These results indicate that the developmental toxicity of n-hexane in F1 ovaries is accompanied by the altered methylation of promoters of genes associated with apoptotic processes and steroid hormone biosynthesis.

  1. 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

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

    PubMed

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

    2017-02-08

    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.

  3. A novel follicle-stimulating hormone receptor mutation causing primary ovarian failure: a fertility application of whole exome sequencing

    PubMed Central

    Bramble, Matthew S.; Goldstein, Ellen H.; Lipson, Allen; Ngun, Tuck; Eskin, Ascia; Gosschalk, Jason E.; Roach, Lara; Vashist, Neerja; Barseghyan, Hayk; Lee, Eric; Arboleda, Valerie A.; Vaiman, Daniel; Yuksel, Zafer; Fellous, Marc; Vilain, Eric

    2016-01-01

    STUDY QUESTION Can whole exome sequencing (WES) and in vitro validation studies be used to find the causative genetic etiology in a patient with primary ovarian failure and infertility? SUMMARY ANSWER A novel follicle-stimulating hormone receptor (FSHR) mutation was found by WES and shown, via in vitro flow cytometry studies, to affect membrane trafficking. WHAT IS KNOWN ALREADY WES may diagnose up to 25–35% of patients with suspected disorders of sex development (DSD). FSHR mutations are an extremely rare cause of 46, XX gonadal dysgenesis with primary amenorrhea due to hypergonadotropic ovarian failure. STUDY DESIGN, SIZE, DURATION A WES study was followed by flow cytometry studies of mutant protein function. PARTICIPANTS/MATERIALS, SETTING, METHODS The study subjects were two Turkish sisters with hypergonadotropic primary amenorrhea, their parents and two unaffected sisters. The affected siblings and both parents were sequenced (trio-WES). Transient transfection of HEK 293T cells was performed with a vector containing wild-type FSHR as well as the novel FSHR variant that was discovered by WES. Cellular localization of FSHR protein as well as FSH-stimulated cyclic AMP (cAMP) production was evaluated using flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE Both affected sisters were homozygous for a previously unreported missense mutation (c.1222G>T, p.Asp408Tyr) in the second transmembrane domain of FSHR. Modeling predicted disrupted secondary structure. Flow cytometry demonstrated an average of 48% reduction in cell-surface signal detection (P < 0.01). The mean fluorescent signal for cAMP (second messenger of FSHR), stimulated by FSH, was reduced by 50% in the mutant-transfected cells (P < 0.01). LIMITATIONS, REASONS FOR CAUTION This is an in vitro validation. All novel purported genetic variants can be clinically reported only as ‘variants of uncertain significance’ until more patients with a similar phenotype are discovered with the same variant. WIDER

  4. The association of bisphenol-A urinary concentrations with antral follicle counts and other measures of ovarian reserve in women undergoing infertility treatments.

    PubMed

    Souter, Irene; Smith, Kristen W; Dimitriadis, Irene; Ehrlich, Shelley; Williams, Paige L; Calafat, Antonia M; Hauser, Russ

    2013-12-01

    In this prospective cohort of women undergoing infertility treatments, we measured specific-gravity adjusted urinary BPA (SG-BPA) concentrations and used regression models to evaluate the association of BPA with antral follicle count (AFC), day-3 serum follicle stimulating hormone levels (FSH), and ovarian volume (OV). BPA, detected in >80% of women, had a geometric mean (±GSD) of 1.6±2.0, 1.7±2.1, and 1.5±1.8μg/L for the women contributing to the AFC (n=154), day-3 FSH (n=120), and OV (n=114) analyses, respectively. There was an average decrease in AFC of 12% (95% CI: -23%, -0.6%), 22% (95% CI: -31%, -11%), and 17% (95% CI: -27%, -6%), in the 2nd, 3rd, and 4th SG-BPA quartile compared to the 1st quartile, respectively (p-trend: <0.001). No association of SG-BPA with FSH or OV was observed. Among women from an infertility clinic, higher urinary BPA concentrations were associated with lower AFC, raising concern for possible accelerated follicle loss and reproductive aging.

  5. The Association of Bisphenol-A Urinary Concentrations with Antral Follicle Counts and Other Measures of Ovarian Reserve in Women Undergoing Infertility Treatments.☆

    PubMed Central

    Souter, Irene; Smith, Kristen W; Dimitriadis, Irene; Ehrlich, Shelley; Williams, Paige L; Calafat, Antonia M; Hauser, Russ

    2015-01-01

    In this prospective cohort of women undergoing infertility treatments, we measured specific-gravity adjusted urinary BPA (SG-BPA) concentrations and used regression models to evaluate the association of BPA with antral follicle count (AFC), day-3 serum follicle stimulating hormone levels (FSH), and ovarian volume (OV). BPA, detected in >80% of women, had a geometric mean (±GSD) of 1.6±2.0, 1.7±2.1, and 1.5±1.8µg/L for the women contributing to the AFC (n=154), day-3 FSH (n=120), and OV (n=1 14) analyses, respectively. There was an average decrease in AFC of 12% (95% CI: −23%, −0.6%), 22% (95% CI: −31%, −11%), and 17% (95% CI: −27%, −6%), in the 2nd, 3rd, and 4th SG-BPA quartile compared to the 1st quartile, respectively (p-trend: <0.001). No association of SG-BPA with FSH or OV was observed. Among women from an infertility clinic, higher urinary BPA concentrations were associated with lower AFC, raising concern for possible accelerated follicle loss and reproductive aging. PMID:24100206

  6. Regulation of the ovarian oxidative status by leptin during the ovulatory process in rats.

    PubMed

    Bilbao, María Guillermina; Di Yorio, María Paula; Galarza, Rocío Alejandra; Varone, Cecilia Laura; Faletti, Alicia Graciela

    2015-04-01

    Leptin exerts both stimulatory and inhibitory effects on the ovulatory process. In this study, we investigated whether these opposite effects involve changes in the oxidative status in response to different levels of leptin. To this end, we performed both in vivo and in vitro assays using ovaries of immature rats primed with gonadotropins to induce ovulation. Superoxide dismutase (SOD) and catalase (CAT) activity, lipid peroxidation, glutathione (GSH) content, and reactive oxygen species (ROS) were studied as oxidative damage-related parameters. The expression of BCL2, BAX, and caspase 3 were measured by western blot as apoptosis-related biomarkers. The acute treatment with leptin, which inhibits ovulation, decreased SOD activity and increased active caspase 3 expression. No differences were found in CAT activity, lipid peroxidation, or total GSH. In contrast, the daily administration of leptin, which induces ovulation, decreased GSH content, ROS levels, and Bax and active caspase 3 expression, but caused no changes in other parameters. In addition, the daily administration of leptin induced follicular growth, measured by the number of antral follicles in ovarian sections. Using ovarian explant cultures, we found increased BCL2 expression and decreased SOD activity at low and high concentrations of leptin respectively. Thus, leptin can modulate the oxidative status of the ovarian tissue, during the ovulatory process, by acting on different targets according to its circulating levels. At low concentration, leptin seems to play a protective role against the oxidative stress, whereas at high concentrations, this protein seems to be involved in cell death. © 2015 Society for Reproduction and Fertility.

  7. Altered ovarian responsiveness to gonadotropins in neonatally irradiated immature rats

    SciTech Connect

    Freud, A.; Sod-Moriah, U.A.

    1988-01-01

    Female rats which were exposed to a single low dose of gamma irradiation (6R or 15R) at the age of 8 days produce smaller litters when mature than untreated controls. In order to study the possibility that such an impaired reproductive performance could result from a reduced ovulation rate, neonatally irradiated females were treated with PMSG (12 iu/rat) at the age of 26 days. Another group of rats, similarly treated, was further injected with hCG (5 iu/rat) 48 hours later. Animals were killed 48, 55, 60 and 72 hours after PMSG treatment or 72 and 120 after hCG injection. The results indicated that PMSG treatment increased the ovarian weight of non-irradiated controls as well as of irradiated rats and in all animals induced a proestrus like profile of LH. Only a combined treatment of PMSG and hCG resulted in ovulation and corpora lutea formation with significantly increased numbers of corpora lutea in the ovaries of the irradiated rats. The latter was associated with higher progesterone plasma levels not correlated to the number of corpora lutea. The gradual decrease in the number of ovarian binding sites for hCG with increased radiation dose and the increased association constant in the 15R group could not explain the increased sensitivity of the ovary to exogenous gonadotropins which results from neonatal exposure to low doses of gamma irradiation.

  8. Movento influences development of granulosa cells and ovarian follicles and FoxO1 and Vnn1 gene expression in BALB/c mice

    PubMed Central

    Kafshgiri, Sakineh Kaboli; Parivar, Kazem; Baharara, Javad; Kerachian, Mohammad Amin; Roodbari, Nasim Hayati

    2016-01-01

    Objective(s): Pesticides has wide range of infertility in female reproductive. This study was done to evaluate the effect of movento pesticide on development of granulosa cells and ovarian follicles and FoxO1 and Vnn1 gene expression in BALB/c mice. Materials and Methods: In this study 40 healthy BALB/c mice 5-6 weeks age were used. Animals were randomly allocated into four groups. Control (without any intervention), three experimental groups received 25, 50 and 100 mg/kg movento dissolved in PBS by gavage for 21 days. Animals scarified after three weeks. For determining the effects of movento on granulosa cells in culture, treatments were conducted to movento (125, 250, 500 μg/ml) for 24 hr. We surveyed the expression of the FoxO1 and Vnn1 in granulosa cells in vitro, and its relation to cell death by flowcytometer and DAPI. Levels of FoxO1 and Vnn1 were analyzed by real-time PCR. Results: Exposure to movento significantly decreased ovarian weight and the number of primary, secondary and antral follicles. Further, treatment with different concentration of movento induced apoptosis on granulosa cells. Gene expression analysis showed the transcriptional expression of FoxO1 and vnn1 in granulosa cells. Level of Vnn1 mRNA in granulosa cells was decreased in granulosa cells and expression of FoxO1 significantly increased in treated groups in compare to controls (P-value <0.05). Conclusion: Exposure to movento significantly reduced the number of follicles and increased apoptosis of granulosa cells leading disruption of the reproductive system. Also movento reduced expression of Vnn1 and increased FoxO1 genes in a dose dependent manner. PMID:27917277

  9. 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.

  10. The modulatory role of transforming growth factor beta1 and androstenedione on follicle-stimulating hormone-induced gelatinase secretion and steroidogenesis in rat granulosa cells.

    PubMed

    Ke, Ferng-Chun; Chuang, Li-Chung; Lee, Ming-Ting; Chen, Yun Ju; Lin, Sui-Wen; Wang, Paulus S; Stocco, Douglas M; Hwang, Jiuan-Jiuan

    2004-05-01

    To investigate the potential roles of matrix metalloproteinases (MMPs) in ovarian granulosa cell differentiation, we studied the interactive effects of FSH and local ovarian factors, transforming growth factor beta1 (TGFbeta1) and androstenedione, on gelatinase secretion and progesterone production in rat ovarian granulosa cells. Granulosa cells of eCG-primed immature rats were treated once with various doses of FSH and TGFbeta1 and androstenedione alone or in combinations for 2 days. Conditioned media were analyzed for gelatinase activity using gelatin-zymography/densitometry and progesterone levels using enzyme immunoassay. Cell lysates were analyzed for steroidogenic acute regulatory (StAR) and cholesterol side-chain-cleavage (P450scc) enzyme protein levels. This study demonstrates for the first time that FSH dose-dependently increased the secretion of a major 63-kDa gelatinase and minor 92- and 67-kDa gelatinases. TGFbeta1 also dose-dependently increased the secretion of 63-kDa gelatinase, while androstenedione alone had no effect. The 92-kDa gelatinase was identified as the pro-MMP9 that could be cleaved by aminophenylmercuric acetate into the 83-kDa active form. Importantly, we show that TGFbeta1 and androgen act in an additive manner to enhance FSH stimulatory effects both on the secretion of gelatinases and the production of progesterone. We further show by immunoblotting that the enhancing effect of TGFbeta1 and androstenedione on FSH-stimulated steroidogenesis is partly mediated through the increased level of StAR protein and/or P450scc enzyme. In conclusion, this study indicates that, during antral follicle development, TGFbeta1 and androgen act to enhance FSH promotion of granulosa cell differentiation and that the process may involve the interplay of modulating cell- to-matrix/cell-to-cell interaction and steroidogenic activity.

  11. Amitriptyline plays important roles in modifying the ovarian morphology and improving its functions in rats with estradiol valerate-induced polycystic ovary.

    PubMed

    Li, Xinqiang; Wang, Shufen; Zhang, Li; Zhang, Lei; Liu, Jiali; Luo, Haoshu; Gou, Kemian; Cui, Sheng

    2017-09-08

    Previous studies demonstrated that depression is more prevalent in women with polycystic ovary syndrome (PCOS). In this study, we aimed to determine whether amitr