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Sample records for immature ovarian follicles

  1. 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.

  2. Extracellular matrix in ovarian follicles.

    PubMed

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

    2000-05-25

    A lot is known about the control of the development of ovarian follicles by growth factors and hormones, but less is known about the roles of extracellular matrix in the control of follicular growth and development. In this review we focus on the specialized extracellular matrix of the basal laminas that are present in ovarian follicles. These include the follicular basal lamina itself, the Call-Exner bodies of the membrana granulosa, the subendothelial and arteriole smooth muscle basal laminas in the theca, and the basal lamina-like material of the thecal matrix. We discuss the evidence that during follicle development the follicular basal lamina changes in composition, that many of its components are produced by the granulosa cells, and that the follicular basal laminas of different follicles have different ultrastructural appearances, linked to the shape of the aligning granulosa cells. All these studies suggest that the follicular basal lamina is extremely dynamic during follicular development. PMID:10963877

  3. Bioengineering the Ovarian Follicle Microenvironment

    PubMed Central

    Shea, Lonnie D.; Woodruff, Teresa K.; Shikanov, Ariella

    2014-01-01

    Chemo- and radiation therapies used to treat cancer can have the unintended effect of making patients infertile. Clinically established fertility preservation methods, such as egg and embryo cryopreservation, are not applicable to all patients, which has motivated the development of strategies that involve ovarian tissue removal and cryopreservation before the first sterilizing treatment. To restore fertility at a later date, the early-stage follicles present in the tissue must be matured to produce functional oocytes, a process that is not possible using existing cell culture technologies. This review describes the application of tissue engineering principles to promote ovarian follicle maturation and produce mature oocytes through either in vitro culture or transplantation. The design principles for these engineered systems are presented, along with identification of emerging opportunities in reproductive biology. PMID:24849592

  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. Xenobiotic effects on ovarian preantral follicles.

    PubMed

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

    2011-11-01

    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

  6. 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. PMID:26408854

  7. In vitro development of ovarian follicles.

    PubMed

    Telfer, Evelyn E; McLaughlin, Marie

    2011-01-01

    Tissue banking of ovarian material is being increasingly offered to a variety of patients as a means of fertility preservation. This tissue comprises thin cortical surface biopsies that contain predominantly primordial follicles, and currently the only option to restore fertility is by transplantation. However, this is not a viable option for all patients. The potential of this tissue could be realized by the development of in vitro systems to support complete growth from the early primordial stages through to maturity. This technology would have many therapeutic applications including the production of competent oocytes for assisted reproduction technologies, determination of toxicological effects on germ cell development, assessment of cryopreserved ovarian tissue before transplantation for fertility preservation as well as providing an experimental model to address basic scientific questions concerning human oocyte development. Complete oocyte development in vitro from the primordial stage has been achieved in mice, but the larger size and longer growth period of human follicles has made the interspecies translation of these techniques difficult. Recently progress has been made in defining conditions that support different stages of follicle development in vitro that make a complete in vitro system from primordial to maturation a possible reality. This article deals with our current understanding of in vitro development. PMID:21207331

  8. Oocyte-derived R-spondin2 promotes ovarian follicle development.

    PubMed

    Cheng, Yuan; Kawamura, Kazuhiro; Takae, Seido; Deguchi, Masashi; Yang, Qing; Kuo, Calvin; Hsueh, Aaron J W

    2013-06-01

    R-spondin proteins are adult stem cell growth factors capable of stimulating gut development by activating LGR4, 5, and 6 receptors to promote Wnt signaling. Although multiple Wnt ligands and cognate Frizzled receptors are expressed in the ovary, their physiological roles are unclear. Based on bioinformatic and in situ hybridization analyses, we demonstrated the exclusive expression of R-spondin2 in oocytes of ovarian follicles. In cultured somatic cells from preantral follicles, R-spondin2 treatment (ED50: 3 ng/ml) synergized with Wnt3a to stimulate Wnt signaling. In cultured ovarian explants from prepubertal mice containing preantral follicles, treatment with R-spondin2, similar to follicle stimulating hormone, promoted the development of primary follicles to the secondary stage. In vivo administration of an R-spondin agonist stimulated the development of primary follicles to the antral stage in both immature mice and gonadotropin releasing hormone antagonist-treated adult mice. Subsequent treatment with gonadotropins allowed the generation of mature oocytes capable of undergoing early embryonic development and successful pregnancy. Furthermore, R-spondin agonist treatment of immune-deficient mice grafted with human cortical fragments stimulated the development of primary follicles to the secondary stage. Thus, oocyte-derived R-spondin2 is a paracrine factor essential for primary follicle development, and R-spondin agonists could provide a new treatment regimen for infertile women with low responses to the traditional gonadotropin therapy. PMID:23407710

  9. Follicle-Stimulating Hormone and Estradiol Interact to Stimulate Glutathione Synthesis in Rat Ovarian Follicles and Granulosa Cells1

    PubMed Central

    Hoang, Yvonne D.; Nakamura, Brooke N.; Luderer, Ulrike

    2009-01-01

    Glutathione (GSH), the most abundant intracellular nonprotein thiol, is critical for many cellular functions. The rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL), a heterodimer composed of a catalytic (GCLC) and a modifier (GCLM) subunit. The tissue-specific regulation of GSH synthesis is poorly understood. We showed previously that gonadotropin hormones regulate ovarian GSH synthesis. In the present study, we sought to clarify the ovarian cell type-specific effects of follicle-stimulating hormone (FSH) and estradiol on GSH synthesis. Immature female rats were treated with estradiol to stimulate development of small antral follicles. Granulosa cells (GCs) from these follicles or whole follicles were cultured in serum-free media, with or without FSH and 17beta-estradiol. The GSH and GCLC protein and mRNA levels increased in GCs treated with FSH alone. The effects of FSH on GCLC and GCLM protein and mRNA levels, GCL enzymatic activity, and GSH concentrations in GCs were significantly enhanced by the addition of estradiol. Estradiol alone had no effects on GSH. Dibromo-cAMP mimicked and protein kinase A (PKA) inhibitors prevented FSH stimulation of GCL subunit protein levels. In cultured small antral follicles, FSH stimulated estradiol synthesis and robustly increased GCL subunit mRNA and protein levels and GSH concentrations. The GCL subunit mRNA expression increased in both the granulosa cells and theca cells of follicles with FSH stimulation. These data demonstrate that maximal stimulation of GSH synthesis by FSH in granulosa cells and follicles requires estradiol. Without estradiol, FSH causes lesser increases in GCL subunit expression via a PKA-dependent pathway. PMID:19516019

  10. Oocyte-derived R-spondin2 promotes ovarian follicle development

    PubMed Central

    Cheng, Yuan; Kawamura, Kazuhiro; Takae, Seido; Deguchi, Masashi; Yang, Qing; Kuo, Calvin; Hsueh, Aaron J. W.

    2013-01-01

    R-spondin proteins are adult stem cell growth factors capable of stimulating gut development by activating LGR4, 5, and 6 receptors to promote Wnt signaling. Although multiple Wnt ligands and cognate Frizzled receptors are expressed in the ovary, their physiological roles are unclear. Based on bioinformatic and in situ hybridization analyses, we demonstrated the exclusive expression of R-spondin2 in oocytes of ovarian follicles. In cultured somatic cells from preantral follicles, R-spondin2 treatment (ED50: 3 ng/ml) synergized with Wnt3a to stimulate Wnt signaling. In cultured ovarian explants from prepubertal mice containing preantral follicles, treatment with R-spondin2, similar to follicle stimulating hormone, promoted the development of primary follicles to the secondary stage. In vivo administration of an R-spondin agonist stimulated the development of primary follicles to the antral stage in both immature mice and gonadotropin releasing hormone antagonist-treated adult mice. Subsequent treatment with gonadotropins allowed the generation of mature oocytes capable of undergoing early embryonic development and successful pregnancy. Furthermore, R-spondin agonist treatment of immune-deficient mice grafted with human cortical fragments stimulated the development of primary follicles to the secondary stage. Thus, oocyte-derived R-spondin2 is a paracrine factor essential for primary follicle development, and R-spondin agonists could provide a new treatment regimen for infertile women with low responses to the traditional gonadotropin therapy.—Cheng, Y., Kawamura, K., Takae, S., Deguchi, M., Yang, Q., Kuo, C., Hsueh, A. J. W. Oocyte-derived R-spondin2 promotes ovarian follicle development. PMID:23407710

  11. Gossypol Promotes Degeneration of Ovarian Follicles in Rats

    PubMed Central

    Gadelha, Ivana Cristina Nunes; Fernandes de Macedo, Michelly; Oloris, Sílvia Catarina Salgado; Melo, Marilia Martins

    2014-01-01

    The present study aimed to determine if gossypol interferes with ovarian follicles in rats. Twenty-four female Wistar rats were assigned to two equal groups: one control group and the other dosed with gossypol (25 mg/kg/day, subcutaneously) for 15 days. Ovarian follicles were histologically classified according to the stage of development and as normal or atretic. Gossypol treatment reduced the length of estrous with an increase in the duration of the diestrus phase. This compound was responsible for reduced serum levels of T4 and progesterone. Treatment with gossypol was responsible for a significant reduction in the number of normal ovarian follicles and a significant increase in the number of atretic follicles, both in all stages of development. Thus, treatment of rats with gossypol was responsible for reduction in the number of viable follicles and changes in hormone levels that resulted in interference of the estrous cycle. PMID:25540815

  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. 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.

  14. 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

  15. Follicle Depletion Provides a Permissive Environment for Ovarian Carcinogenesis.

    PubMed

    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; Xu, Xiang-Xi

    2016-09-15

    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

  16. WNT4 is required for normal ovarian follicle development and female fertility

    PubMed Central

    Boyer, Alexandre; Lapointe, Évelyne; Zheng, Xiaofeng; Cowan, Robert G.; Li, Huaiguang; Quirk, Susan M.; DeMayo, Francesco J.; Richards, JoAnne S.; Boerboom, Derek

    2010-01-01

    To study the role of WNT4 in the postnatal ovary, a mouse strain bearing a floxed Wnt4 allele was created and mated to the Amhr2tm3(cre)Bhr strain to target deletion of Wnt4 to granulosa cells. Wnt4flox/−;Amhr2tm3(cre)Bhr/+ mice had reduced ovary weights and produced smaller litters (P<0.05). Serial follicle counting demonstrated that Wnt4flox/−;Amhr2tm3(cre)Bhr/+ mice were born with a normal ovarian reserve and maintained normal numbers of small follicles until puberty but had only 25.2% of the normal number of healthy antral follicles. Some Wnt4flox/−;Amhr2tm3(cre)Bhr/+ mice had no antral follicles or corpora lutea and underwent premature follicle depletion. RT-PCR analyses of Wnt4flox/−;Amhr2tm3(cre)Bhr/+ granulosa cells and cultured granulosa cells that overexpress WNT4 demonstrated that WNT4 regulates the expression of Star, Cyp11a1, and Cyp19, steroidogenic genes previously identified as downstream targets of the WNT signaling effector CTNNB1. Decreased serum progesterone levels were found in immature, gonadotropin-treated Wnt4flox/−;Amhr2tm3(cre)Bhr/+ mice (P<0.05). WNT4- and CTNNB1-overexpressing cultured granulosa cells were analyzed by microarray for alterations in gene expression, which showed that WNT4 regulates additional genes involved in late follicle development via the WNT/CTNNB1 signaling pathway. Together, these data indicate that WNT4 is required for normal antral follicle development and may act by regulating granulosa cell functions including steroidogenesis.—Boyer, A., Lapointe, E., Zheng, X., Cowan, R. G., Li, H., Quirk, S. M., DeMayo, F. J., Richards, J. S., Boerboom, D. WNT4 is required for normal ovarian follicle development and female fertility. PMID:20371632

  17. Morphological classification of bovine ovarian follicles.

    PubMed

    Rodgers, R J; Irving-Rodgers, H F

    2010-02-01

    Follicle classification is an important aid to the understanding of follicular development and atresia. Some bovine primordial follicles have the classical primordial shape, but ellipsoidal shaped follicles with some cuboidal granulosa cells at the poles are far more common. Preantral follicles have one of two basal lamina phenotypes, either a single aligned layer or one with additional layers. In antral follicles <5 mm diameter, half of the healthy follicles have columnar shaped basal granulosa cells and additional layers of basal lamina, which appear as loops in cross section ('loopy'). The remainder have aligned single-layered follicular basal laminas with rounded basal cells, and contain better quality oocytes than the loopy/columnar follicles. In sizes >5 mm, only aligned/rounded phenotypes are present. Dominant and subordinate follicles can be identified by ultrasound and/or histological examination of pairs of ovaries. Atretic follicles <5 mm are either basal atretic or antral atretic, named on the basis of the location in the membrana granulosa where cells die first. Basal atretic follicles have considerable biological differences to antral atretic follicles. In follicles >5 mm, only antral atresia is observed. The concentrations of follicular fluid steroid hormones can be used to classify atresia and distinguish some of the different types of atresia; however, this method is unlikely to identify follicles early in atresia, and hence misclassify them as healthy. Other biochemical and histological methods can be used, but since cell death is a part of normal homoeostatis, deciding when a follicle has entered atresia remains somewhat subjective. PMID:19786400

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

    PubMed

    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

  19. 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

  20. 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

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

    PubMed

    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

  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. 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. PMID:26289531

  4. 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. PMID:25819501

  5. Colour Doppler sonography of cystic ovarian follicles in cows.

    PubMed

    Rauch, Alexandra; Krüger, Lars; Miyamoto, Akio; Bollwein, Heinrich

    2008-12-01

    The goals of the present study were to investigate whether colour Doppler sonography can be used to differentiate temporary from persistent ovarian follicles and follicles with luteal tissue from follicles without luteal tissue and to assess the response of follicular cysts to administration of a gonadotropin releasing hormone (GnRH) analogue. Fifty-four cows having ovarian follicular structures with a diameter of >15 mm but no corpus luteum were included. These cows were examined via B-mode and colour Doppler sonography. The same examinations were repeated 10 to 12 days later, and the cows with follicular cysts (n=17) received a GnRH analogue. Blood flow was measured before and 30 min after treatment. Ten to 12 days later, the response to treatment was assessed using B-mode sonography. While 31 of 54 follicles disappeared spontaneously (temporary follicles), 23 follicles persisted and were diagnosed as cystic ovarian follicles (COFs). There was no difference between temporary follicles and COFs in regard to total area, wall thickness or the perfused area. In the luteinized follicles (n=13), based on the plasma progesterone concentration, total area was twice as large, wall thickness was three times greater and the perfused area was 4.5 times larger than those of the non-luteinized follicles (n=41). The sensitivity of diagnosing luteinized follicles was 61.5% using B-mode sonography and 92.3% using colour Doppler sonography. Twelve cows responded to GnRH, and five cows did not. There was a trend (P=0.07) toward higher (59.3%) blood flow in the cyst wall 30 min after treatment in the responding cows compared with the non-responding cows. Our results showed that the perfused area more accurately reflects active luteal tissue than wall thickness. Thus, colour Doppler sonography is superior to B-mode sonography for differentiating follicular and luteal cysts and aids in the selection of treatment. However, exact prediction of COFs destined to regress or persist and the

  6. WNT4 is required for normal ovarian follicle development and female fertility.

    PubMed

    Boyer, Alexandre; Lapointe, Evelyne; Zheng, Xiaofeng; Cowan, Robert G; Li, Huaiguang; Quirk, Susan M; DeMayo, Francesco J; Richards, JoAnne S; Boerboom, Derek

    2010-08-01

    To study the role of WNT4 in the postnatal ovary, a mouse strain bearing a floxed Wnt4 allele was created and mated to the Amhr2(tm3(cre)Bhr) strain to target deletion of Wnt4 to granulosa cells. Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice had reduced ovary weights and produced smaller litters (P<0.05). Serial follicle counting demonstrated that Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice were born with a normal ovarian reserve and maintained normal numbers of small follicles until puberty but had only 25.2% of the normal number of healthy antral follicles. Some Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice had no antral follicles or corpora lutea and underwent premature follicle depletion. RT-PCR analyses of Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) granulosa cells and cultured granulosa cells that overexpress WNT4 demonstrated that WNT4 regulates the expression of Star, Cyp11a1, and Cyp19, steroidogenic genes previously identified as downstream targets of the WNT signaling effector CTNNB1. Decreased serum progesterone levels were found in immature, gonadotropin-treated Wnt4(flox/-);Amhr2(tm3(cre)Bhr/+) mice (P<0.05). WNT4- and CTNNB1-overexpressing cultured granulosa cells were analyzed by microarray for alterations in gene expression, which showed that WNT4 regulates additional genes involved in late follicle development via the WNT/CTNNB1 signaling pathway. Together, these data indicate that WNT4 is required for normal antral follicle development and may act by regulating granulosa cell functions including steroidogenesis. PMID:20371632

  7. Extracellular matrix of the developing ovarian follicle.

    PubMed

    Irving-Rodgers, Helen F; Rodgers, Raymond J

    2006-09-01

    There are many different types of extracellular matrices in the different follicle compartments. These have different roles in follicle development and atresia, and they change in composition during these processes. This review focuses on basal lamina matrix in particular, and considers follicular fluid, the newly identified focimatrix, and thecal matrices. When follicles commence growing, the follicular basal lamina changes in its composition from containing all six alpha chains of type IV collagen to only alpha1 and alpha2. Perlecan and nidogen-1 and -2 subsequently become components of the follicular basal lamina, and there is an increase in the amount of laminin chains alpha1, beta2, and gamma1, in the bovine at least. Late in follicular development and on atresia some follicles contain laminin alpha2. On atresia the follicular basal lamina is not degraded, as occurs in ovulation, but can be breached by cells from the thecal layer when it is not aligned by granulosa cells. A novel type of basal lamina-like matrix, called focimatrix (abbreviated from focal intraepithelial matrix), develops between the cells of the membrana granulosa as aggregates of basal lamina material. It does not envelop cells and so cannot perform functions of basal lamina as currently understood. It is hypothesized that focimatrix assists or initiates depolarization of the membrana granulosa necessary for the transformation into luteal cells. The largest osmotically active molecules in follicular fluid are hyaluronan and chondroitin sulfate proteoglycans, including versican and inter-alpha trypsin inhibitor. It has been suggested that these might be responsible for the formation of follicular fluid by creating an osmotic gradient across the follicular wall. The formation, development, and then either ovulation or regression of follicles requires considerable tissue remodeling, cellular replication, and specialization. The expectation of researchers is that extracellular matrix will be

  8. Immunohistochemical localization of basic fibroblast growth factor in bovine ovarian follicles.

    PubMed

    van Wezel, I L; Umapathysivam, K; Tilley, W D; Rodgers, R J

    1995-12-29

    Basic fibroblast growth factor (bFGF, FGF2) controls cell proliferation and differentiation in many organs and tissues. In the ovary, cells proliferate and differentiate during folliculogenesis and during formation of the corpus luteum. While previous studies have inferred a role for bFGF in these processes, the precise contribution of bFGF to follicular activation or recruitment has not been established. For this reason, bFGF was immunolocalized in bovine follicles, using anti-bFGF immunoglobulin specific for the 1-24-amino acid terminus of the 18-kDa peptide. Basic FGF was immunolocalized to the cytoplasm of oocytes from bovine primordial and primary follicles. Strong immunostaining was also observed in corpora lutea, the ovarian surface epithelium, and smooth muscle cells surrounding blood vessels, while substantial levels of immunostaining were also present in cells of the theca interna. In most of the healthy antral follicles examined, the three or so layers of granulosa cells which were closest to the basement membrane were also stained, with greatest levels of staining at the most basal region of each cell. Atretic antral follicles had significant and uniform levels of immunostaining throughout the theca interna and the membrana granulosa. Immunostaining as described above was reduced to background levels when the primary specific immunoglobulin was preabsorbed with a 350 molar excess of peptide comprising the NH2-terminal 24 amino acids of bFGF. Based upon our previous observations and those reported here, we propose that basic fibroblast growth factor is synthesized by immature oocytes, especially those from primordial and primary follicles, and that bFGF has a potential role in activating follicle growth via stimulation of granulosa cell proliferation and follicular basement membrane synthesis. PMID:8824888

  9. 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

  10. Prediagnostic circulating follicle stimulating hormone concentrations and ovarian cancer risk.

    PubMed

    McSorley, Meghan A; Alberg, Anthony J; Allen, Diane S; Allen, Naomi E; Brinton, Louise A; Dorgan, Joanne F; Kaaks, Rudolf; Rinaldi, Sabina; Helzlsouer, Kathy J

    2009-08-01

    Gonadotropins have been indicted in ovarian carcinogenesis but direct evidence has been limited and inconsistent. The aim of this study was to determine the association between prediagnostic levels of follicle stimulating hormone (FSH) and subsequent development of invasive epithelial ovarian cancer. A nested case-control study was conducted using cases and controls drawn from three cohorts: CLUE I and CLUE II of Washington County, MD, and the Island of Guernsey Study, United Kingdom. In total, 67 incident invasive epithelial ovarian cancer cases were each matched to 1 to 2 controls on age, menopausal status, time since last menstrual period, current hormone use and other relevant factors. FSH concentrations were classified into ranked thirds of low, medium or high based on the distribution among controls. Conditional logistic regression was used to estimate the odds ratio (OR) across increasing thirds of FSH concentrations. Results of the analysis showed that ovarian cancer risk decreased with higher FSH concentrations (p-trend = 0.005). Compared with the lowest third of FSH concentrations, the OR among those in the middle and highest thirds were 0.45 [95% Confidence Interval (CI): 0.20-1.00] and 0.26 (95% CI: 0.10-0.70), respectively. Associations persisted after excluding cases diagnosed within 5 years of follow-up. In conclusion, a reduction in subsequent risk of invasive epithelial ovarian cancer was observed among women with higher circulating FSH concentrations. These findings contradict the hypothesized role of FSH as a risk factor in ovarian carcinogenesis. PMID:19444906

  11. Proliferating cell nuclear antigen--a marker for ovarian follicle counts.

    PubMed

    Muskhelishvili, Levan; Wingard, Susan K; Latendresse, John R

    2005-01-01

    Enumerating ovarian follicles is an effective way to estimate the extent of ovarian toxicity in female rodents exposed to xenobiotics. Differential follicle counts are useful in safety assessment bioassays and in interspecies extrapolation of ovarian toxicity. Counting the follicles in H&E-stained sections is labor intensive, tedious, and costly. In the present study we demonstrated that in rat formalin-fixed, paraffin-embedded ovary sections follicles of all degrees of maturity can be visualized by the use of antibody directed against proliferating cell nuclear antigen (PCNA). Follicles are easily distinguished from ovarian background with the ability to detect and identify primordial follicles being enhanced. This translates into a significant decrease in variability of follicle counts, labor, and cost. Specifically, variability dropped from 11% to 0.2%, the counting time was reduced by 46%, and the cost by 48%. PMID:15805074

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

    PubMed

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

    2015-05-01

    Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. Follicular cell steroidogenesis and proliferation in ovulatory follicles is stimulated by hormones such as insulin and its necessary post-receptor response. The aim of this study was to determine the expression of insulin receptor (IR), IR substrate-1 (IRS1) and phosphatidylinositol 3-kinase (PI3K), key intermediates in the insulin pathway, in control cows and cows with spontaneous COD and ACTH-induced COD. IR and IRS1 mRNA levels were greater in granulosa cells and lower in follicular cysts than in control tertiary follicles. PI3K mRNA levels were similar in all follicles evaluated, whereas the expression of IR, IRS1 and PI3K was similar in theca cells. Protein expression of IR was higher in control tertiary follicles than in the same structures in animals with COD and with cysts. IRS1 and PI3K protein expression showed the same pattern in tertiary and cystic follicles. However, the protein expression of subunit alpha p85 of PI3K was greater in theca cells from tertiary follicles than in cystic follicles. These results provide new insights into the insulin response in cows with COD. The lower gene and protein expressions of some insulin downstream effectors at an early stage of the signaling pathway could negatively influence the functionality of ovaries and contribute to follicle persistence. PMID:25813700

  13. Steroid receptor mRNA expression in the ovarian follicles of cows with cystic ovarian disease.

    PubMed

    Alfaro, Natalia S; Salvetti, Natalia R; Velazquez, Melisa M; Stangaferro, Matías L; Rey, Florencia; Ortega, Hugo H

    2012-06-01

    Steroid receptors have been demonstrated to be important intra-ovarian regulators of follicular development and ovulatory processes. The aim of the present study was to determine the expression of steroid receptor mRNA in ovarian follicular structures from cows with cystic ovarian disease (COD) compared with ovarian structures from regularly cycling cows using reverse transcription polymerase chain reaction (RT-PCR). The cystic follicles showed a higher estrogen receptor α (ESR1) mRNA expression in the theca and granulosa and a lower estrogen receptor β (ESR2) expression. The cystic follicles also showed a strong expression of androgen receptor mRNA in the granulosa. No changes were observed in total progesterone receptor mRNA, but a very significant increase in the B isoform was found in the granulosa of the cystic follicles. The findings of the current study provide evidence that an altered steroid signaling system may be present in bovine follicular cysts, and we suggest that in conditions characterized by altered ovulation, such as COD, changes in the expression of ovarian steroid receptors could play a fundamental role in the pathogeny of this disease. PMID:21536311

  14. 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. PMID:27151372

  15. 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. PMID:23567219

  16. Ultrastructure of the theca interna of ovarian follicles in sheep.

    PubMed

    O'Shea, J D; Cran, D G; Hay, M F; Moor, R M

    1978-03-13

    The theca interna of non-atretic ovarian follicles from 2.0 mm in diameter up to the stage shortly following ovulation was studied by light and electron microscopy. In follicles less than 3.0 mm in diameter, the theca interna consisted of about 8-12 layers of flattened cells, together with many capillaries and small bundles of collagen. Two main forms of cellular differentiation were seen. These were towards either fibroblast-like cells or presumed steroidogenic cells whose cytoplasm contained large amounts of predominantly smooth tubular endoplasmic reticulum, to which some ribosomes were attached. The majority of cells were of relatively undifferentiated or intermediate structure. In larger follicles up to the early stages of oestrus the theca interna cells became larger and less flattened, and cells rich in tubular endoplasmic reticulum became proportionately more numerous. By 18 h after the onset of oestrus the theca interna was oedematous, and many cells possessed pseudopodia. Many cells also contained numerous lipid droplets, but there were no signs of thecal cell degeneration or death. Shortly after ovulation the basal lamina of the membrana granulosa was incomplete, and it became more difficult to distinguish between theca and granulosa layers. Structural heterogeneity, with two major cell types and cells of intermediate structure, was present at all stages. It was concluded that: (1) the theca interna of 2.0-2.9 mm follicles contained many cells whose structure was compatible with a steroidogenic capacity; (2) changes in the differentiated thecal cells up to the early stages of oestrus were quantitative rather than qualitative, and suggestive of an increased steroidogenic capacity; (3) the accumulation of lipid in many cells of the theca interna by 18 h after the onset of oestrus probably reflected a reduction in steroidogenic activity; and (4) there was no evidence of any structural specialization to facilitate the transport of steroids from the theca

  17. Fibrin-Mediated Delivery of an Ovarian Follicle Pool in a Mouse Model of Infertility

    PubMed Central

    Smith, Rachel M.; Shikanov, Ariella; Kniazeva, Ekaterina; Ramadurai, Deepa; Woodruff, Teresa K.

    2014-01-01

    The cryopreservation and autotransplantation of ovarian tissue is emerging as a powerful approach for preserving fertility. However, for cancer patients, it may not be possible to transplant ovarian tissue due to the risk of re-seeding disease. We investigated strategies for transplantation of individually isolated follicles to minimize the risk of re-introducing cancer cells present within the vasculature of ovarian stroma. Procedures for large-scale isolation of early-stage follicles and their encapsulation into fibrin hydrogels were developed. For in vivo validation studies, mice were ovariectomized and transplanted with encapsulated follicles into the ovarian bursa. A substantial increase in the number of secondary follicles was observed in the graft at 9 days after transplantation, and antral follicles by day 21, demonstrating primordial follicle recruitment into the growing pool. Initially, elevated follicle-stimulating hormone levels declined substantially by day 21, indicating feedback from the graft; presence of corpora lutea showed the graft's capability of restoring hormone cyclicity. Taken together, the transplanted follicles were able to engraft, mature, and restore ovarian function in an infertile mouse. This biomaterial may, thus, provide a platform for follicle transplantation with a low risk of cancer contamination and for developing strategies that preserve fertility for women facing a cancer diagnosis. PMID:24802617

  18. Development of an automatic follicle isolation tool for ovarian ultrasonographic images

    NASA Astrophysics Data System (ADS)

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

    1997-04-01

    PURPOSE: To develop an automatic computer algorithm for isolating the follicles in ovarian ultrasonographic images. METHODS: A semi-automatic algorithm has been developed as the first step in the development of a totally automatic follicle isolation tool for use with ovarian ultrasonographic imaging. The algorithm is knowledge-based and depends upon the use of a priori information about the structure of the follicle. Graph searching techniques are used along with a method of assigning graph node costs that represent edge information combined with the a priori knowledge.Interactive identification of the follicle of interest is followed by, in some cases, a manual editing of an automatically defined interior boundary. After the interior boundary has been defined, the outer follicle wall border is found without further human intervention. RESULTS: Based on a test with 31 ultrasonographic images of women's ovaries made in vivo, the algorithm is able to locate the outer follicle wall to an rms accuracy of 0.59 mm +/- 0.28 mm in comparison with human expert manual boundary tracing. BREAKTHROUGHS: The isolation of ovarian follicles in ultrasonographic imaging has heretofore only been accomplished by manual tracing. Our semi-automated follicle border finding algorithm is, to the best of our knowledge, the first computerized method capable of finding the outer wall boundary of the follicle. CONCLUSIONS: The success of our semi-automatic follicle isolation algorithm clearly demonstrates the feasibility of a totally automatic tool that should have wide application in ultrasonographic studies of ovarian follicular dynamics.

  19. Multifactorial regulation of prostaglandin synthesis in preovulatory goldfish ovarian follicles.

    PubMed

    Kellner, R G; Van der Kraak, G

    1992-04-01

    Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (PG) production. The protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 micrograms/ml), and 1,2-dioctanoylglycerol (10 and 50 micrograms/ml) stimulated PGE production; the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23187 (0.25-4.0 microM) stimulated PGE production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23187. The direct activator of phospholipase A2, melittin (0.1-1.0 microM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 microM), a putative inhibitor of phospholipase A2, blocked basal and PMA + A23187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), forskolin (10 microM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23187-stimulated PGE production. Melittin-stimulated production of PGE was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian follicles. PMID:1315582

  20. 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

  1. The crucial role of mechanical heterogeneity in regulating follicle development and ovulation with engineered ovarian microtissue

    PubMed Central

    Choi, Jung Kyu; Agarwal, Pranay; Huang, Haishui; Zhao, Shuting; He, Xiaoming

    2014-01-01

    Contemporary systems for in vitro culture of ovarian follicles do not recapitulate the mechanical heterogeneity in mammalian ovary. Here we report microfluidic generation of biomimetic ovarian microtissue for miniaturized three-dimensional (3D) culture of early secondary preantral follicles by using alginate (harder) and collagen (softer) to fabricate the ovarian cortical and medullary tissues, respectively. This biomimetic configuration greatly facilitates follicle development to antral stage. Moreover, it enables in vitro ovulation of cumulus-oocyte complex (COC) from the antral follicles in the absence of luteinizing hormone (LH) and epidermal growth factor (EGF) that are well accepted to be responsible for ovulation in contemporary literature. These data reveal the crucial role of mechanical heterogeneity in the mammalian ovary in regulating follicle development and ovulation. The biomimetic ovarian microtissue and the microfluidic technology developed in this study are valuable for improving in vitro culture of follicles to preserve fertility and for understanding the mechanism of follicle development and ovulation to facilitate the search of cures to infertility due to ovarian disorders. PMID:24702961

  2. 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

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

    PubMed

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

    1981-01-01

    A rapid technique for counting and classifying large ovarian follicles of domestic animals is described. Using a cryostat, 250-micrograms thick sections were cut from the frozen ovary; an image of the surface of each ovarian section was recorded on videotape. By replaying the videotape, the largest profile of each follicle larger than 1 mm in diameter was readily identified and measured. The presence or absence of atresia was determined by applying standard histological methods to fragments of individual follicles taken from the frozen sections. The results obtained are similar to those found using previous methods and demand only one-quarter of the time. PMID:6760292

  4. Bisphenol A inhibits cultured mouse ovarian follicle growth partially via the aryl hydrocarbon receptor signaling pathway

    PubMed Central

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

    2013-01-01

    Bisphenol A (BPA) is an endocrine disruptor that inhibits growth of mouse ovarian follicles and disrupts steroidogenesis at a dose of 438 μM. However, the effects of lower doses of BPA and its mechanism of action in ovarian follicles are unknown. We hypothesized that low doses of BPA inhibit follicular growth and decrease estradiol levels through the aryl hydrocarbon receptor (AHR) pathway. Antral follicles from wild-type and Ahr knock-out (AhrKO) mice were cultured for 96 hours. Follicle diameters and estradiol levels then were compared in wild-type and AhrKO follicles ± BPA (0.004 - 438 μM). BPA inhibited follicle growth (110 - 438 μM) and decreased estradiol levels (43.8 - 438 μM) in wild-type and AhrKO follicles. However, at BPA 110 μM, inhibition of growth in AhrKO follicles was attenuated compared to wild-type follicles. These data suggest that BPA may inhibit follicle growth partially via the AHR pathway, whereas its effects on estradiol synthesis likely involve other mechanisms. PMID:23928317

  5. Sex steroids level in blood plasma and ovarian follicles of the chimeric chicken.

    PubMed

    Sechman, A; Lakota, P; Wojtysiak, D; Hrabia, A; Mika, M; Lisowski, M; Czekalski, P; Rzasa, J; Kapkowska, E; Bednarczyk, M

    2006-12-01

    The study was performed to determine the hormonal status of mature germline chimeras obtained by blastodermal cell transfer from chicken embryos of a donor breed [Green-legged Partridgelike breed (GP) x Araucana (AR)] to those of a recipient breed [White Leghorn (WL)] being at the same stage of embryonic development. The egg-laying chimeras and WL hens (control) of the same age were used in the experiment. At first, blood samples were taken from each bird at 0.5, 5, 12.5 and 18.5 h following oviposition. Subsequently, the chimeras and the WL hens were decapitated 1-2 h after ovulation. A stroma and the following follicles were isolated from the ovary: white normal (1-4, 4-6 and 6-8 mm), white atretic and yellow preovulatory follicles (F4-F1). Sex hormones, progesterone (P4), testosterone (T) and oestradiol (E2) in blood plasma and ovarian follicles were determined radioimmunologically. The activity of the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in the granulosa and theca layers of the follicles was analysed histochemically. In chimeric chickens, a higher level of T in blood plasma during the ovulatory cycle was noticed. However, in the stroma, white prehierarchical and medium-size preovulatory ovarian follicles the level of T was significantly lower. With respect to E2, its elevated levels were found both in blood and in the ovarian follicles. There were no significant differences in P4 concentrations in blood plasma while in ovarian follicles a higher level was observed only in white 6-8 mm follicles. 3beta-HSD activity in granulosa and theca layers of the ovarian follicles in chimeras was not different from that in the WL hens. In conclusion, the results obtained indicate that germline chimeras exhibit significant alterations in sex hormone levels in the ovary and blood plasma, which in turn may affect their reproductive abilities. PMID:17105570

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

    PubMed Central

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

    2015-01-01

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

  7. Cystogenesis of the ovarian antral follicle of the rat: ultrastructural changes and hormonal profile following the administration of dehydroepiandrosterone.

    PubMed

    Anderson, E; Lee, M T; Lee, G Y

    1992-11-01

    Immature 27-day-old female Sprague-Dawley rats were administered daily subcutaneous injections of dehydroepiandrosterone (DHEA, 5 mg/100 g BW) to induce the formation of ovarian follicular cysts. Groups of rats were killed on days 0, 10, 15, 20, 25, and 30. Ovaries from each group of rats were processed for light and electron microscopy and for follicular or cystic fluid hormone analysis. Normal antral follicle fluid, PMSG-treated preovulatory follicular fluid, and cystic fluids were analyzed for progesterone (P), estrone (E1), estradiol (E2), testosterone (T), delta 4-androstenedione (delta 4-A), 5 alpha-dihydrotestosterone (DHT), luteinizing hormone (LH), follicle stimulating hormone (FSH), and prolactin (PRL). DHEA induced anovulation, acyclicity, and the formation of follicular cysts. In certain antral follicles, there was a dramatic increase in the quantities of smooth endoplasmic reticulum (SER) in the granulosa cells and many mitochondria had tubular cristae. Further depletion of granulosa cell number was associated with intense blebbing of the cytoplasm into the follicle antrum. Formation of the ovarian follicular cyst was completed when the entire cyst was lined by a single layer of transformed granulosa cells in contact via adhering, gap, and tight junctions. These cells had little cytoplasm, mitochondria with lamellar cristae, vast basal and apical bands of microfilaments, and an extensive array of smooth-surfaced endocytotic invaginations on the basal plasma membrane. These endocytotic pits may subsequently form smooth-surfaced vesicles and thereby serve as one mechanism for moving fluid from the ovarian interstitium into the cyst. Theca interna cells were rarely observed in the peripheral regions of the cyst. Abundant smooth muscle cells were located beneath the basement membrane of the epithelial cells comprising the cyst wall. These acquired morphological and physiological features may ensure persistence of the ovarian cyst and thus potentiate a

  8. Influence of varied progestogen treatments on ovarian follicle status and subsequent ovarian superstimulatory responses in cows.

    PubMed

    D'Occhio, M J; Niasari-Naslaji, A; Kinder, J E

    1997-01-01

    The influence of ovarian follicle status and follicle dominance on the response to superstimulatory treatment with FSH was examined in cows. In Experiment 1, oestrus was synchronised using Crestar and on Days 4-6 of the ensuing oestrous cycle cows were assigned to: Group NO (n = 9), control, endogenous CL and no treatment; Group N1 (n = 15), injected with a luteolytic dose of cloprostenol (500 micrograms) and implanted with one implant (3 mg) of the synthetic progestogen, norgestomet; Group N8 (n = 18), injected with 500 micrograms cloprostenol and implanted with eight (24 mg) implants of norgestomet. On Days 9-11, seven implants were removed from six cows in Group N8 and these cows, plus eight Group N1 and all Group N0 cows, were superstimulated with porcine FSH (Folltropin-V) over 4 days (360 mg total dose). The remaining implants were removed from cows in Groups N1 and N8 on Days 11-13, and all cows received 500 micrograms cloprostenol. Numbers and sizes of ovarian follicles, and CL, were recorded by trans-rectal ultrasonography; the largest follicle > 10 mm in diameter was considered morphologically dominant (DF). On Days 9-11, the proportions of cows with a DF were: Group N0, 3/9; Group N1, 14/15; Group N8, 0/18. Total follicles on the 4th day of FSH treatment were greater (P < 0.05) for cows in Group N1 (21.6 +/- 4.2) compared with Group N0 (10.9 +/- 2.4), with cows in Group N8 (13.2 +/- 0.9) not different from the other two groups. Subsequent numbers of CL were lower (P < 0.05) for cows in Group N1 (5.0 +/- 1.3) compared with Group N0 (9.4 +/- 2.0), with cows in Group N8 (8.5 +/- 1.0) not different from the other two groups. In Experiment 2, oestrus was synchronised in cows and on Days 4-6, cows were assigned to: Group C0 (n = 7), control, endogenous CL and no treatment; Group C3 (n = 6), received three CIDR-B intra-vaginal devices that delivered progesterone. On Days 9-11, two CIDR-B were removed from cows in Group C3 and all cows treated with FSH as in

  9. Physiologic classification of bovine ovarian follicles with wavelet packet texture analysis

    NASA Astrophysics Data System (ADS)

    Mannivannan, Ujwala; Sarty, Gordon E.; Sirounis, Heather; Singh, Jaswant; Adams, Gregg P.; Pierson, Roger A.

    1999-05-01

    The purpose of the study was to develop a computer tool that can distinguish between atretic (non-ovulatory) and viable (ovulatory) bovine ovarian follicles on the basis of ultrasonographic image texture. Ovarian follicles of heifers (n equals 14) were removed at four physiologically important time points during the estrous cycle. Regions of interest (ROI) in the center of the follicular antrum of the largest follicle in in vitro images were selected manually. The follicles were classified as viable or atretic on the basis of texture quantified with two metrics: standard deviation (D) and energy (E) of gray-scale values in the wavelet transformed ROI images. The sensitivities S and specificities Sp varied between S equals 0.30 and Sp equals 1.00 and S equals 0.93, Sp equals 0.67 when a minimum distance classifier was used. The computer algorithm was able to distinguish between atretic and viable ovarian follicles based ultrasonographic textures of follicular fluid that were not discernible to the human eye. The ROC analysis demonstrated that the texture classification of follicular fluid may be developed into a clinically useful diagnostic tool. It is anticipated that the computer tool will allow diagnostic assessment of reproductive competence of ovarian follicles in women.

  10. Altered Expression of Pro-inflammatory Cytokines in Ovarian Follicles of Cows with Cystic Ovarian Disease.

    PubMed

    Baravalle, M E; Stassi, A F; Velázquez, M M L; Belotti, E M; Rodríguez, F M; Ortega, H H; Salvetti, N R

    2015-01-01

    A growing body of evidence suggests that ovulation shares many of the features of an inflammatory reaction and that cytokines play many diverse and important roles in reproductive biology. The aim of this study was to examine the expression of the pro-inflammatory cytokines interleukin (IL)-1α, IL-6 and tumour necrosis factor (TNF)-α in ovarian cells from cows with cystic ovarian disease (COD) as compared with that in ovarian structures from regularly cycling cows. Expression of genes encoding IL-1α, IL-6 and TNF-α was detected by real-time polymerase chain reaction in follicular cells from ovaries from healthy cows and cows with COD with no significant differences. However, immunohistochemistry showed increased expression of IL-1α, IL-6 and TNF-α in cystic follicles, suggesting that this expression may be related to the persistence of follicular cysts. The effect of COD was evident for IL-1α and TNF-α, and a follicular structure-disease interaction was observed in the expression of all the cytokines evaluated. Thus, altered expression of these proinflammatory cytokines may be related to ovulation failure and development of follicular cysts. PMID:26065705

  11. Effect of tamoxifen on sex steroid concentrations in chicken ovarian follicles.

    PubMed

    Rzasa, Janusz; Sechman, Andrzej; Paczoska-Eliasiewicz, Helena; Hrabia, Anna

    2009-03-01

    The aim of the present investigation was to study the effect of tamoxifen (TAM), an oestrogen receptor antagonist, on the concentrations of sex hormones in chicken ovarian follicles. The experiment was carried out on Hy-line hens which were randomly divided into two groups (control and experimental). TAM was given at a dose of 4 mg/hen (per os) at first once a day for 7 consecutive days, and subsequently four times a day for the next 6 days. Control hens received placebo. Birds were killed on the day after the last TAM treatment. From the dissected ovaries the following compartments were isolated: stroma with follicles < 1 mm, white non-hierarchical (1-4 mm and 4-8 mm) and yellow hierarchical follicles (F6-F1; 18-35 mm). The concentrations of the sex steroids progesterone (P4), testosterone (T) and oestradiol (E2) in the ovarian follicles were determined by radioimmunoassay. In the TAM-treated group, a gradual decrease in egg-laying rate was observed from the 4th day of the experiment. Eventually, egg laying stopped entirely on the 12th day of the experiment. TAM significantly decreased the weight of the ovary and affected the sex hormone concentrations in the ovarian follicles. Following TAM treatment (1) a significant increase in E2 and T concentrations in the stroma, white follicles and the F4 and F1 follicles, (2) a significant decrease in E2 and T concentrations in the F2 follicle, and (3) a significant decline of P4 in the F4 to F1 follicles were observed. The results indicate that the blockade of oestrogen receptors by TAM significantly modulates the process of chicken ovarian steroidogenesis. PMID:19457777

  12. A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

    PubMed Central

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

    2011-01-01

    The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly

  13. Association of the prion protein and its expression with ovarian follicle development in cattle.

    PubMed

    Forde, N; Rogers, M; Canty, M J; Lonergan, P; Smith, G W; Coussens, P M; Ireland, J J; Evans, A C O

    2008-02-01

    The cellular form of the prion protein (PrP(C)) has been detected in many tissues including reproductive tissues. While its function is unclear, it has been suggested to act as a receptor for an unidentified ligand and/or as an antioxidant agent. We tested the hypothesis that PrP(C) is differentially expressed in dominant, growing, compared to subordinate bovine ovarian follicles. Using both microarray analysis and quantitative real-time PCR, the level of prion protein mRNA (Prnp) in both theca and granulosa cells was measured. We found that levels of Prnp were significantly higher in the theca cells of dominant compared to subordinate follicles but similar among granulosa cells from different follicles. This difference was apparent immediately after selection of the dominant follicle and continued to the dominance stage of the follicle wave. Levels of the protein for PrP(C) were also higher (P < 0.05) in theca cells of dominant compared to subordinate follicles. In conclusion, elevated PrP(C) was associated with ovarian follicle growth and development and we suggest that it may play a role in the success of follicle development. PMID:17595008

  14. Vascular endothelial growth factor and angiopoietins during hen ovarian follicle development.

    PubMed

    Kim, Dongwon; Lee, Jeeyoung; Johnson, A L

    2016-06-01

    Growth and maturation of ovarian follicles in the hen (Gallus gallus) requires a network of blood vessels that increases in complexity during development. The present studies investigate expression of vascular endothelial growth factor A (VEGF), angiopoietin1 (ANGPT1) and ANGPT2 mRNAs together with their associated receptors (VEGFR and TIE2, respectively) during maturation. Elevated expression of VEGF and its receptors is associated with healthy, compared to atretic, follicles. Levels of VEGF significantly increase, while antagonistic ANGPT2 decrease, in granulosa cells (GC) at follicle selection. By comparison, levels of VEGF, VEGFR1, VEGFR2, ANGPT1, ANGPT2 and TIE2 within the theca layer do not change (P>0.05) relative to developmental stages surrounding follicle selection (6-8mm versus 9-12mm follicles). Prior to selection, treatment with transforming growth factor β1 (TGFβ1) significantly increases levels of VEGF in undifferentiated GC from prehierarchal (6-8mm) follicles and actively differentiating GC from selected (9-12 and F4) follicles. Moreover, subsequent to selection follicle stimulating hormone (FSH) increases VEGF expression in GC from 9 to 12mm follicles, and eventually luteinizing hormone (LH) promotes VEGF expression in GC from more mature preovulatory follicles. It is concluded that prior to follicle selection VEGF expression is regulated by autocrine and paracrine actions of TGFβ1 (but not FSH), and that a comparatively limited extent of vasculature is sufficient to maintain prehierarchal follicles in a viable and undifferentiated state. At follicle selection, FSH- and subsequently LH-induced VEGF production within the GC layer enhance angiogenesis within the theca layer, which facilitates the rapid growth of preovulatory follicles via enhanced incorporation of yellow yolk. PMID:26996428

  15. 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. PMID:26505254

  16. Thioredoxin, an antioxidant redox protein, in ovarian follicles of women undergoing in vitro fertilization.

    PubMed

    Kishi, Ikuko; Ohishi, Miho; Akiba, Yasuo; Asada, Hironori; Konishi, Yasuhiro; Nakano, Masao; Kamei, Kiyoshi; Yoshimura, Yasunori; Maruyama, Tetsuo

    2016-01-01

    Oxidative stress has a bidirectional role in the development and maturation of zygotes and embryos. Reduction-oxidation reactions and regulatory proteins, such as thioredoxin (TRX) and thioredoxin reductase (TRXR), are intimately involved in the regulation of oxidative stress. The aim of this study was to determine the levels of TRX mRNA and protein in ovarian follicles collected from women undergoing in vitro fertilization (IVF) and to assess these levels relative to follicle size, presence of oocytes, and responsiveness to superovulation. Follicular fluid (FF) and/or granulosa cells (GCs) from large and small follicles were collected at the time of ovum pick-up from 42 IVF patients enrolled in this study. We divided the patients into normal and poor responders (NR and PR, respectively) based on the serum estradiol levels on the day of human chorionic gonadotropin (hCG) administration. We also compared the TRX concentration in FF (FF-TRX) between oocyte-containing follicles (Oc+) and empty follicles (Oc-). The transcript levels of TRX, but not TRXR, were significantly higher in GCs derived from follicles collected from NR than PR, as determined by semi-quantitative RT-PCR analysis. In NR, the FF-TRX was significantly higher in Oc+ follicles than in Oc- follicles and also in large Oc+ follicles than in large Oc- follicles. Unlike NR, PR exhibited no positive association with elevated FF-TRX and presence of oocytes. Based on its collective anti-oxidative, cytoprotective, and cytokine-like properties of TRX, TRX is likely to be involved in the optimal growth and maturation of ovarian follicles and responsiveness to hyperstimulation. PMID:26536897

  17. Endocrine milieu and developmental dynamics of ovarian cysts and persistent follicles in postpartum dairy cows.

    PubMed

    Roth, Z; Biran, D; Lavon, Y; Dafni, I; Yakobi, S; Braw-Tal, R

    2012-04-01

    Ovarian follicular cysts and persistent follicles are follicular pathologies involved in reduced fertility of dairy cows. Two separate experiments were performed on high-yielding Holstein cows to characterize ovarian cyclicity and evaluate the developmental dynamics of follicle pathologies postpartum. In experiment 1, 58 cows were monitored by ultrasonography twice weekly from d 18±1 to 69±2 postpartum. First ovulation occurred 38±3, 27±2, 20±1, and 25±3 d postpartum in cows with 1 cycle (n=11), 2 cycles (n=21), 3 cycles (n=13), and 4 cycles (n=7), respectively. Follicular pathologies were developed in cows that were either acyclic (n=6) or had 1 or 2 cycles, but not in cows with more than 2 cycles. In experiment 2, 47 cows were monitored twice weekly from 10 d postpartum to second ovulation. Follicles ≥17 mm in diameter in 2 consecutive scans were aspirated, and concentrations of various hormones were measured. Cows were defined as cyclic (n=30; 64%) or with the potential to develop follicular pathology (n=17; 36%). Aspirated follicles (n=27) were classified into 3 main groups based on follicular growth rate, follicular diameter, and ovarian activity before and after follicular aspiration. Dominant follicles (n=4) were defined as large follicles (20 mm in diameter) with growth rate ≤1 mm/d and normal ovarian activity. Persistent follicles (n=6) had the same growth rate and diameter as the dominant follicles, but persisted at the same diameter for ≥10 d. Ovarian cysts (n=17) were defined as the largest follicular structures (19 to 32 mm in diameter), with abnormal growth rate (>1 mm/d) and abnormal ovarian activity. Single or turnover cysts did not differ in their growth parameters and were therefore combined and further classified according to follicular-fluid hormone concentrations. Estradiol-dominant cysts (n=7) were characterized by normal estradiol (284 to 659 ng/mL) and progesterone (20 to 113 ng/mL) concentrations, similar to those of the

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

    PubMed Central

    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

  19. 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

  20. 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. PMID:1228337

  1. 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. PMID:25628263

  2. 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

  3. Effect of growth differentiation factor-9 (GDF-9) on the progression of buffalo follicles in vitrified-warmed ovarian tissues.

    PubMed

    Abdel-Ghani, M A; El-Sherry, T M; Abdelhafeez, H H

    2016-10-01

    To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor-9 (GDF-9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF-9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen-thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF-9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF-9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF-9 was detected in mural granulosa cells and theca cells of pre-antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF-9. In corpora lutea, GDF-9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF-9 and cultured control that indicated the GDF-9 treatment has no effect on the primordial to primary follicle transition. GDF-9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre-antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF-9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF-9. In conclusion, treatment with GDF-9 was found to promote progression of primary follicle that could provide an alternative approach to

  4. Effects of daidzein on messenger ribonucleic Acid expression of gonadotropin receptors in chicken ovarian follicles.

    PubMed

    Liu, H Y; Zhang, C Q

    2008-03-01

    Effects of daidzein on expression of mRNA of gonadotropin receptors [follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR)] were evaluated in ovarian follicles of ISA laying hens that were 13 mo old in the postpeak period of egg laying. The hens were randomly allocated as control and daidzein-treated groups, with daidzein supplemented to the basal diet at the level of 10 mg/kg for 7 wk. The granulosa layers of preovulatory follicles (F1, F2, F3, F4, F5) and follicular layers of the small yellow follicle (SYF), large white follicle (LWF), and atretic follicle were collected. The mRNA expression of related genes was measured by semiquantitative reverse transcription PCR. Results showed that daidzein significantly increased the egg-laying rate (P < 0.05) and the number of SYF and LWF (P < 0.05). The relative abundance of the FSHR mRNA decreased in the granulosa layers from F5 to F1, but LHR mRNA displayed the opposite trend in developmental changes. Treatment with daidzein resulted in increased expression of FSHR mRNA in LWF, SYF, and granulosa layers of F4 to F2 and LHR mRNA in granulosa layers of F4 and F1 (P < 0.05). These results indicated that dietary supplementation of daidzein upregulated mRNA expression of gonadotropin receptors to improve follicle development in chicken developing follicles and laying performance after the peak laying period. PMID:18281582

  5. The Developmental Competence of Oocytes Retrieved from The Leading Follicle in Controlled Ovarian Stimulated Cycles

    PubMed Central

    Braga, Daniela Paes de Almeida Ferreira; Bonetti, Tatiana Carvalho de Souza; da Silva, Ismael Dale Cotrim Guerreiro; Setti, Amanda Souza; Iaconelli, Assumpto Jr; Borges, Edson Jr

    2013-01-01

    Background: This study compares the developmental capacity of gametes retrieved from the largest follicle with small follicles of a cohort in controlled ovarian stimulated cycles. Materials and Methods: This prospective study performed in a private assisted fertilization center included 1016 follicles collected from 96 patients who underwent intra cytoplasmic sperm injection (ICSI). After follicular aspiration, oocytes were assigned to two groups according to the diameter of the derived follicle. The large follicle group (n=96) comprised oocytes derived from the leading follicle of the cohort and the small follicle group (n=920) consisted oocytes derived from the smaller follicles of the cohort. The fertilization and percentage of topquality embryos were compared between groups by Chi-square or Fisher’s exact test, where appropriate. The effect of the follicular diameter on oocyte dimorphism was assessed by binary logistic regression. Results: A significantly higher percentage of oocytes derived from the leading follicle were in the metaphase II (MII) stage (100 vs. 70.0%, p<0.001). However we observed no significant differences regarding the percentage of degenerated oocytes between the large (6.25%) and small follicle (5.0%) groups (p=0.550). Regression analysis demonstrated a nearly two-fold increase in the incidence of vacuoles in oocytes derived from the largest follicle of the cohort (OR: 1.81, p=0.046). The fertilization rate (50.0 vs. 38.8%, p=0.038) and the percentage of top quality embryos (84.7 vs. 76.4%, p=0.040) were significantly higher for oocytes derived from the largest follicle. However, the percentage of abnormal fertilized oocytes was equally distributed between the large follicle (15.0%) and small follicle (12. 8%) groups (p=0.550). Conclusion: Our data suggest that intrafollicular mechanisms within the larger follicle of the cohort may allow for these follicles to amplify the responsiveness to exogenous gonadotropin, which leads to the

  6. Assessment of DNA damage in goat preantral follicles after vitrification of the ovarian cortex.

    PubMed

    Faustino, Luciana R; Carvalho, Adeline A; Silva, Cleidson M G; Rossetto, Rafael; Lopes, Cláudio A P; van Tilburg, Maurício F; Carneiro, Pedro B M; Báo, Sônia N; Moura, Arlindo A A; Bordignon, Vilceu; Figueiredo, José R; Rodrigues, Ana Paula R

    2015-03-01

    Effective methods for gamete preservation should have low impact on DNA integrity. The present study investigated the effects of vitrification of goat ovarian tissues on the occurrence of DNA fragmentation and DNA double-stand breaks using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay and detection of phosphorylated histone H2AX (γH2AX), respectively. Goat ovaries were collected at a local abattoir and 12 tissue fragments were prepared from each ovarian pair. Tissue fragments were used as fresh control samples or were cultured in vitro, vitrified or vitrified and cultured. Vitrification was performed using the Ovarian Tissue Cryosystem. Fragments from all groups (control and treatments) were processed for histology, transmission electron microscopy, TUNEL assay and immunofluorescence. Compared with fresh control samples, a lower percentage of morphologically normal follicles was detected in the vitrification followed by culture treatment group (P<0.05). Normal follicular ultrastructure was observed in all groups. Immunofluorescence revealed the presence of γH2AX foci in few oocytes and ovarian stromal cells. TUNEL-positive follicles were found in samples without significant differences among groups (P>0.05). In conclusion, the vitrification protocol used in the present study did not increase DNA damage in preantral follicles enclosed in goat ovarian tissues. PMID:25481978

  7. Effects of Maternal Caffeine Consumption on Ovarian Follicle Development in Wistar Rats Offspring

    PubMed Central

    Dorostghoal, Mehran; Mahabadi, Mahmood Khaksari; Adham, Sahar

    2011-01-01

    Background In recent years concerns have been raised about human reproductive disorders, specially the effects of environmental factors on human fertility and pregnancy outcome. Therefore, the present study was designed to assess the effects of maternal caffeine consumption on ovarian follicles development in rat offspring. Methods 60 pregnant female rats were randomly divided into a control and two experimental groups. The rats in the two experimental groups received caffeine via drinking water during gestation (26 and 45 mg/kg) and lactation (25 and 35 mg/kg). The ovaries of the offspring were removed at 7, 14, 28, 60, 90 and 120 days after birth, and fixed in Bouin's solution. By preparing serial tissue sections, structural changes in ovarian follicles and corpora lutea were studied during postnatal development. Results The weight of ovaries decreased significantly (p<0.05) in the high dose caffeine-treated group at all stages of postnatal development. Significant (p<0.05) decreases were seen in the number of primordial follicles from day 7 to 120 after birth in the high dose caffeine-treated group. Moreover, the number of primary and secondary follicles decreased significantly on days 7, 14 and 28 as did the number of antral follicles on days 14 and 28 after birth (p<0.05) in the high dose caffeine-treated group. The diameter of secondary and antral follicles decreased significantly (p<0.05) in high dose caffeine-treated group on the early days of postnatal development. No statistically significant differences were seen in the number of corpora lutea between the groups. Conclusion The present study shows that caffeine consumption during gestation and lactation affects the early stages of ovarian follicle development and reduces reproductive efficiency in the offspring of Wistar rats. PMID:23926495

  8. Association of follicle stimulating hormone receptor promoter with ovarian response in IVF-ET patients

    PubMed Central

    Dan, Wang; Jing, Gao; Liangbin, Xia; Ting, Zhang; Ying, Zeng

    2015-01-01

    Background: Poor ovarian response phenomenon has been observed in some of the in vitro fertilization-embryo transfer patients. Some investigations found that follicle stimulating hormone receptor (FSHR) gene plays a role in the process, but no direct evidence shows the correlation between genotypes of FSHR and ovarian response. Objective: Exploring the molecular mechanism behind the mutation of FSHR promoter association with ovarian granulosa cells and poor ovarian response. Materials and Methods: This cross sectional study was performed using 158 women undergoing the controlled short program ovarian stimulation for IVF treatment. The 263 bp DNA fragments before the follicle stimulating hormone (FSH) receptor 5' initiation site were sequenced in the patients under IVF cycle, 70 of which had poor ovarian response and 88 showed normal ovarian responses. Results: With a mutation rate of 40%, 63 in 158 cases showed a 29th site G→A point mutation; among the mutated cases, the mutation rate of the poor ovarian responders was significantly higher than the normal group (60% versus 23.9%; χ2=21.450, p<0.001). Besides, the variability was also obvious in antral follicle count, and ovum pick-ups. The estradiol peak values and the number of mature eggs between the two groups had significant difference. However, there was no obvious variability (t=0.457, p=0.324) in the basic FSH values between the two groups (normal group, 7.2±2.3 U/L; mutation group, 7.1±2.0 U/L). Conclusion: The activity of FSHR promoter is significantly affected by the 29th site G→A mutation that will weaken promoter activity and result in poor response to FSH. PMID:26730247

  9. Maternal acrylamide treatment reduces ovarian follicle number in newborn guinea pig offspring.

    PubMed

    Hułas-Stasiak, Monika; Dobrowolski, Piotr; Tomaszewska, Ewa; Kostro, Krzysztof

    2013-12-01

    Acrylamide is an industrial chemical which has toxic effects on reproduction. In this study, we investigated whether acrylamide administered prenatally can induce follicular atresia in the newborn guinea pig ovary. Another aim was to describe the localization of vimentin filaments and determine their participation in atresia. After prenatal acrylamide treatment, the pool of primordial and primary follicles was significantly reduced. The number of caspase 3 and TUNEL positive oocytes increased compared to the control group. There were no differences in Lamp1 (autophagy marker) staining. A vimentin immunosignal was present in the granulosa cells of primordial, primary and secondary follicles. Interestingly, in contrast to the control group, the oocytes from all follicles in the ACR-treated females were negative for vimentin. These data suggest that prenatal exposure to acrylamide reduced the number of ovarian follicles by inducing follicular atresia mediated by oocyte apoptosis. Acrylamide-induced apoptosis may be associated with destruction of vimentin filaments. PMID:23994668

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

    PubMed

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

    2016-04-15

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

  11. 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

  12. Lipopolysaccharide in ovarian follicular fluid influences the steroid production in large follicles of dairy cows.

    PubMed

    Magata, Fumie; Horiuchi, Maya; Echizenya, Riku; Miura, Ryotaro; Chiba, Shiori; Matsui, Motozumi; Miyamoto, Akio; Kobayashi, Yoshiyasu; Shimizu, Takashi

    2014-01-10

    In postpartum dairy cows, various inflammatory diseases depress reproductive performance. Lipopolysaccharide (LPS) derived from infections of the uterus or mammary gland with Gram-negative bacteria was shown to suppress steroid production in the granulosa cells of follicles in vitro. The aim of the study was to investigate the relationship between LPS in ovarian follicular fluid and steroidogenesis by the theca and granulosa cells of the large follicles in vivo. Bovine ovaries were collected from a slaughterhouse, and the largest (F1) and the second largest (F2) follicles were used (>8 mm in diameter, n=38). LPS concentration in the follicular fluid was measured using quantitative kinetic assay. Follicular steroidogenesis was evaluated by measuring the estradiol (E2) and progesterone (P4) concentration in follicular fluid and by analysing transcription levels of steroidogenesis-related genes in theca and granulosa cells. LPS concentration detected in follicular fluid ranged from 0.2 to 2.0 EU/mL. In follicles with a high level of LPS (>0.5 EU/mL, n=15), the concentration of E2 was lower and that of P4 was higher when compared to those in follicles with a low level of LPS (<0.5 EU/mL, n=23), which was observed both in F1 and F2 follicles. Furthermore, in follicles with a high level of LPS, transcripts of steroidogenic enzymes such as CYP17 and P450arom were lower. In those follicles, the expression of caspase-3 was high, suggesting an association with follicular atresia. These findings indicate that LPS present in follicular fluid may cause ovarian dysfunction by inhibiting follicular activity. PMID:24321186

  13. Ecdysone signalling and ovarian development in insects: from stem cells to ovarian follicle formation.

    PubMed

    Belles, Xavier; Piulachs, Maria-Dolors

    2015-02-01

    Although a great deal of information is available concerning the role of ecdysone in insect oogenesis, research has tended to focus on vitellogenesis and choriogenesis. As such, the study of oogenesis in a strict sense has received much less attention. This situation changed recently when a number of observations carried out in the meroistic polytrophic ovarioles of Drosophila melanogaster started to unravel the key roles played by ecdysone in different steps of oogenesis. Thus, in larval stages, a non-autonomous role of ecdysone, first in repression and later in activation, of stem cell niche and primordial germ cell differentiation has been reported. In the adult, ecdysone stimulates the proliferation of germline stem cells, plays a role in stem cell niche maintenance and is needed non-cell-autonomously for correct differentiation of germline stem cells. Moreover, in somatic cells ecdysone is required for 16-cell cyst formation and for ovarian follicle development. In the transition from stages 8 to 9 of oogenesis, ecdysone signalling is fundamental when deciding whether or not to go ahead with vitellogenesis depending on the nutritional status, as well as to start border cell migration. This article is part of a Special Issue entitled: Nuclear receptors in animal development. PMID:24939835

  14. 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

  15. Changes in ovarian follicles following acute infection with bovine viral diarrhea virus.

    PubMed

    Grooms, D L; Brock, K V; Pate, J L; Day, M L

    1998-02-01

    Bovine viral diarrhea virus (BVDV) has been associated with several reproductive problems in cattle, including poor fertility, early embryonic deaths, abortion and congenital anomalies. Little is known about the cause of poor fertility in cows acutely infected with BVDV. The purpose of this study was to identify changes in ovarian function following acute infection with noncytopathic BVDV. The ovaries of 5 BVDV sero-negative and virus-negative pubertal heifers were monitored daily for 4 consecutive estrous cycles. The position and diameter of all follicles (> 5 mm) and luteal structures were recorded. Daily plasma samples were collected to measure peripheral progesterone and estradiol levels. Each heifer was infected intranasally with noncytopathic BVDV following ovulation of the second estrous cycle. The maximum diameter and growth rate of dominant anovulatory and ovulatory follicles were significantly reduced following acute BVDV infection. Similarly, the number of subordinate follicles associated with both the anovulatory and ovulatory follicle was reduced following infection. There were no significant differences in other follicle or luteal dynamic parameters or in peripheral progesterone or estradiol levels. Ovarian follicular growth was different during the first 2 estrous cycles following acute infection with BVDV when compared with the 2 estrous cycles preceding infection. These differences may be important in explaining reduced fertility in herds with acute BVDV infection. PMID:10732038

  16. Aromatase activity in ovarian follicles of the golden hamster.

    PubMed

    Matson, P L; Collins, W P

    1984-09-01

    The aromatizing ability of recombined granulosa and thecal cells in culture, isolated from hamsters 72-78 h and 96-102 h after PMSG-stimulation, was assessed by the addition to the culture medium of androstenedione, testosterone, dehydroepiandrosterone (DHEA) or 5 alpha-dihydrotestosterone (DHT), and measuring the output of oestradiol 4 h later. The cells from all follicles taken after 96-102 h had a reduced oestradiol output compared to those isolated after 72-78 h (P less than 0.02). Recombined cells from the unluteinized follicles at 96-102 h (Group I) showed similar oestradiol output in the presence of androstenedione, testosterone and DHEA to the cells from follicles taken at 72-78 h. However, the recombined cells from the luteinized follicles (Group II) showed a reduced output of oestradiol in the presence of androstenedione, testosterone and DHEA when compared to the recombined cells from the previous period cultured with the corresponding C19 steroid. The results show that a reduced oestradiol output can be caused by (1) the reduced availability of aromatizable substrate and (2) a reduced potential aromatase activity. PMID:6471042

  17. Transcriptome Profiling of the Theca Interna in Transition from Small to Large Antral Ovarian Follicles

    PubMed Central

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

    2014-01-01

    The theca interna layer of the ovarian follicle forms during the antral stage of follicle development and lies adjacent to and directly outside the follicular basal lamina. It supplies androgens and communicates with the granulosa cells and the oocyte by extracellular signaling. To better understand developmental changes in the theca interna, we undertook transcriptome profiling of the theca interna from small (3–5 mm, n = 10) and large (9–12 mm, n = 5) healthy antral bovine follicles, representing a calculated >7-fold increase in the amount of thecal tissue. Principal Component Analysis and hierarchical classification of the signal intensity plots for the arrays showed no clustering of the theca interna samples into groups depending on follicle size or subcategories of small follicles. From the over 23,000 probe sets analysed, only 76 were differentially expressed between large and small healthy follicles. Some of the differentially expressed genes were associated with processes such as myoblast differentiation, protein ubiquitination, nitric oxide and transforming growth factor β signaling. The most significant pathway affected from our analyses was found to be Wnt signaling, which was suppressed in large follicles via down-regulation of WNT2B and up-regulation of the inhibitor FRZB. These changes in the transcriptional profile could have been due to changes in cellular function or alternatively since the theca interna is composed of a number of different cell types it could have been due to any systematic change in the volume density of any particular cell type. However, our study suggests that the transcriptional profile of the theca interna is relatively stable during antral follicle development unlike that of granulosa cells observed previously. Thus both the cellular composition and cellular behavior of the theca interna and its contribution to follicular development appear to be relatively constant throughout the follicle growth phase examined

  18. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. 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. PMID:26489629

  1. Hedgehog signaling pathway in small bovine ovarian follicles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  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. 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. PMID:11387873

  4. Ontogeny of the long form of leptin receptor gene expression in the porcine ovarian follicles.

    PubMed

    Smolinska, N; Kaminski, T; Siawrys, G; Przala, J

    2013-01-01

    Leptin is a polypeptide hormone produced predominantly in adipocytes. It has been found to be implicated in the regulation of satiety and energy homeostasis. A role for leptin in reproduction was later suggested by findings that this hormone may be involved in the regulation of the hypothalamic-pituitary-gonadal axis via endocrine, paracrine and/or autocrine pathways. The objective of the study was to investigate the ontogeny of the long isoform of leptin receptor (OB-Rb) gene in porcine ovarian follicles. The expression of OB-Rb gene was detected in porcine primordial, primary, secondary and antral follicles by in situ hybridization. In summary, our data suggest that leptin might have a direct effect on porcine follicles and plays an important role in the follicular development. PMID:23691582

  5. 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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Ishikawa, Takayuki; Kyoya, Toshihiko; Nakamura, Yusuke; Sato, Eimei; Tomiyama, Tatsuhiro; Kyono, Koichi

    2014-01-01

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

  8. Prepubertal bisphenol A exposure interferes with ovarian follicle development and its relevant gene expression.

    PubMed

    Li, Yuchen; Zhang, Wenchang; Liu, Jin; Wang, Wenxiang; Li, Hong; Zhu, Jianling; Weng, Shaozheng; Xiao, Shihua; Wu, Tingting

    2014-04-01

    Bisphenol A (BPA) is recognized as one of several environmental estrogens. Pre-puberty is an important part of reproductive system development, and even a short-term exposure to BPA during this period may cause serious damage to the reproductive system. In this study, Pre-puberty female Wistar rats were exposed to BPA for one week. The effects of BPA on ovarian structure and function were assessed. The expression levels of follicle development-related genes were analyzed. Our study showed that BPA reduced rat ovarian weights and follicle numbers, and interferes with the constituent ratio of follicles. With increasing doses of BPA, the expression of factor in the germline alpha (FIGLA) and oocyte-specific histone H1 variant (H1FOO) genes decreased, and anti-mullerian hormone (AMH) genes expression increased, suggesting that BPA exposure during the pre-pubertal period may inhibit the development of ovaries, and follicle development-related genes may play certain roles in this process. PMID:24051130

  9. A study on the vitrification of stage III zebrafish (Danio rerio) ovarian follicles.

    PubMed

    Godoy, Leandro Cesar; Streit, Danilo P; Zampolla, Tiziana; Bos-Mikich, Adriana; Zhang, Tiantian

    2013-10-24

    Attempts to cryopreserve fish embryos have been conducted over the past three decades, nevertheless successful cryopreservation protocol for long-term storage still remains elusive. Fish oocytes offer some advantages when compared to embryos, which may help in improving the chances of cryopreservation. In the present study, a series of cryo-solutions were designed and tested for their vitrifying ability using different devices (0.25 ml plastic straw, vitrification block and fibreplug(TM)). Toxicity of vitrification solutions was evaluated by assessing follicle membrane integrity with trypan blue staining. In addition, the effect of vitrification protocol on stage III zebrafish ovarian follicles was investigated by measuring the cytoplasmic ATP content and the mitochondrial distribution and activity using JC-1 probe and confocal microscopy. After vitrification, follicles showed membrane integrity of 59.9 ± 18.4% when fibreplug and V16 (1.5 M methanol + 4.5 M propylene glycol) solution were employed. When vitrified in V2 (1.5 M methanol + 5.5 M Me2SO) the membrane integrity decreased to 42.0 ± 21.0%. It was observed that follicles located in the middle of the fragments were more protected from injuries and some of them showed good morphological appearance two hours post-warming. Mitochondria integrity of granulosa cells layer was clearly damaged by the vitrification protocol and ATP level in the follicles declined significantly after warming. Vitrification of zebrafish follicles in ovarian tissue fragments and its effect at sub-cellular level is reported here for the first time. Information gained from this study will help in guiding development of optimal protocol for cryopreservation of fish oocytes. PMID:24513460

  10. Histopathological alterations in the antral ovarian follicles in dairy cows with a tendency to emaciation.

    PubMed

    Pivko, J; Makarevich, A V; Kubovicova, E; Ostro, A; Hegedusová, Z; Louda, F

    2012-09-01

    The aim of the study was to define interrelationships between histopathological alterations in ovarian antral follicles and body condition in dairy cows with a tendency to emaciation (BCS 1 and 2) compared with dairy cows with normal body condition (BCS 3). The ovaries were recovered from slaughtered cyclic dairy cows (at the luteal phase of the cycle) of Czech Fleckvieh and Holstein breeds at different times of the post-partum period. The animals were estimated as belonging to certain grade of body condition score (BCS) according to a 5-point scale. Only dairy cows with BCS1 (emaciation; n=6), BCS2 (tendency to emaciation; n=5) and BCS3 (optimal body condition status; n=6) were available for the experiment. The ovarian samples were embedded into Technovit 7100 resin; the tissue sections were stained with buffered basic fuchsine with toluidine blue. For acidic mucopolysaccharides (aMPS) a combination of PAS-technique with Alcian blue was used. Histological analysis showed that emaciation was associated with an increased occurrence of late (cystic) and luteinization-related atresia in granulosa and theca cells and increased levels of aMPS in small atretic follicles. Our observations indicate that dairy cows with a tendency to emaciation (BCS 2) or emaciated (BCS 1) have elevated occurrence of late atresia and atresia with luteinization, while initial atresia is less. This expands our basic knowledge of ovarian histopathology providing new insight into the association of antral follicle atresia and body condition status in dairy cows. PMID:22806908

  11. Structural changes occurring during atresia in sheep ovarian follicles.

    PubMed

    Hay, M R; Cran, D G; Moor, R M

    1976-07-01

    The structural changes that characterize primary, secondary and tertiary atresia in sheep Graafian follicles have been studied by means of histological, histochemical and ultrastructural techniques. In primary atresia vacuoles representing swollen endoplasmic reticulum are prominent along the antral border together with disorganized granulosa cells containing pyknotic nuclei. Phagocytic cells, which increase in number as atresia progresses, were seen within the membrana granulosa and are considered to be transformed granulosa cells. Even in follicles classified as nonatretic, a few antral vacuoles and occasional pyknotic nuclei are present. During secondary atresia there is a large increase in the number of cells with pyknotic nuclei; many of these nuclei had been extruded and had fused to form the characteristic Feulgen-positive atretic bodies found along the edge of the antral cavity. These bodies usually have a diameter of up to 15 mum but occasionally reached as much as 400 mum. A second area of degeneration is frequently present in the membrana granulosa, two or three cell layers from the basal lamina, and it is at this level that exfoliation of granulosa cells occurs in tertiary atresia. In contrast to the membrana granulosa, there are during secondary atresia, only slight indications of degeneration in the cumulus. In tertiary atresia the membrana granulosa is highly disorganized; the atretic bodies are often fewer in number than at earlier stages. The basal lamina remains essentially intact. It is at this stage that the first clear signs of degeneration occur in the theca interna. Despite some disintegration of the cumulus, the integrity of the oocyte is maintained and its nucleus remains vesicular. Changes in the thecal microcirculation may plan a key role in atresia: adjacent to the basal lamina of non-atretic follicles, there is a well-developed capillary network which is significantly reduced as atresia progresses. PMID:991198

  12. In vitro development of secondary follicles from cryopreserved rhesus macaque ovarian tissue after slow-rate freeze or vitrification

    PubMed Central

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

    2011-01-01

    BACKGROUND Ovarian tissue cryopreservation is the only option for preserving fertility in prepubertal girls and cancer patients requiring immediate treatment. Following ovarian tissue cryopreservation, fertility can be restored after tissue transplant or in vitro follicle maturation. METHODS Macaque (n= 4) ovarian cortex was cryopreserved using slow-rate freezing (slow freezing) or vitrification. Tissues were fixed for histology or phosphohistone H3 (PPH3) analysis, cultured with bromodeoxyuridine (BrdU) or used for three-dimensional secondary follicle culture. Follicular diameter and steroid hormones were measured weekly. RESULTS Slow freezing induced frequent cryo-injuries while vitrification consistently maintained morphology of the stroma and secondary follicles. PPH3 was similar in fresh and vitrified, but sparse in slow-frozen tissues. BrdU uptake appeared diminished following both methods compared with that in fresh follicles. In vitro follicle survival and growth were greater in fresh than in cryopreserved follicles. Antrum formation appeared similar after vitrification compared with the fresh, but was reduced following slow freezing. Steroid production was delayed or diminished following both methods compared with fresh samples. CONCLUSIONS Secondary follicle morphology was improved after vitrification relative to slow freezing. Following vitrification, stroma was consistently more compact with intact cells typical to that of fresh tissue. BrdU uptake demonstrated follicle viability post-thaw/warming. For the first time, although not to the extent of fresh follicles, macaque follicles from cryopreserved tissue can survive, grow, form an antrum and produce steroid hormones, indicating some functional preservation. The combination of successful ovarian tissue cryopreservation with in vitro maturation of follicles will offer a major advancement to the field of fertility preservation. PMID:21705370

  13. 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. PMID:20207405

  14. Progesterone receptor membrane component 1 and its role in ovarian follicle growth.

    PubMed

    Peluso, John J

    2013-01-01

    Progesterone (P4) is synthesized in the ovary and acts directly on granulosa cells of developing ovarian follicles to suppress their rate of mitosis and apoptosis. Granulosa cells do not express nuclear progesterone receptor (PGR) but rather progesterone receptor membrane component-1 (PGRMC1). PGRMC1 binds P4 and mediates P4's actions, as evidenced by PGRMC1 siRNA studies. PGRMC1 acts by binding plasminogen activator inhibitor 1 RNA-binding protein and regulating gene expression. Specifically, PGRMC1 suppresses some genes that promote cell death (i.e., Bad, Caspase-3, Caspase-4). P4 regulates gene expression in part by inhibiting PGRMC1 binding to Tcf/Lef transcription sites, thereby reducing Tcf/Lef transcriptional activity. Since Tcf/Lef transcription sites are located within the promoters of genes that initiate mitosis and/or apoptosis (i.e., c-jun and c-myc), P4-PGRMC1 mediated suppression of these Tcf/Lef regulated genes could account for P4's actions. PGRMC1 expression is also altered in women with polycystic ovarian syndrome, premature ovarian failure and infertility. Collectively, these observations support a role for PGRMC1 in regulating human ovarian follicle development. PMID:23781168

  15. 3-Nitropropionic acid induces ovarian oxidative stress and impairs follicle in mouse.

    PubMed

    Zhang, Jia-Qing; Shen, Ming; Zhu, Cheng-Cheng; Yu, Feng-Xiang; Liu, Ze-Qun; Ally, Nazim; Sun, Shao-Chen; Li, Kui; Liu, Hong-Lin

    2014-01-01

    Oxidative stress induces many serious reproductive diseases in female mammals and thus poses a serious threat to reproductive health. However, the relationship between reactive oxygen species (ROS)-induced oxidative stress and follicular development, oocyte and embryo quality is not clear. The aim of this study was to investigate the effect of ovarian oxidative stress on the health of follicle and oocyte development. Female ICR mice were dosed with 3-nitropropionic acid (3-NPA) at three different concentrations (6.25, 12.5 and 25 mg/kg) and saline (control) via continuous intraperitoneal injection for 7 days. The treatment with 12.5 mg/kg reduced the weight of mouse ovaries, and significantly increased ROS levels and the activities of antioxidant enzymes--total superoxide dismutase (T-SOD), glutathione peroxidase (GPx) and catalase (CAT)--in granulosa cells and ovarian tissues, but not in other tissues (brain, liver, kidney and spleen). The same treatment significantly increased the percentage of atretic large follicles, and reduced the number of large follicles, the number of ovulated oocytes, and the capacity for early embryonic development compared with controls. It also significantly decreased the ratio of Bcl-2 to Bax, while causing an increase in the mRNA expression of (SOD2, CAT and GP X) and ROS levels in granulosa cells. Collectively, these data indicate that 3-NPA induces granulosa cell apoptosis, large follicle atresia, and an increase of ROS levels in the ovary. Therefore, we have established an in vivo model of ovarian oxidative stress for studying the mechanism of resulting damage induced by free radicals and for the screening of novel antioxidants. PMID:24505260

  16. 3-Nitropropionic Acid Induces Ovarian Oxidative Stress and Impairs Follicle in Mouse

    PubMed Central

    Zhang, Jia-Qing; Shen, Ming; Zhu, Cheng-Cheng; Yu, Feng-Xiang; Liu, Ze-Qun; Ally, Nazim; Sun, Shao-Chen; Li, Kui; Liu, Hong-Lin

    2014-01-01

    Oxidative stress induces many serious reproductive diseases in female mammals and thus poses a serious threat to reproductive health. However, the relationship between reactive oxygen species (ROS)—induced oxidative stress and follicular development, oocyte and embryo quality is not clear. The aim of this study was to investigate the effect of ovarian oxidative stress on the health of follicle and oocyte development. Female ICR mice were dosed with 3-nitropropionic acid (3-NPA) at three different concentrations (6.25, 12.5 and 25 mg/kg) and saline (control) via continuous intraperitoneal injection for 7 days. The treatment with 12.5 mg/kg reduced the weight of mouse ovaries, and significantly increased ROS levels and the activities of antioxidant enzymes—total superoxide dismutase (T-SOD), glutathione peroxidase (GPx) and catalase (CAT) — in granulosa cells and ovarian tissues, but not in other tissues (brain, liver, kidney and spleen). The same treatment significantly increased the percentage of atretic large follicles, and reduced the number of large follicles, the number of ovulated oocytes, and the capacity for early embryonic development compared with controls. It also significantly decreased the ratio of Bcl-2 to Bax, while causing an increase in the mRNA expression of (SOD2, CAT and GPX) and ROS levels in granulosa cells. Collectively, these data indicate that 3-NPA induces granulosa cell apoptosis, large follicle atresia, and an increase of ROS levels in the ovary. Therefore, we have established an in vivo model of ovarian oxidative stress for studying the mechanism of resulting damage induced by free radicals and for the screening of novel antioxidants. PMID:24505260

  17. [Isolation and expression of novel expressed sequence tags (ESTs) from ovarian follicles of Shaoxing ducks].

    PubMed

    Shu, Gang; Chen, Jie; Ni, Ying-Dong; Zhou, Yu-Chuan; Zhao, Ru-Qian

    2004-10-01

    Three expressed sequence tags ( ESTs), SXDF0201 (271 bp), SXDF0202 (200 bp) and SXDF0203 (173 bp), were isolated from ovarian follicles of Shaoxing ducks by using silver staining mRNA differential display. GenBank/BLAST analysis revealed that SXDF0201 was not homologous to any of the published sequences from all species, indicating that it was a novel EST and was then registered in GenBank (GenBank Accession No.: CB072629), while SXDF0202 and SXDF0203 were found to be highly homologous to seven known chicken ESTs and chicken mRNA for gizzard smooth muscle myosin heavy chain. 5'-RACE was employed to extend the SXDF0201 to 544 bp which was confirmed as novel in BLAST search. The temporal and spatial expression of SXDF0201 and SXDF0202 were also investigated with semi-quantitative RT-PCR. The result showed that: both SXDF0201 and SXDF0202 were found to be expressed in hypothalamus, pituitary, muscle, liver, and fat tissues of Shaoxing ducks; SXDF0201 was expressed significantly higher in ovaries of 30-day-old Shaoxing ducks compared with that of 60-day-old (P < 0.05) and 90-day-old (P = 0.015), but the expression of SXDF0202 showed no difference throughout the ovarian development; granulose layers expressed higher SXDF0201 than theca layers in almost all hierarchical follicles, the expression of SXDF0202 in granulose layers increased along with follicular maturation (P < 0.01) from Fw to F3 follicles, but decreased dramatically to the lowest in F1 follicles (P < 0.01). In theca layers, the highest expression of SXDF0202 was found in Fw follicles (P < 0.01). PMID:15552044

  18. A correlative microscopical analysis of differentiating ovarian follicles of mammals.

    PubMed

    Anderson, E; Wilkinson, R F; Lee, G; Meller, S

    1978-06-01

    The mammalian ovary has been studied by optical microscopy and by scanning and transmission electron microscopy with the purpose of presenting an integrated view of the differentiating mammalian follicle. During follicular development, changes in the granulosa cells are particularly noteworthy and include dramatic modifications in cell shape coincident with antrum formation. The cytoplasmic processes of those granulosa cells immediately surrounding the oocyte, as well as the more peripheral granulosa cells comprising a second and third layer, traverse the zona pellucida, infrequently interdigitate with the microvilli of the egg, and make both desmosomal and gap junction contacts with the oocyte. The zona pellucida is thus distinguished by numerous fenestrations of varying diameters. The membrana limitans (basal lamina) is a bipartite structure composed of (a) a homogenous stratum upon which the peripheral layer of granulosa cells rests, and (b) an outer region of collagen-like fibers. The specific advantages and limitations of the different methodologies utilized to study folliculo-genesis are discussed. PMID:671553

  19. 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.

  20. Persistent cAMP Signaling by Internalized LH Receptors in Ovarian Follicles.

    PubMed

    Lyga, Sandra; Volpe, Silvia; Werthmann, Ruth C; Götz, Konrad; Sungkaworn, Titiwat; Lohse, Martin J; Calebiro, Davide

    2016-04-01

    A crucial event in female reproduction occurs at midcycle, when a LH peak induces the final maturation of ovarian follicles. LH signals via a G protein-coupled receptor selectively expressed in the outermost follicular cell layers. However, how LH signals are relayed inside these cells and finally to the oocyte is incompletely understood. Here, we monitored LH signaling in intact ovarian follicles of transgenic mice expressing a fluorescent cAMP sensor. We found that LH stimulation induces 2 phases of cAMP signaling in all cell layers surrounding the oocyte. Interfering with LH receptor internalization abolished the second, persistent cAMP phase and partially inhibited oocyte meiosis resumption. These data suggest that persistent cAMP signals from internalized LH receptors contribute to transmitting LH effects inside follicle cells and ultimately to the oocyte. Thus, this study indicates that the recently proposed paradigm of cAMP signaling by internalized G protein-coupled receptors is implicated in receptor function and is physiologically relevant. PMID:26828746

  1. Oocyte production and ovarian steroid concentrations of immature rats in response to some commercial gonadotrophin preparations.

    PubMed

    Henderson, K M; Weaver, A; Wards, R L; Ball, K; Lun, S; Mullin, C; McNatty, K P

    1990-01-01

    Four commercial gonadotrophin preparations, namely Folligon, F.S.H.-P., Folltropin and Ovagen, were examined for their effects on oocyte production and ovarian steroid concentrations in immature rats. The ratios of the FSH to LH concentrations of the preparations, determined by radioreceptor assays, were Folligon 5, F.S.H.-P. 18, Folltropin 49 and Ovagen 1090. Forty-eight hours after administering each gonadotrophin preparation to immature rats, ovulation was induced by injection of chorionic gonadotrophin. Twenty-four hours later, oocytes were recovered from the oviducts and counted. Oocytes were produced after injection of chorionic gonadotrophin following a single injection of Folligon (10-50 i.u.). However, no oocytes were produced in response to the other gonadotrophin preparations unless they were administered by continuous infusion (30-1000 micrograms day-1). When given by injection (Folligon) or infusion (others), the gonadotrophin preparations all promoted a dose-dependent increase in mean oocyte production, except at the highest doses when mean oocyte numbers either remained unchanged or declined significantly in the cases of Folligon and F.S.H.-P. The highest mean numbers of oocytes produced in response to Folltropin (48 +/- 9 oocytes, mean +/- s.e.m.) and Ovagen (47 +/- 7) were greater than those attained with Folligon (21 +/- 6) or F.S.H.-P. (31 +/- 5). Mean ovarian weights also increased in a dose-dependent fashion in response to each of the gonadotrophin preparations. Measurements of ovarian steroid concentrations 48 h after the onset of gonadotrophin treatment (i.e. immediately prior to ovulation induction with chorionic gonadotrophin) showed that the gonadotrophin preparations markedly influenced the ratios of ovarian oestradiol-17 beta and androgen (androstenedione plus testosterone) concentrations. At low doses the gonadotrophin preparations increased the ratio of oestradiol-17 beta to androgens, but at the highest doses, with the exception of

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

    PubMed

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

    2016-04-01

    This study investigated the effect of adding different concentrations of bovine recombinant follicle-stimulating hormone on the IVC of equine preantral follicles enclosed in ovarian tissue fragments. Randomized ovarian fragments were fixed immediately (fresh noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0, 10, 50, and 100 ng/mL FSH and subsequently analyzed by classical histology. Culture media collected on Day 1 or Day 7 and were analyzed for steroids (estradiol and progesterone) and reactive oxygen species (ROS). After Day 1 and Day 7 of culture, 50-ng/mL FSH treatment had a greater (P < 0.05) percentage of morphologically normal follicles when compared to the other groups, except the 10-ng/mL FSH treatment at Day 1 of culture. The percentage of developing follicles (transition, primary, and secondary), and follicular and oocyte diameters were higher (P < 0.05) in the 50-ng/mL FSH treatment compared to the other groups after Day 7 of culture. Furthermore, estradiol secretion and ROS production were maintained (P > 0.05) throughout the culture in the 50-ng/mL FSH treatment. In conclusion, the addition of 50 ng/mL of FSH promoted activation of primordial follicles to developing follicles, improved survival of preantral follicles, and maintained estradiol and ROS production of equine ovarian tissue after 7 days of culture. PMID:26723132

  3. 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

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

    PubMed

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

    2016-04-01

    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

  5. Effects of an agonist of gonadotropin-releasing hormone on ovarian follicles in cattle.

    PubMed

    Macmillan, K L; Thatcher, W W

    1991-12-01

    Three experiments were conducted to examine effects of Buserelin, a potent agonist of gonadotropin-releasing hormone, on characteristics of ovarian follicles in cycling cows and heifers. In experiment 1, heifers were injected once with 10 micrograms Buserelin on Day 11, 12, or 13 of the estrous cycle (estrus = Day 0), or once with 20 micrograms of Buserelin on Day 12. Additionally, two groups were injected with a luteolytic dose of prostaglandin F2 alpha (PGF2 alpha) on Day 13 preceded with or without a Buserelin injection (10 micrograms) on Day 12. A control group did not receive a Buserelin injection. Ovaries were recovered and weighed after animals were slaughtered on Day 15. Follicle diameters were measured with calipers. Follicles for all experiments were classified as small (class 1: 3-5 mm diameter), medium (class 2: 6-9 mm), or large (class 3: greater than 9 mm). Heifers receiving only Buserelin had an increased number of medium-sized follicles compared to controls. Buserelin injection administered 24 h before PGF2 alpha reduced the decline in the average weight of the ovaries containing the corpus luteum (7.8 g for Buserelin before PGF2 alpha vs. 6.7 g for no Buserelin before PGF2 alpha). Buserelin pretreatment appeared to delay or prevent complete luteolysis by the injected PGF2 alpha. In experiment 2, 0, or 10 micrograms Buserelin was injected on Day 12 and follicle development was monitored by ultrasonography in situ from Day 12 to estrus. Follicles also were classified as clear or cloudy; cloudy was associated with flocculent material in the follicular fluid or with an indistinct follicular wall.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1805991

  6. Interpenetrating Fibrin-Alginate Matrices for in vitro Ovarian Follicle Development

    PubMed Central

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

    2010-01-01

    In this report, we investigate the fibrin-alginate inter penetrating network (FA-IPN) to provide dynamic cell-responsive mechanical properties, which we apply to the in vitro growth of ovarian follicles. The mechanical properties and polymerization rate of the gels were investigated by rheology, and the fiber structure was imaged by electron microscopy. Using a mouse model, two-layered secondary follicles were encapsulated in FA-IPNs, and growth, morphology, hormone production, fibrin degradation rate and the numbers of competent eggs were assessed. The initial mechanics of the FA-IPN are determined by the composite material, and subsequent degradation of fibrin by the encapsulated cells would produce a material with mechanical properties due to the alginate alone. The rate of meotically competent oocytes produced by culture in FA-IPN was 82%, which was significantly greater than in alginate alone. This increase in oocyte quality is an important step in identifying 3D culture systems that can provide a fundamental tool to investigate follicle maturation, and may be applied to promote the growth of human follicles, which can be used to provide reproductive options for women facing a cancer diagnosis PMID:19616843

  7. 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

  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. PMID:20071017

  9. X-Ray Binary Stars and Ovarian Follicle Growth: Math Models in Astrophysics and Physiology

    NASA Astrophysics Data System (ADS)

    Sarty, Gordon E.; Saxton, Curtis; Wu, Kinwah; Pierson, Roger

    2010-10-01

    The scientific investigation of Nature relies on mathematics. While mathematics itself is precise, its application to the understanding of nature always involves an approximation. The approximate nature of mathematical application ultimately limits the scientific approach to Nature, but the freedom to make appropriate approximations allows us to understand complex phenomena on the basis of a few simple principles in approaches that are limited only by imagination. Here we review and compare two mathematical models of natural phenomena in astrophysics and physiology. The astrophysical model describes the emission of polarized radiation from a magnetic cataclysmic variable; the physiological model describes growth and competition among ovarian follicles under the influence of circulating hormones. In each case, the understanding offered by the mathematical model must be verified by fitting the model to data; this is part of the usual scientific method. In the follicle model case, we show how model fits may also be used for medical diagnostic purposes.

  10. Connexin 37 and 43 gene and protein expression and developmental competence of isolated ovine secondary follicles cultured in vitro after vitrification of ovarian tissue.

    PubMed

    Sampaio da Silva, Andréa Moreira; Bruno, Jamily Bezerra; de Lima, Laritza Ferreira; Ribeiro de Sá, Naíza Arcângela; Lunardi, Franciele Osmarini; Ferreira, Anna Clara Accioly; Vieira Correia, Hudson Henrique; de Aguiar, Francisco Léo Nascimento; Araújo, Valdevane Rocha; Lobo, Carlos Henrique; de Alencar Araripe Moura, Arlindo; Campello, Cláudio Cabral; Smitz, Johan; de Figueiredo, José Ricardo; Ribeiro Rodrigues, Ana Paula

    2016-05-01

    Cryoinjuries caused by vitrification of tissues and organs lead to the loss of membrane proteins that mediate intercellular communications, such as connexins 37 (Cx37) and 43 (Cx43). Thus, the present study aimed to evaluate ovine Cx37 and Cx43 gene and protein expressions and developmental competence by in vitro-cultured secondary follicles retrieved from vitrified ovarian tissue. Ovarian fragments for the same ovary pair were distributed into six treatments: (1) fresh ovarian tissue (FOT); (2) vitrified ovarian tissue (VOT); (3) isolated follicles from fresh ovarian tissue (FIF); (4) isolated follicles from vitrified ovarian tissue; (5) isolated follicles from fresh ovarian tissue followed by in vitro culture (CFIF); (6) isolated follicles from vitrified ovarian tissue followed by in vitro culture (CVIF). In all treatments, Cx37 and Cx43 gene and protein expression patterns were evaluated by reverse transcription polymerase chain reaction and immunocytochemistry. In addition, secondary follicles were analyzed according to follicular integrity and growth, apoptosis, and cell proliferation. In vitro-cultured secondary follicles (CFIF and CVIF) were evaluated based on morphology (extruded follicles), antrum formation, and viability. The percentage of intact follicles was higher, whereas antrum formation, oocyte extrusion rate, and follicle viability were lower in CVIF than in CFIF treatment (P < 0.05). Terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphates nick end-labeling assay demonstrated that apoptosis was absent in FIF, whereas follicles from all other treatments showed positive labeling. Cell proliferation index was higher in isolated follicles from vitrified ovarian tissue and CVIF treatments than in follicles from FIF. Expression of Cx43 messenger RNA was lower in CVIF treatment when compared with follicles from all other treatments (P < 0.05). Follicle Cx37 messenger RNA levels did not show alterations in any

  11. 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. PMID:26842836

  12. Alteration in localization of steroid hormone receptors and coregulatory proteins in follicles from cows with induced ovarian follicular cysts.

    PubMed

    Salvetti, Natalia R; Alfaro, Natalia S; Velázquez, Melisa M L; Amweg, Ayelen N; Matiller, Valentina; Díaz, Pablo U; Ortega, Hugo H

    2012-12-01

    Cystic ovarian disease (COD) is an important cause of infertility in cattle. The altered follicular dynamics and cellular differentiation observed in COD may be mediated through a disruption of the expression of steroid receptors and their associated transcriptional cofactors. The aim of this study was to determine the protein expression profiles of ESR1, ESR2, PGR, AR, NCOA3, NCOR2, and PHB2 (REA) in ovarian follicles in an experimental model of COD induced by the administration of ACTH. Ovaries were collected and follicles were dissected from heifers during the follicular phase (control) or from heifers treated with ACTH to induce the formation of ovarian follicular cysts. Ovaries were fixed, sectioned, and stained immunohistochemically for steroid receptors and the associated transcription factors. The relative expression of ESR1 was similar in follicular cysts and in tertiary follicles from both control and cystic cows and was significantly higher than in secondary follicles. The expression of ESR2 in the granulosa was higher in cystic follicles. No differences were seen for PGR. The expression of androgen receptor was significantly increased in tertiary follicles with lower immunostaining in cysts. The expression of NCOA3 was observed in the granulosa and theca with a significantly increased expression in the theca interna of cystic follicles. The highest levels of NCOR2 expression in granulosa, theca interna, and theca externa were observed in cysts. In granulosa cells, NCOR2 levels increase progressively as follicles mature and the treatment had no effect. In summary, ovaries from animals with induced COD exhibited altered steroid receptor expression compared with normal animals, as well as changes in the expression of their regulators. It is reasonable to suggest that in conditions characterized by altered ovulation and follicular persistence, such as COD, changes in the intra-ovarian expression of these proteins could play a role in their pathogenesis

  13. 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.

  14. Influence of triiodothyronine (T(3)) on secretion of steroids and thyroid hormone receptor expression in chicken ovarian follicles.

    PubMed

    Sechman, A; Pawlowska, K; Rzasa, J

    2009-08-01

    The present study was designed to (1) assess the role of triiodothyronine (T(3)) with regard to in vitro steroid hormone secretion by chicken ovarian follicles; (2) determine whether T(3) influences the in vivo function of the pituitary-ovarian axis in the hen; and (3) detect expression of thyroid hormone receptor (TR) mRNA in chicken ovarian follicles. In the first experiment, laying hens were decapitated 22.5h before ovulation. White prehierarchical follicles (1-8mm) and fragments of theca and granulosa layers of the 3 largest yellow preovulatory follicles F3-F1 (22-35mm) were incubated in a medium supplemented with T(3) (0, 0.1, 1, 10, 100, or 1000ng/mL) or ovine luteinizing hormone (LH) (10ng/mL) in combination with doses of T(3) (1, 10, and 100ng/mL). Triiodothyronine decreased basal and LH-stimulated estradiol secretion by white follicles and the theca layer of all preovulatory follicles. On the other hand, it increased progesterone secretion by F2 and F1 follicles. In the second experiment, hens were injected 1h after ovulation with saline (control) or T(3) (10microg/100g body weight, intraperitoneally). Results indicated that exogenous T(3) decreased plasma concentrations of LH and estradiol and increased plasma concentrations of progesterone. In the third experiment, using reverse transcription polymerase chain reaction (RT-PCR) analysis, expression of thyroid hormone receptor (TRalpha and TRbeta0), mRNA was detected in all of the ovarian compartments. The expression of TRalpha mRNA was relatively greater in comparison with TRbeta0. There were no differences between white ovarian follicles in the expression of TRalpha and TRbeta0 mRNA. A considerably higher TRalpha and lower TRbeta0 expression was detected in the granulosa layer of preovulatory follicles in comparison with the theca layer. In conclusion, the data indicate that thyroid hormones acting via nuclear receptors are involved in regulation of the pituitary-ovarian axis and processes associated

  15. Immunolocalization of zona pellucida antigens in the ovarian follicle of dogs, cats, horses and elephants.

    PubMed

    Barber, M R; Lee, S M; Steffens, W L; Ard, M; Fayrer-Hosken, R A

    2001-05-01

    A comparative evaluation of the location of immunoreactive porcine zona pellucida (pZP) glycoproteins was performed with polyclonal rabbit anti-pZP antibodies on ovarian sections of the dog, cat, horse, and elephant. For this, formalin (light microscopy) and glutaraldehyde (transmission electron microscopy [TEM]) fixed ovarian sections were incubated with antibodies raised against highly purified pZP. Staining patterns were determined with diaminobenzidine (DAB) at the light level. The dog ZP had a distinct staining distribution that is characterized by intense staining around the periphery of the ZP and the oolemma and less dense staining throughout the width of the ZP. In dog follicles that contained multiple oocytes, there were oocytes of identical and dissimilar stages. Cat ovarian sections showed uniform staining of the ZP. Horse results showed uniform staining of ZP and ooplasm, and granulosa cells (GC). Elephant sections showed staining of the ZP with dense staining at the oolemma, as well as staining of the ooplasm. In all species the staining of the ZP was not evident until GC differentiation. In all cases there was no staining of ovarian tissue with control normal rabbit serum. Specific staining patterns of ZP were evaluated by TEM and immunogold staining. The immunogold-linked anti-pZP antibodies stained the ZP matrix in all species. There was staining of ooplasm organelles suggesting that ZP secretion originates from the oocyte of the dog and cat. In addition, follicular and ZP measurements were taken that allowed accurate characterization of follicle stage. These findings suggest that in all four species the ZP is recognized by anti-pZP antibodies and there is also evidence to suggest the possible origins of ZP glycoproteins. PMID:11393221

  16. 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

  17. Development of Follicle-Stimulating Hormone Receptor Binding Probes to Image Ovarian Xenografts

    PubMed Central

    Lee, Chung-Wein; Guo, Lili; Matei, Daniela; Stantz, Keith

    2015-01-01

    The Follicle-Stimulating Hormone Receptor (FSHR) is used as an imaging biomarker for the detection of ovarian cancer (OC). FSHR is highly expressed on ovarian tumors and involved with cancer development and metastatic signaling pathways. A decapeptide specific to the FSHR extracellular domain is synthesized and conjugated to fluorescent dyes to image OC cells in vitro and tumors xenograft model in vivo. The in vitro binding curve and the average number of FSHR per cell are obtained for OVCAR-3 cells by a high resolution flow cytometer. For the decapeptide, the measured EC50 was 160 μM and the average number of receptors per cell was 1.7 × 107. The decapeptide molecular imaging probe reached a maximum tumor to muscle ratio five hours after intravenous injection and a dose-dependent plateau after 24–48 hours. These results indicate the potential application of a small molecular weight imaging probe specific to ovarian cancer through binding to FSHR. Based on these results, multimeric constructs are being developed to optimize binding to ovarian cells and tumors. PMID:26779384

  18. Pre-diagnostic circulating follicle stimulating hormone (FSH) concentrations and ovarian cancer risk

    PubMed Central

    McSorley, MA; Alberg, AJ; Allen, DS; Allen, NE; Brinton, LA; Dorgan, JF; Kaaks, R; Rinaldi, S; Helzlsouer, KJ

    2009-01-01

    Gonadotropins have been indicted in ovarian carcinogenesis but direct evidence has been limited and inconsistent. The aim of this study was to determine the association between pre-diagnostic levels of follicle stimulating hormone (FSH) and subsequent development of invasive epithelial ovarian cancer. A nested case-control study was conducted using cases and controls drawn from three cohorts: CLUE I and CLUE II of Washington County, MD, and the Island of Guernsey Study, UK. In total, 67 incident invasive epithelial ovarian cancer cases were each matched to one to two controls on age, menopausal status, time since last menstrual period, current hormone use, and other relevant factors. FSH concentrations were classified into ranked thirds of low, medium, or high based on the distribution among controls. Conditional logistic regression was used to estimate the odds ratio (OR) across increasing thirds of FSH concentrations. Results of the analysis showed that ovarian cancer risk decreased with higher FSH concentrations (p-trend =0.005). Compared to the lowest third of FSH concentrations, the odds ratio among those in the middle and highest thirds were 0.45 (95% Confidence Interval (CI) 0.20– 1.00) and 0.26 (95% CI 0.10–0.70), respectively. Associations persisted after excluding cases diagnosed within five years of follow-up. In conclusion, a reduction in subsequent risk of invasive epithelial ovarian cancer was observed among women with higher circulating FSH concentrations. These findings contradict the hypothesized role of FSH as a risk factor in ovarian carcinogenesis. PMID:19444906

  19. 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. PMID:26968252

  20. Suppression in the secretion of follicle-stimulating hormone and luteinizing hormone, and ovarian follicle development in heifers continuously infused with a gonadotropin-releasing hormone agonist.

    PubMed

    Gong, J G; Campbell, B K; Bramley, T A; Gutierrez, C G; Peters, A R; Webb, R

    1996-07-01

    A novel experimental model was developed in cattle to investigate the requirement for FSH and LH during ovarian follicle growth and development. On Day 5 of the estrous cycle, 7 heifers were each implanted with an osmotic minipump containing a GnRH agonist (GnRHa), Buserelin (release rate, 2.5 micrograms/h). Another 7 heifers served as controls. Each minipump was replaced 28 days later with a second pump, which was left in place for a further 20 days. Blood samples were collected daily throughout the experimental period, and frequent samples were also collected on both days of minipump insertion and at 10 days after insertion of the second pump. The ovaries of all heifers were scanned daily by real-time ultrasonography to monitor follicular dynamics. All controls displayed 2 or 3 waves of FSH and follicular development per estrous cycle during the experiment. Insertion of the first minipump produced a large LH and FSH surge and induced ovulation in all 7 animals. Within 8 days of the start of treatment, serum LH concentrations fell to basal levels; they then remained constant at this level throughout the infusion period, only beginning to recover 4-5 days after the termination of infusion. After the initial increase, FSH returned to basal levels before showing a normal wave that was coincident with the emergence, growth, and regression of a dominant follicle. However, despite the peak levels of FSH, dominant follicles from the next wave failed to grow beyond 7-9 mm; they remained at this size for 3 wk until 3-4 days after insertion of the second minipump, when FSH fell precipitously to reach low levels that were maintained throughout the remainder of the infusion. After this fall in FSH concentrations, these follicles regressed rapidly, and no antral follicles > 4 mm were detected until after the termination of treatment. Thereafter, FSH concentrations increased significantly; the increase was accompanied by the emergence of a follicular wave and development of a

  1. 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.

  2. Adrenocorticotropic Hormone Suppresses Gonadotropin-Stimulated Estradiol Release from Zebrafish Ovarian Follicles

    PubMed Central

    Alsop, Derek; Ings, Jennifer S.; Vijayan, Mathilakath M.

    2009-01-01

    While stress is known to impact reproductive performance, the pathways involved are not entirely understood. Corticosteroid effects on the functioning of the hypothalamus-pituitary-gonadal axis are thought to be a key aspect of stress-mediated reproductive dysfunction. A vital component of the stress response is the pituitary secretion of adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. We recently reported MC2R mRNA abundance in fish gonads leading to the hypothesis that ACTH may be directly involved in gonadal steroid modulation. Using zebrafish (Danio rerio) ovarian follicles, we tested the hypothesis that acute ACTH stimulation modulates cortisol and estradiol (E2) secretion. ACTH neither affected cortisol nor unstimulated E2 release from ovarian follicles. However, ACTH suppressed human chorionic gonadotropin (hCG)-stimulated E2 secretion in a dose-related manner, with a maximum decrease of 62% observed at 1 I.U. ACTH mL−1. This effect of ACTH on E2 release was not observed in the presence of either 8-bromo-cAMP or forskolin, suggesting that the mechanism(s) involved in steroid attenuation was upstream of adenylyl cyclase activation. Overall, our results suggest that a stress-induced rise in plasma ACTH levels may initiate a rapid down-regulation of acute stimulated E2 biosynthesis in the zebrafish ovary, underscoring a novel physiological role for this pituitary peptide in modulating reproductive activity. PMID:19649243

  3. 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. PMID:23962763

  4. 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

  5. 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

  6. 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

  7. Transcriptomic Diversification of Developing Cumulus and Mural Granulosa Cells in Mouse Ovarian Follicles1

    PubMed Central

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

    2014-01-01

    ABSTRACT 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. PMID:25376232

  8. Preservation of bovine preantral follicle viability and ultra-structure after cooling and freezing of ovarian tissue.

    PubMed

    Celestino, Juliana Jales de Hollanda; dos Santos, Regiane Rodrigues; Lopes, Cláudio Afonso Pinho; Martins, Fabrício Sousa; Matos, Maria Helena Tavares; Melo, Mônica Aline Parente; Báo, Sônia Nair; Rodrigues, Ana Paula Ribeiro; Silva, José Roberto Viana; de Figueiredo, José Ricardo

    2008-11-01

    Bovine preantral follicles within ovarian fragments were exposed and cryopreserved in absence or presence of 1.5M glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO), undergoing a previous cooling at 20 degrees C for 1h (protocol 1) or at 4 degrees C for 24h (protocol 2) in 0.9% saline solution. At the end of each treatment, preantral follicles were classified as non-viable/viable when they were stained/not stained with trypan blue, respectively. To confirm viability staining, ultra-structure of the follicles was evaluated by transmission electronic microscopy (TEM). Data were compared by Chi-square test (P<0.05). The storage of the ovaries at 20 degrees C for 1h (78%) and 4 degrees C for 24h (80%) did not reduce significantly the percentage of viable preantral follicles when compared to the control (75%). Similar results were obtained when ovarian fragments, respectively, for protocols 1 and 2, were exposed to MEM (78 and 77%), 1.5M EG (78 and 71%), as well as frozen in 1.5M EG (74 and 77%). Percentages of viable follicles in control were similar to those observed after exposure (75%) and freezing (76%) in presence of 1.5M DMSO only when protocol 1 was used. The increase of the concentration from 1.5 to 3.0M, for all cryoprotectants, reduced significantly the percentage of viable preantral follicles after freezing. Ultra-structural analysis has confirmed trypan blue results, showing that not only basement membrane, but also organelles, were intact in viable preantral follicles. In conclusion, ovarian tissue cooling at 4 degrees C for 24h before cryopreservation (protocol 2) does not affect the viability of bovine preantral follicles when 1.5M EG is present in the cryopreservation medium. PMID:17945440

  9. Growing Teratoma Syndrome Secondary to Ovarian Giant Immature Teratoma in an Adolescent Girl

    PubMed Central

    Li, Song; Liu, Zhenzhen; Dong, Chengyong; Long, Fei; Liu, Qinlong; Sun, Deguang; Gao, Zhenming; Wang, Liming

    2016-01-01

    Abstract Growing teratoma syndrome (GTS) is a rare clinical entity first described by Logothetis et al in 1982. Although it is unusual for GTS to be located in the ovary, this report is of a case of an adolescent girl who underwent a complete surgical resection of the mass. Histopathology confirmed only an immature teratoma had originated from the ovary and so she received adjuvant chemotherapy with blemycin, etopside, and cisplatin over 4 cycles. Results from an abdominal enhanced CT (computed tomography) 9 years later revealed a giant mass had compressed adjacent tissues and organs. Laparotomy was performed and a postoperative histopathology showed the presence of a mature teratoma, and so the diagnosis of ovarian GTS was made. One hundred one cases of ovarian GTS from English literature published between 1977 and 2015 were collected and respectively analyzed in large samples for the first time. The median age of diagnosis with primary immature teratoma was 22 years (range 4–48 years, n = 56). GTS originating from the right ovary accounted for 57% (27/47, n = 47) whereas the left contained 43% (20/47, n = 47). Median primary tumor size was 18.7 cm (range 6–45 cm, n = 28) and median subsequent tumor size was 8.6 cm (range 1–25 cm, n = 25). From the primary treatment to the diagnosis of ovarian GTS, median tumor growth speed was 0.94 cm/month (range 0.3–4.3 cm/month, n = 21). Median time interval was 26.6 months (range 1–264 months, n = 41). According to these findings, 5 patients did have a pregnancy during the time interval between primary disease and GTS, making our patient the first case of having a pregnancy following the diagnosis of ovarian GTS. Because of its high recurrence and insensitiveness to chemotherapy, complete surgical resection is the preferred treatment and fertility-sparing surgery should be considered for women of child-bearing age. Anyhow GTS of the ovary has an excellent prognosis. Patients with GTS had

  10. Temporal expression of GDF-9 and BMP-15 mRNAs in canine ovarian follicles.

    PubMed

    Palomino, Jaime; De Los Reyes, Monica

    2016-10-01

    This study aimed to investigate the expression profiles of growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) mRNA in canine oocytes and follicular cells throughout development at the different phases of the estrus cycle. Ovarian structures (follicles and CL) and plasma progesterone concentration were used to confirm the physiological status of each donor. Denuded oocytes and their follicular cells were recovered from follicles (n = 675) distributed into 4 types (preantral, small antral ∼0.2-0.39 mm, medium antral ∼0.4-5.9 mm, and large antral ∼6-8 mm). Total RNA was extracted and reverse transcribed, and the levels of expression for these 2 genes were determined using a quantitative real-time polymerase chain reaction technique; the data were evaluated by ANOVA. Relative expressions levels of GDF-9 and BMP-15 transcripts were detected in the oocyte and follicular cells in all follicular stages evaluated, showing differential changes (P < 0.05) during development over the estrus cycle. The expression patterns of both transcripts were highly correlated between follicles cells and oocytes (r > 0.8; P < 0.05 for GDF-9 and BMP-15), although GDF-9 was expressed at higher levels (P < 0.05) in the oocyte compared with the follicle cells. All cell types showed more GDF-9 mRNA abundance at early developing stages, mainly in the anestrus phase, and declining levels in the later stages (P < 0.05), whereas BMP-15 mRNA levels increased (P < 0.05) in follicular cells and oocytes from the preantral to the later stages, and remained constant during the final preovulatory stage. In conclusion, these two genes were detected in follicular cells and oocytes and were differentially expressed during the follicular development across the estrus cycle. PMID:27341772

  11. Insulin-like growth factor I in sera, ovarian follicles and follicular fluid of cows with spontaneous or induced cystic ovarian disease.

    PubMed

    Ortega, Hugo H; Palomar, Martin M; Acosta, Juan C; Salvetti, Natalia R; Dallard, Bibiana E; Lorente, Juan A; Barbeito, Claudio G; Gimeno, Eduardo J

    2008-06-01

    The objective of this research was to determine changes in IGF-I levels in serum and follicular fluid, and immunoreactivity of the follicle wall of cows with spontaneous (slaughter specimens) or ACTH-induced follicular cysts, and to compare results to normal cycling (control) cows after selection of the ovulatory follicle. Concentrations of IGF-I in serum did not differ between control and cystic animals (p=0.76). Fluid from the ovulatory follicle in control cows had 41% higher concentrations of IGF-I than that from cystic follicles collected at slaughter (spontaneous cysts; p<0.05) and 70% higher than that in induced follicular cysts (p<0.05). An intense positive immunostaining with anti-IGF-I was observed in granulosa cells (p<0.05) and in the theca interna (p<0.05) of secondary and tertiary follicles in all three groups of animals, but staining was less intense in cystic (p<0.05) and atretic follicles (p<0.05). This study provides evidence to suggest that cystic ovarian disease in cattle is associated with decreased concentrations of IGF-I in follicular fluid, but not in serum, and decreased production of IGF-I in the follicular wall. These data support the notion that IGF-I plays a role in the regulation of folliculogenesis, and may participate in the pathogenesis of cystic ovarian disease in cattle. PMID:17631370

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

    PubMed

    Ganesan, Shanthi; Keating, Aileen F

    2016-02-01

    Phosphoramide mustard (PM) is an ovotoxic metabolite of cyclophosphamide and destroys primordial and primary follicles potentially by DNA damage induction. The temporal pattern by which PM induces DNA damage and initiation of the ovarian response to DNA damage has not yet been well characterized. This study investigated DNA damage initiation, the DNA repair response, as well as induction of follicular demise using a neonatal rat ovarian culture system. Additionally, to delineate specific mechanisms involved in the ovarian response to PM exposure, utility was made of PKC delta (PKCδ) deficient mice as well as an ATM inhibitor (KU 55933; AI). Fisher 344 PND4 rat ovaries were cultured for 12, 24, 48 or 96h in medium containing DMSO ±60μM PM or KU 55933 (48h; 10nM). PM-induced activation of DNA damage repair genes was observed as early as 12h post-exposure. ATM, PARP1, E2F7, P73 and CASP3 abundance were increased but RAD51 and BCL2 protein decreased after 96h of PM exposure. PKCδ deficiency reduced numbers of all follicular stages, but did not have an additive impact on PM-induced ovotoxicity. ATM inhibition protected all follicle stages from PM-induced depletion. In conclusion, the ovarian DNA damage repair response is active post-PM exposure, supporting that DNA damage contributes to PM-induced ovotoxicity. PMID:26708502

  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. PMID:26503557

  14. Different follicle stimulating hormone/luteinizing hormone ratios for ovarian stimulation.

    PubMed

    Duijkers, I J; Vemer, H M; Hollanders, J M; Willemsen, W N; Bastiaans, L A; Hamilton, C J; Thomas, C M; Borm, G F

    1993-09-01

    The aim of the present study was to investigate whether reducing the amount of luteinizing hormone (LH) in gonadotrophic preparations impairs follicular growth in in-vitro fertilization (IVF) cycles during suppression of endogenous LH levels. A selected group of 20 IVF patients was randomly divided into two groups. One group was treated with Org 31338 [follicle stimulating hormone (FSH)/LH 3:1], the other group with Metrodin (purified FSH), both during pituitary down-regulation with buserelin. A fixed daily dose of 150 IU FSH i.m. was given. Serum concentrations of FSH, LH, oestradiol and progesterone were determined frequently and serial ultrasound examinations were performed. Multiple follicular growth with concomitant rise of oestradiol levels was observed in all cycles. The duration of the stimulation phase was shorter in the group treated with Org 31338 than in the group treated with Metrodin. The number of follicles and oocytes and the fertilization rate was larger and the mean embryo quality was higher in the Org 31338 group, but the differences did not reach statistical significance. No significant differences were found in hormonal values. In women with normal endocrine profiles, lowering of the LH activity in gonadotrophic preparations during gonadotrophin-releasing hormone agonist treatment results in adequate ovarian stimulation. However, a preparation with some LH needed a shorter stimulation than a purified FSH preparation. Whether the other beneficial effects of Org 31338 also occur in a larger population needs further investigation. PMID:8253923

  15. Rate of decline of cortisol concentrations in ovarian follicles following ACTH treatment in the sow.

    PubMed Central

    Montgomery, A; Viveiros, M; Cummings, E; Liptrap, R

    1997-01-01

    The rates of decline in cortisol concentrations in blood and ovarian follicular fluid were assessed in cyclic sows (n = 30) after treatment with saline or a depot form of adrenocorticotrophic hormone (ACTH). After a single injection of ACTH (0.5 iu/kg, BW, i.m.), peak cortisol values were achieved in blood within 3 to 4 h followed by a half-life net clearance rate (t1/2 of 2.40 +/- 0.29 (SE) h. The same dose of ACTH was then given at 12 h intervals from days 9 to 13 of the estrous cycle. On day 14 the concentrations of cortisol in follicular fluid were higher (P < 0.05) in ACTH-injected sows than in saline-injected controls. A t1/2 value of 37.81 h was determined for cortisol based on the decline in concentrations in follicular fluid collected on days 14, 16 and 18. This relatively slow rate of removal from developing ovarian follicles may have implications for the previously observed detrimental effects of increased cortisol concentrations on follicular development. PMID:9342457

  16. 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. PMID:17637101

  17. Imprint cytology of high-grade immature ovarian teratoma: a case report, literature review, and distinction from other ovarian small round cell tumors.

    PubMed

    Ramalingam, Preetha; Teague, Daniel; Reid-Nicholson, Michelle

    2008-08-01

    Immature ovarian teratoma (IOT) is a rare and aggressive malignant neoplasm characterized by immature neural tissue. The cytomorphologic features have only rarely been described. We herein describe an additional case and review the literature regarding this entity. To the best of our knowledge, this is the first reported case with imprint cytology. A 35-year-old woman presented with a pelvic mass which was resected and sent for frozen section evaluation. Imprint smears and frozen section of the mass were diagnostic of IOT. IOT has diagnostic cytologic features which show complete concordance with histology. Differential diagnoses include other small round cell neoplasms such as ovarian neuroblastoma, small cell carcinoma of hypercalcemic type, primitive neuroectodermal tumor, Wilm's tumor, desmoplastic small round cell tumor, and Non-Hodgkin lymphoma. Distinguishing IOT from these tumors can be challenging however if diligent morphologic study and/or ancillary studies are performed accurate diagnosis is possible. PMID:18618728

  18. 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

  19. 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.

  20. Luteinizing hormone and follicle stimulating hormone synergy: A review of role in controlled ovarian hyper-stimulation

    PubMed Central

    Raju, Gottumukkala Achyuta Rama; Chavan, Rahul; Deenadayal, Mamata; Gunasheela, Devika; Gutgutia, Rohit; Haripriya, Geetha; Govindarajan, Mirudhubashini; Patel, Nayana Hitesh; Patki, Ameet Shashikant

    2013-01-01

    Luteinizing hormone (LH) in synergy with follicle stimulating hormone (FSH) stimulates normal follicular growth and ovulation. FSH is frequently used in assisted reproductive technology (ART). Recent studies have facilitated better understanding on the complementary role of the LH to FSH in regulation of the follicle; however, role of LH in stimulation of follicle, optimal dosage of LH in stimulation and its importance in advanced aged patients has been a topic of discussion among medical fraternity. Though the administration of exogenous LH with FSH is obligatory for controlled ovarian stimulation in patients with hypogonadotropic hypogonadism, there is still a paucity of information of its usage in other patient population. In this review we looked in to the multiple roles that LH plays complementary to FSH to better understand the LH requirement in patients undergoing ART. PMID:24672160

  1. 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

  2. Cisplatin and Doxorubicin Induce Distinct Mechanisms of Ovarian Follicle Loss; Imatinib Provides Selective Protection Only against Cisplatin

    PubMed Central

    Morgan, Stephanie; Lopes, Federica; Gourley, Charlie; Anderson, Richard A.; Spears, Norah

    2013-01-01

    Purpose Chemotherapy treatment in premenopausal women has been linked to ovarian follicle loss and premature ovarian failure; the exact mechanism by which this occurs is uncertain. Here, two commonly used chemotherapeutic agents (cisplatin and doxorubicin) were added to a mouse ovary culture system, to compare the sequence of events that leads to germ cell loss. The ability of imatinib mesylate to protect the ovary against cisplatin or doxorubicin-induced ovarian damage was also examined. Experimental design Newborn mouse ovaries were cultured for a total of six days, exposed to a chemotherapeutic agent on the second day: this allowed for the examination of the earliest stages of follicle development. Cleaved PARP and TUNEL were used to assess apoptosis following drug treatment. Imatinib was added to cultures with cisplatin and doxorubicin to determine any protective effect. Results Histological analysis of ovaries treated with cisplatin showed oocyte-specific damage; in comparison doxorubicin preferentially caused damage to the granulosa cells. Cleaved PARP expression significantly increased for cisplatin (16 fold, p<0.001) and doxorubicin (3 fold, p<0.01). TUNEL staining gave little evidence of primordial follicle damage with either drug. Imatinib had a significant protective effect against cisplatin-induced follicle damage (p<0.01) but not against doxorubicin treatment. Conclusion Cisplatin and doxorubicin both induced ovarian damage, but in a markedly different pattern, with imatinib protecting the ovary against damage by cisplatin but not doxorubicin. Any treatment designed to block the effects of chemotherapeutic agents on the ovary may need to be specific to the drug(s) the patient is exposed to. PMID:23922929

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

    PubMed Central

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

    2016-01-01

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

  4. Mechanisms of action of two different natural mixtures of persistent organic pollutants (POPs) in ovarian follicles.

    PubMed

    Gregoraszczuk, E L; Ptak, A; Skaare, J U; Mularz, K; Chmielowiec, A; Wojtowicz, A; Ropstad, E

    2009-01-01

    The present work investigated the effects of two different natural mixtures on aryl hydrocarbon receptor (AhR) and oestrogen receptor (ER)beta protein levels, as well as on the activity of cytochrome P450 (CYP) 1A1 and CYP2B. Consequently, the authors observed the effects of these mixtures on gonadotropine-stimulated steroid secretion by ovarian follicles. The natural mixtures that were studied were 'Mjosa' extracted from burbot liver, which contains a high level of PBDEs, and 'Marine mix', extracted from Atlantic cod liver, which contains a high level of polychlorinated biphenyls (PCBs). Follicular cells were exposed in vitro to 'Marine mix' and 'Mjosa mix' at doses of 3.6 and 1.4 microg ml(-1), respectively. Media were collected and used for steroid analysis and cell viability assays. Cells were used to estimate aromatase activity (CYP19), AhR and ER protein levels, and CYP1A1 and CYP2B1 activity. Western blot analysis indicated down-regulation of AhR by 'Marine mix' and down-regulation of ERbeta by Mjosa mix. Up-regulation of CYP1A1 expression and activity were seen following treatment with Marine mix, but not Mjosa mix. Increased CYP2B1 activity was noted after treatment with both 'Marine mix' and Mjosa mix. Both mixtures increased luteinizing hormone (LH)-stimulated progesterone and testosterone secretion, follicle-stimulating hormone (FSH)-stimulated oestradiol secretion, and CYP19 activity. These results suggest that: (1) 'Marine mix' is a mixed-type CYP inducer; (2) 'Mjosa mix' is an inducer of ERbeta and CYP2B; and (3) both 'Marine mix' and 'Mjosa mix' stimulate aromatase activity as a consequence of oestradiol secretion through activation of CYP19. PMID:19219750

  5. 17beta-estradiol affords protection against 4-vinylcyclohexene diepoxide-induced ovarian follicle loss in Fischer-344 rats.

    PubMed

    Thompson, Kary E; Sipes, I Glenn; Greenstein, Ben D; Hoyer, Patricia B

    2002-03-01

    Repeated dosing with 4-vinylcyclohexene diepoxide (VCD) accelerates atresia via apoptosis in primordial and primary follicles in ovaries of rats. The mechanisms that control atresia and VCD-induced toxicity are unknown; however, they could involve 17beta-E2. Atresia slows as animals enter puberty, whereas circulating E2 levels increase with the the onset of cyclicity. This inverse relationship suggests that E2 may be involved in the control of atresia. Therefore, this study was designed to determine whether treatment of immature rats with E2 could protect follicles normally destroyed by VCD-induced apoptosis. Female F344 rats were treated daily with E2, ER analogs, and/or VCD for 15 d. VCD alone caused a 50% reduction in primordial and primary follicles. Coinjection of E2 (0.1 mg/kg) and VCD (80 mg/kg) selectively protected primary follicles from VCD-induced follicle loss. This protection was mimicked by an ER agonist, genistein (0.1 mg/kg), and prevented by an ER antagonist, 4-hydroxytamoxifen (2 mg/kg). VCD treatment increased caspase-3-like activity, whereas concurrent treatment with genistein and VCD restored caspase-3-like activity to control levels. VCD treatment had no effect on circulating E2 levels, uterine weight, or E2 binding to the ER, nor could it directly displace E2 from ERbeta. These observations support the idea that ER-mediated protection against VCD-induced follicle toxicity is obtained by reducing apoptosis in small preantral follicles, although VCD does not appear to directly interact with ER. PMID:11861533

  6. Supplemented αMEM/F12-based medium enables the survival and growth of primary ovarian follicles encapsulated in alginate hydrogels

    PubMed Central

    Tagler, David; Makanji, Yogeshwar; Anderson, Nicholas R.; Woodruff, Teresa K.; Shea, Lonnie D.

    2013-01-01

    Hydrogel-encapsulating culture systems for ovarian follicles support the in vitro growth of secondary follicles from various species including mouse, non-primate human, and human; however, the growth of early stage follicles (primary and primordial) has been limited. While encapsulation maintains the structure of early stage follicles, feeder cell populations, such as mouse embryonic fibroblasts (MEFs), are required to stimulate growth and development. Hence, in this report, we investigated feeder-free culture environments for early stage follicle development. Mouse ovarian follicles were encapsulated within alginate hydrogels and cultured in various growth medium formulations. Initial studies employed embryonic stem cell medium formulations as a tool to identify factors that influence the survival, growth, and meiotic competence of early stage follicles. The medium formulation that maximized survival and growth was identified as αMEM/F12 supplemented with fetuin, insulin, transferrin, selenium, and follicle stimulating hormone (FSH). This medium stimulated the growth of late primary (average initial diameter of 80 µm) and early secondary (average initial diameter of 90 µm) follicles, which developed antral cavities and increased to terminal diameters exceeding 300 µm in 14 days. Survival ranged from 18% for 80 µm follicles to 36% for 90 µm follicles. Furthermore, 80% of the oocytes from surviving follicles with an initial diameter of 90–100 µm underwent germinal vesicle breakdown (GVBD), and the percentage of metaphase II (MII) eggs was 50%. Follicle/oocyte growth and GVBD/MII rates were not significantly different from MEF co-culture. Survival was reduced relative to MEF co-culture, yet substantially increased relative to the control medium that had been previously used for secondary follicles. Continued development of culture medium could enable mechanistic studies of early stage folliculogenesis and emerging strategies for fertility preservation. PMID

  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. Effect of Antiprogesterone RU486 on VEGF Expression and Blood Vessel Remodeling on Ovarian Follicles before Ovulation

    PubMed Central

    Berardinelli, Paolo; Russo, Valentina; Bernabò, Nicola; Di Giacinto, Oriana; Mattioli, Mauro; Barboni, Barbara

    2014-01-01

    Background The success of ovarian follicle growth and ovulation is strictly related to the development of an adequate blood vessel network required to sustain the proliferative and endocrine functions of the follicular cells. Even if the Vascular Endothelial Growth Factor (VEGF) drives angiogenesis before ovulation, the local role exerted by Progesterone (P4) remains to be clarified, in particular when its concentration rapidly increases before ovulation. Aim This in vivo study was designed to clarify the effect promoted by a P4 receptor antagonist, RU486, on VEGF expression and follicular angiogenesis before ovulation, in particular, during the transition from pre to periovulatory follicles induced by human Chorionic Gonadotropins (hCG) administration. Material and Methods Preovulatory follicle growth and ovulation were pharmacologically induced in prepubertal gilts by combining equine Chorionic Gonadotropins (eCG) and hCG used in the presence or absence of RU486. The effects on VEGF expression were analyzed using biochemical and immunohistochemical studies, either on granulosa or on theca layers of follicles isolated few hours before ovulation. This angiogenic factor was also correlated to follicular morphology and to blood vessels architecture. Results and Conclusions VEGF production, blood vessel network and follicle remodeling were impaired by RU486 treatment, even if the cause-effect correlation remains to be clarified. The P4 antagonist strongly down-regulated theca VEGF expression, thus, preventing most of the angiogenic follicle response induced by hCG. RU486-treated follicles displayed a reduced vascular area, a lower rate of endothelial cell proliferation and a reduced recruitment of perivascular mural cells. These data provide important insights on the biological role of RU486 and, indirectly, on steroid hormones during periovulatory follicular phase. In addition, an in vivo model is proposed to evaluate how periovulatory follicular angiogenesis may

  9. Histological populations and atresia of ovarian follicles in postpartum cattle treated with an agonist of gonadotropin-releasing hormone.

    PubMed

    Twagiramungu, H; Guilbault, L A; Proulx, J; Ramkumar, R; Dufour, J J

    1994-01-01

    Surface and histological changes of ovarian follicles and degree of atresia were characterized 3 and 6 d after a treatment with a GnRH agonist (buserelin) in postpartum beef cows. Sixteen cyclic cows on d 7 of their estrous cycle and eight acyclic cows were used. On d 0 of the experimental period, eight cyclic cows were injected with 2 mL of physiological saline and the remaining eight cyclic cows and the acyclic cows were injected i.m. with 8 micrograms of buserelin. Ovaries were collected 3 or 6 d later (n = 4 cows per group per day). All follicles (> 1.57 mm) were observed microscopically, measured using routine histological techniques, and classified as either nonatretic or atretic, with early or late atresia, and grouped into Class 1, 1.57 to 3.67 mm, Class 2, 3.68 to 8.56 mm, or Class 3, > 8.56 mm. Buserelin induced formation of new corpora lutea in all cows but one (acyclic cow). Class 1 follicles decreased on d 3 but increased on d 6 (day x treatment, P < .004), whereas those of Class 2 increased (P < .07) on both d 3 and 6 after buserelin treatment. However, within Class 2 follicles, we detected an increase on d 3 and 6 in the number (P < .01) and proportion (P < .07) of atretic follicles, an increase in the number of follicles in late atresia on d 6 (day x treatment, P < .03), and a decrease in the number (P < .06) and proportion (P < .03) of nonatretic follicles on d 6 (day x treatment).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8138489

  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. PMID:23330011

  11. Differences between follicular waves 1 and 2 in patterns of emergence of 2-mm follicles, associated FSH surges, and ovarian vascular perfusion in heifers.

    PubMed

    Ginther, O J; Siddiqui, M A R; Baldrighi, J M; Wolf, C A; Greene, J M

    2015-10-01

    The emergence (first detection) of 2-mm follicles, FSH surges, and ovarian vascular perfusion for follicular wave 1 and surge 1 (n = 26) and wave 2 and surge 2 (n = 25) were studied daily in heifers. The day the future dominant follicle was closest to 5.5 mm was designated Day 0 for each wave. In wave 1, many 2-mm follicles (41%) emerged on Days -5 to -3, whereas FSH surge 1 did not begin until Day -3. Concentration of FSH increased abruptly in 1 day to a peak on the day of maximal number of emerging 2-mm follicles, although the day of maximal number relative to Day 0 differed among individuals. The first emergence of 2-mm follicles in wave 2 occurred concurrently with the first increase in the FSH of surge 2. In wave 1, ovarian resistance to vascular perfusion was negatively correlated (r = -0.48, P < 0.05) with a number of 2-mm follicles on Days -4 to -1 for ovaries that did not contain the preovulatory follicle; vascular perfusion increased with an increase in the number of small follicles. The following hypotheses were supported for wave 1 but not for wave 2: (1) an increase in the number of emerging 2-mm follicles of a follicular wave occurs before the beginning of an increase in FSH, (2) the day of maximal number of emerging 2-mm follicles occurs concurrently with an abrupt FSH increase on different days among individuals, and (3) the association between the number of emerging 2-mm follicles and the extent of ovarian vascular perfusion is positive. PMID:26159090

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

    PubMed Central

    Choi, Jung Kyu; Agarwal, Pranay

    2013-01-01

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

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

    PubMed

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

    1999-08-01

    We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells. PMID:10411512

  14. 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

  15. Levels of heat shock protein transcripts in normal follicles and ovarian follicular cysts.

    PubMed

    Velázquez, Melisa M L; Alfaro, Natalia S; Salvetti, Natalia R; Stangaferro, Matías L; Rey, Florencia; Panzani, Carolina G; Ortega, Hugo H

    2011-11-01

    In the study, the gene expression of several heat shock proteins (HSPs) was determined in normal follicles and cystic follicles from cattle. A lower expression of HSP10 and HSP40 was observed in granulosa and theca cells of cysts compared to normal follicles. HSP27 was significantly less expressed in granulosa cells in cystic and large antral follicles than in other follicular categories. HSP60 and HSP90a expressions were highest in theca cells of cysts. However, HSP70 and HSP90b exhibited a lower expression in cysts than in healthy follicles. PMID:22139340

  16. 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. PMID:10864785

  17. Effects of 5α-Dihydrotestosterone and 17β-Estradiol on the Mouse Ovarian Follicle Development and Oocyte Maturation

    PubMed Central

    Tarumi, Wataru; Itoh, Masanori T.; Suzuki, Nao

    2014-01-01

    We have previously reported that androstenedione induces abnormalities of follicle development and oocyte maturation in the mouse ovary. In granulosa cells of the ovarian follicle, androstenedione is aromatized to 17β-estradiol (E2). To determine whether the androgen or estrogen acts directly on the follicle to induce the above-mentioned abnormalities, we compared the effects of a non-aromatizable androgen, 5α-dihydrotestosterone (DHT), with those of E2 on murine follicular development and oocyte maturation in a single follicle culture system. The high dose (10−6 M) of DHT prompted normal follicular development, and there was no effect on oocyte meiotic maturation after stimulation with human chorionic gonadotropin (hCG) and epidermal growth factor (EGF). In contrast, culture with the high dose (10−6 M) of E2 delayed follicular growth and also suppressed proliferation of granulosa cells and antrum formation. Furthermore, culture with E2 delayed or inhibited oocyte meiotic maturation, such as chromosome alignment on the metaphase plate and extrusion of the first polar body, after addition of hCG and EGF. In conclusion, these findings demonstrate that E2, but not DHT, induces abnormalities of follicular development, which leads to delay or inhibition of oocyte meiotic maturation. PMID:24911314

  18. Ovarian volume and antral follicle count for the prediction of low and hyper responders with in vitro fertilization

    PubMed Central

    Kwee, Janet; Elting, Mariet E; Schats, Roel; McDonnell, Joseph; Lambalk, Cornelis B

    2007-01-01

    Background The current study was designed to compare antral follicle count (AFC) and basal ovarian volume (BOV), the exogenous FSH ovarian reserve test (EFORT) and the clomiphene citrate challenge test (CCCT), with respect to their ability to predict poor and hyper responders. Methods One hundred and ten regularly menstruating patients, aged 18–39 years, participated in this prospective study, randomized, by a computer designed 4-blocks system study into two groups. Fifty six patients underwent a CCCT, and 54 patients underwent an EFORT. All patients underwent a transvaginal sonography to measure the basal ovarian volume and count of basal antral follicle. In all patients, the test was followed by a standard IVF treatment. The result of ovarian hyperstimulation during IVF treatment, expressed by the total number of follicles, was used as gold standard. Results The best prediction of ovarian reserve (Y) was seen in a multiple regression prediction model that included, AFC, Inhibin B-increment in the EFORT and BOV simultaneously (Y = -3.161 + 0.805 × AFC (0.258-1.352) + 0.034 × Inh. B-incr. (0.007-0.601) + 0.511 BOV (0.480-0.974) (r = 0.848, p < 0.001). Univariate logistic regression showed that the best predictors for poor response were the CCCT (ROC-AUC = 0.87), the bFSH (ROC-AUC = 0.83) and the AFC (ROC-AUC = 0.83). Multiple logistic regression analysis did not produce a better model in terms of improving the prediction of poor response. For hyper response, univariate logistic regression showed that the best predictors were AFC (ROC-AUC = 0.92) and the inhibin B-increment in the EFORT (ROC-AUC = 0.92), but AFC had better test characteristics, namely a sensitivity of 82% and a specificity 89%. Multiple logistic regression analysis did not produce a better model in terms of predicting hyper response. Conclusion In conclusion AFC performs well as a test for ovarian response being superior or at least similar to complex expensive and time consuming endocrine tests

  19. 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. PMID:23503663

  20. 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

  1. 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. PMID:17345639

  2. 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. PMID:26861074

  3. The impact of antiandrogen 2-hydroxyflutamide on the expression of steroidogenic enzymes in cultured porcine ovarian follicles.

    PubMed

    Duda, Malgorzata; Grzesiak, Malgorzata; Knet, Malgorzata; Knapczyk-Stwora, Katarzyna; Tabarowski, Zbigniew; Michna, Agata; Slomczynska, Maria

    2014-07-01

    We used our model system for agonism and antagonism of the androgen receptor (AR), in which the porcine ovarian follicles were exposed on the excessive concentration of an AR agonist- testosterone (T) or an AR antagonist- 2-hydroxyflutamide (2-Hf) to: (1) analyze the spatiotemporal expression of ovarian 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 17α-hydroxylase/c17,20-lyase (P450c17) and cytochrome P450 aromatase (P450arom); (2) to determine the contribution of AR-mediated action during steroidogenesis and (3) to establish some correlations between the onset and expression pattern of the investigated proteins. Whole follicles (6-8 mm in diameter) isolated from mature porcine ovaries have been incubated (for 24 h) in an organ culture system in the presence of T (10(-7 )M), 2-Hf (1.7 × 10(-4) M) or both T and 2-hydroxyflutamide (T+2-Hf, at the same concentrations as when added separately). Thereafter, sections obtained from cultured follicles were processed for main steroidogenic enzymes detection by immunohistochemistry. Moreover, expression of their mRNA and protein was determined by real-time PCR and Western blot analysis. Progesterone, androgens and estradiol concentrations in the culture media were measured by radioimmunoassays (RIA). Our results demonstrated that 2-Hf can influence the steroidogenic activity of porcine follicles in vitro through the blockade of AR. It was shown that follicular 2-Hf treatment brought about dramatic decline in the production of the investigated steroids. What is more the addition of 2-Hf separately caused a negative effect on 3β-HSD and P450c17 mRNA and protein expression by ovarian follicles, while it was without effect on P450arom mRNA level. Quite opposite effect was observed in case of the simultaneous addition of 2-Hf and T. It caused high increase, in both P450arom mRNA and its protein. What was interesting, addition T+2-Hf evoked 3β-HSD and P450c17 increase on mRNA level, but decreased their protein

  4. Low numbers of ovarian follicles ≥3 mm in diameter are associated with low fertility in dairy cows.

    PubMed

    Mossa, F; Walsh, S W; Butler, S T; Berry, D P; Carter, F; Lonergan, P; Smith, G W; Ireland, J J; Evans, A C O

    2012-05-01

    The total number of ovarian follicles ≥ 3mm in diameter (antral follicle count, AFC) during follicular waves varies among cattle of similar age, but AFC is highly repeatable within individuals. We hypothesized that lower AFC could be associated with reduced fertility in cattle. The AFC was assessed by ultrasonography for 2 d consecutively during the first wave of follicular growth of the estrous cycle, 4.6±1.43 d (mean ± SD) after estrus, in 306 Holstein-Friesian dairy cows approximately 70 d postpartum. Cows were classified into 3 groups based on AFC: low (AFC ≤15), intermediate (AFC=16 to 24), and high (AFC ≥25). During the cycle in which AFC was assessed and in subsequent cycles, cows were artificially inseminated (AI) following detection of estrus, and pregnancy status was assessed using ultrasonography. Cows with high AFC had 3.34 times greater odds of being pregnant at the end of the breeding season compared with cows with low AFC; the odds of a successful pregnancy at first service were 1.75 times greater in the intermediate compared with the low group. The predicted probability of a successful pregnancy by the end of the breeding period (length of breeding season was 86±16.3 d) was 94, 88, and 84% for the high, intermediate, and low AFC groups, respectively. No difference was evident among groups in 21-d submission rate (proportion of all cows detected in estrus and submitted for AI in the first 21 d of the breeding season), but the interval from calving to conception was shorter in the high (109.5±5.1 d) versus low (117.1±4 d) group, and animals with intermediate AFC received fewer services during the breeding season (2.3±0.1) compared with animals with low AFC (2.7±0.1). Lactating cows with ≤15 ovarian follicles have lower reproductive performance compared with cows with higher numbers of follicles, but the existence of a positive association between high numbers of ovarian follicles and fertility is yet to be established. PMID:22541464

  5. 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. PMID:26335038

  6. 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

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

    PubMed Central

    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.

    2014-01-01

    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. PMID

  8. 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. PMID:957803

  9. Rescue in vitro maturation (IVM) of immature oocytes in stimulated cycles in women with low functional ovarian reserve (LFOR).

    PubMed

    Lee, Ho-Joon; Barad, David H; Kushnir, Vitaly A; Shohat-Tal, Aya; Lazzaroni-Tealdi, Emanuela; Wu, Yan-Guang; Gleicher, Norbert

    2016-04-01

    Rescue in vitro maturation (IVM) is currently not a routine procedure in association with in vitro fertilization (IVF). We compared in a prospectively cohort study of 10 patients with normal functional ovarian reserve (NFOR) and of 25 with low functional ovarian reserve (LFOR), defined by abnormally high FSH and/or abnormally low AMH levels), IVM dynamics of immature oocytes. Following controlled ovarian hyperstimulation in IVF cycles, only immature oocytes underwent rescue IVM (for up to 48 h). Oocyte maturation dynamics, fertilization rates, embryo development, and pregnancy rates were then compared between NFOR and LFOR patients. Though proportion of MI and GV oocytes reaching MII stages within 48 h and rate of maturation of MI oocytes did not differ, in women with LFOR significantly more GV oocytes reached MII stage within 24 h (30.4 vs. 66.9 %; P = 0.013), while fertilization rates and embryo generation numbers were similar between both groups. Rescue IVM, thus, produced 1.5 additional embryos for transfer in women with LFOR and 1.6 in patients with NFOR, a highly significant difference in relative improvement in available embryo numbers for LFOR (+60.0 %) and NFOR women (+16.5 %). Rescue IVM, thus, not only demonstrates different time dynamics between women with LFOR and NFOR but also disproportionate efficacy in improving available embryo numbers for transfer in favor of LFOR patients. 1/7 patients, who reached embryo transfer with only embryos produced via rescue IVF conceived and delivered, proving that rescue IVF in women with LFOR also improves pregnancy and delivery chances. Because of the small number of embryos LFOR patients produce, every additional embryo is of considerable potential clinical significance for them, suggesting that rescue IVM in women with LFOR should become routine practice. PMID:26419849

  10. Functional significance of the signal transduction pathways Akt and Erk in ovarian follicles: in vitro and in vivo studies in cattle and sheep

    PubMed Central

    Ryan, Kate E; Glister, Claire; Lonergan, Pat; Martin, Finian; Knight, Phil G; Evans, Alexander CO

    2008-01-01

    Background The intracellular signalling mechanisms that regulate ovarian follicle development are unclear; however, we have recently shown differences in the Akt and Erk signalling pathways in dominant compared to subordinate follicles. The aim of this study was to investigate the effects of inhibiting Akt and Erk phosphorylation on IGF- and gonadotropin- stimulated granulosa and theca cell function in vitro, and on follicle development in vivo. Methods Bovine granulosa and theca cells were cultured for six days and stimulated with FSH and/or IGF, or LH in combination with PD98059 (Erk inhibitor) and/or LY294002 (Akt inhibitor) and their effect on cell number and hormone secretion (estradiol, activin-A, inhibin-A, follistatin, progesterone and androstenedione) determined. In addition, ovarian follicles were treated in vivo with PD98059 and/or LY294002 in ewes on Day 3 of the cycle and follicles were recovered 48 hours later. Results We have shown that gonadotropin- and IGF-stimulated hormone production by granulosa and theca cells is reduced by treatment with PD98059 and LY294002 in vitro. Furthermore, treatment with PD98059 and LY294002 reduced follicle growth and oestradiol production in vivo. Conclusion These results demonstrate an important functional role for the Akt and Erk signalling pathways in follicle function, growth and development. PMID:19014654

  11. 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

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

    PubMed Central

    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

  13. 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

  14. 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

  15. Effect of 9-cis retinoic acid (RA) on progesterone and estradiol secretion and RA receptor expression in the chicken ovarian follicles.

    PubMed

    Pawłowska, Katarzyna; Sechman, Andrzej; Suchanek, Iwona; Grzegorzewska, Agnieszka; Rzasa, Janusz

    2008-01-01

    Several lines of evidence indicate that retinoids, derivates of vitamin A, affect reproductive function in birds, however, the mechanism of their action in the ovary is still unknown. Therefore, the present study was designed (i) to show whether in the domestic hen 9-cis retinoic acid (9-cis RA), one of the retinoids, influences steroid secretion in vitro by white and yellow chicken ovarian follicles, and (ii) to detect expression of retinoic acid RXR receptor mRNA in these follicles. The white follicles (small: 1-4 mm, medium: 4-6 mm and large 6-8 mm in diameter) and the three largest yellow preovulatory follicles (F3-F1; 25-37 mm) were isolated from the ovary 3 h before ovulation. The granulosa layer was separated from the theca layer in the preovulatory follicles, which were subsequently divided into 4 equal pieces. The isolated whole white follicles or parts of the granulosa or theca layers were incubated for 24 h at 38 degrees C in Eagle's medium in the following 4 groups: control, ovine LH (oLH; 10 ng/ml), 9-cis RA (100 ng/ml) and 9-cis RA + oLH. After incubation, the medium was collected for estradiol (E2) and progesterone (P4) determination while tissues were saved for protein assay. It was found that 9-cis RA affects steroid secretion from chicken ovarian follicles. It decreased E2 secretion from white follicles and from the theca layer of the two largest (F2 and F1) preovulatory follicles. 9-cis RA had no effect on oLH-stimulated E2 secretion by the white follicles and yellow F2 and F1 follicles, but it diminished E2 secretion by F3 follicles. As regards P4, the effect of 9-cis RA was opposite; it increased P4 secretion from the granulosa layer of all preovulatory follicles. 9-cis RA did not change oLH-stimulated P4 secretion by granulosa layers ofF3 and F2 follicles, however, it inhibited oLH-enhanced P4 secretion from the F1 granulosa layer. In a separate experiment, the presence of mRNA encoding RXR was found in the stroma and all follicles of the

  16. 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. PMID:26777561

  17. Oocyte cryopreservation for fertility preservation in post-pubertal female children at risk for premature ovarian failure due to accelerated follicle loss in Turner Syndrome or cancer treatments

    PubMed Central

    Oktay, K; Bedoschi, G

    2014-01-01

    Objective To preliminarily study the feasibility of oocyte cryopreservation in post-pubertal girls aged between 13 and 15 years who were at risk for premature ovarian failure due to the accelerated follicle loss associated with Turner’s Syndrome or cancer treatments. Design Retrospective cohort and review of literature. Setting Academic fertility preservation unit. Participants Three girls diagnosed with Turner syndrome, one girl diagnosed with germ-cell tumor and one girl diagnosed with lymphoblastic leukemia. Interventions Assessment of ovarian reserve, ovarian stimulation, oocyte retrieval, in vitro maturation, and mature oocyte cryopreservation. Main Outcome Measure Response to ovarian stimulation, number of mature oocytes cryopreserved and complications, if any. Results Mean AMH, baseline FSH, 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. Conclusions 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. PMID:25214440

  18. Ultrasound image characteristics of ovarian follicles in relation to oocyte competence and follicular status in cattle.

    PubMed

    Vassena, R; Adams, G P; Mapletoft, R J; Pierson, R A; Singh, J

    2003-03-20

    Assessment of the quality of the female gamete has become paramount for in vitro procedures. There is a need to identify reliable indicators of oocyte competence and develop a simple, non-invasive method to assess competence. The aim of this study was to investigate the relationships among ultrasonographic attributes of a follicle, its stage of development and the competence of the oocyte that it contains. We tested the hypotheses that follicular echotexture characteristics are related to: (1) the phase of development of the follicle, (2) the presence of the corpus luteum (CL) and/or the dominant follicle in the ovary, and (3) developmental competence of cumulus oocyte complexes (COC) from the same ovary. Crossbred beef cows (n=143), age 4-14 years, were given a luteolytic dose of dinoprost to cause ovulation. Ultrasound-guided ablation of all follicles > or = 4mm was done 8 days later to induce new follicular wave emergence during a luteal phase. Ultrasonographic images of dominant follicles and the three largest subordinate follicles (n=402 follicles; 84 cows) were acquired on Days 2, 3, 5 or 7 of the follicular wave (Day 0: wave emergence), i.e. growing, early-static, late static, and regressing phases of subordinate follicle development, respectively. From a subset of these animals (n=33), ovaries were collected within 30 min of slaughter and COC from subordinate follicles > or = 3mm underwent in vitro maturation, fertilization and culture to the blastocyst stage.Image analysis revealed differences in echotexture between dominant and subordinate follicles among Days 2-7 of the follicular wave. Images of dominant and subordinate follicles at Day 7 of the wave displayed consistently lower grey-scale values (P<0.05) in the peripheral antrum, follicular wall and perifollicular stroma than all other days. Follicle images displayed a consistent pattern of variation in echotexture among follicular phases. Data did not support the hypothesis of a local effect of the CL

  19. 1 Secondary follicle growth and oocyte maturation by culture in alginate hydrogel following cryopreservation of the ovary or individual follicles

    PubMed Central

    Xu, Min; Banc, Anna; Woodruff, Teresa K.; Shea, Lonnie D.

    2009-01-01

    An option for fertility preservation for women facing a cancer diagnosis involves the cryopreservation of ovarian tissue for later re-transplantation or in vitro culture, with in vitro culture preferred to avoid reintroduction of the cancer. Small, immature follicles survive the freeze-thaw process, and can be matured through in follicle maturation (IFM) that involves an initial growth of the follicle and subsequent maturation of the oocyte. The ovarian tissue can be cryopreserved in two forms: (i) cortical strips consisting of follicles and surrounding stroma (Cryo-Ov) or (ii) individually isolated follicles (Cryo-In). The aim of this study was to assess the follicle growth and oocyte maturation for follicles that were cryopreserved either as strips or individually using a slow-freezing cryopreservation method. The two follicle groups, together with non-cryopreserved control follicles, were grown in an alginate-based three-dimensional culture system for twelve days. The overall survival, size increase and antrum formation rates were comparable among the three groups. At day 12 of culture, Androstenedione levels were decreased in the Cryo-Ov group relative to the other two, and the ratio of progesterone to estradiol was increased in the two cryopreserved groups relative to the control. Both Gja1 (known as connexin 43) and Gja4 (known as connexin 37) mRNA expression were decreased at day 6 in the cryopreserved groups relative to controls, and by day 12, Gja1 was similar for all 3 groups. Moreover, Cryo-In resulted in lower GVBD rate indicating some impaired oocyte development. Overall, the present study demonstrated that mouse preantral follicles, either within ovarian tissues or individually isolated, could be successfully cryopreserved by the slow-freezing method, as evidenced by post-thaw follicle development and steroidgenesis, oocyte maturation and molecular markers for oocyte and/or granulosa cells connection. PMID:19191350

  20. Transcriptome comparisons identify new cell markers for theca interna and granulosa cells from small and large antral ovarian follicles.

    PubMed

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

    2015-01-01

    In studies using isolated ovarian granulosa and thecal cells it is important to assess the degree of cross contamination. Marker genes commonly used for granulosa cells include FSHR, CYP19A1 and AMH while CYP17A1 and INSL3 are used for thecal cells. To increase the number of marker genes available we compared expression microarray data from isolated theca interna with that from granulosa cells of bovine small (n = 10 for both theca and granulosa cells; 3-5 mm) and large (n = 4 for both theca and granulosa cells, > 9 mm) antral follicles. Validation was conducted by qRT-PCR analyses. Known markers such as CYP19A1, FSHR and NR5A2 and another 11 genes (LOC404103, MGARP, GLDC, CHST8, CSN2, GPX3, SLC35G1, CA8, CLGN, FAM78A, SLC16A3) were common to the lists of the 50 most up regulated genes in granulosa cells from both follicle sizes. The expression in theca interna was more consistent than in granulosa cells between the two follicle sizes. Many genes up regulated in theca interna were common to both sizes of follicles (MGP, DCN, ASPN, ALDH1A1, COL1A2, FN1, COL3A1, OGN, APOD, COL5A2, IGF2, NID1, LHFP, ACTA2, DUSP12, ACTG2, SPARCL1, FILIP1L, EGFLAM, ADAMDEC1, HPGD, COL12A1, FBLN5, RAMP2, COL15A1, PLK2, COL6A3, LOXL1, RARRES1, FLI1, LAMA2). Many of these were stromal extracellular matrix genes. MGARP, GLDC, CHST8, GPX3 were identified as new potential markers for granulosa cells, while FBLN5, OGN, RAMP2 were significantly elevated in the theca interna. PMID:25775029

  1. Involvement of the KIT/KITL signaling pathway in 4-vinylcyclohexene diepoxide-induced ovarian follicle loss in rats.

    PubMed

    Fernandez, Shannon M; Keating, Aileen F; Christian, Patricia J; Sen, Nivedita; Hoying, James B; Brooks, Heddwen L; Hoyer, Patricia B

    2008-08-01

    Repeated daily dosing of rats with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) depletes the ovary of primordial and primary follicles through an increase in the natural process of atresia. Additionally, in vitro exposure of Postnatal Day 4 (PND 4) rat ovaries to VCD causes similar follicular depletion. This study was designed to investigate survival signaling pathways that may be associated with VCD-induced ovotoxicity in small preantral follicles. Female Fischer 344 rats (PND 28) were dosed daily (80 mg/kg/day VCD i.p.; 12 days in vivo), and PND 4 ovaries were cultured (VCD 20 or 30 microM; 8 days in vitro). Microarray analysis identified a subset of 14 genes whose expression was increased or decreased by VCD in both experiments (i.e., via both exposure routes). Particularly, the analysis showed that relative to controls, VCD did not affect mRNA expression of growth and differentiation factor 9 (Gdf9), whereas there were decreases in mRNA encoding bone morphogenic protein receptor 1a (Bmpr1a) and Kit. To confirm findings from microarray, the genes Gdf9, Bmpr1a, and Kit were further examined. When growth factors associated with these pathways were added to ovarian cultures during VCD exposure, GDF9 and BMP4 had no effect on VCD-induced ovotoxicity; however, KITL attenuated this follicle loss. Additionally, there was a decrease in Kit and an increase in Kitl expression (mRNA and protein) following VCD exposure, relative to control. These results support that VCD compromises KIT/KITL signaling, which is critical for follicular survival in primordial and primary follicles. PMID:18448842

  2. Transcriptome Comparisons Identify New Cell Markers for Theca Interna and Granulosa Cells from Small and Large Antral Ovarian Follicles

    PubMed Central

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

    2015-01-01

    In studies using isolated ovarian granulosa and thecal cells it is important to assess the degree of cross contamination. Marker genes commonly used for granulosa cells include FSHR, CYP19A1 and AMH while CYP17A1 and INSL3 are used for thecal cells. To increase the number of marker genes available we compared expression microarray data from isolated theca interna with that from granulosa cells of bovine small (n = 10 for both theca and granulosa cells; 3-5 mm) and large (n = 4 for both theca and granulosa cells, > 9 mm) antral follicles. Validation was conducted by qRT-PCR analyses. Known markers such as CYP19A1, FSHR and NR5A2 and another 11 genes (LOC404103, MGARP, GLDC, CHST8, CSN2, GPX3, SLC35G1, CA8, CLGN, FAM78A, SLC16A3) were common to the lists of the 50 most up regulated genes in granulosa cells from both follicle sizes. The expression in theca interna was more consistent than in granulosa cells between the two follicle sizes. Many genes up regulated in theca interna were common to both sizes of follicles (MGP, DCN, ASPN, ALDH1A1, COL1A2, FN1, COL3A1, OGN, APOD, COL5A2, IGF2, NID1, LHFP, ACTA2, DUSP12, ACTG2, SPARCL1, FILIP1L, EGFLAM, ADAMDEC1, HPGD, COL12A1, FBLN5, RAMP2, COL15A1, PLK2, COL6A3, LOXL1, RARRES1, FLI1, LAMA2). Many of these were stromal extracellular matrix genes. MGARP, GLDC, CHST8, GPX3 were identified as new potential markers for granulosa cells, while FBLN5, OGN, RAMP2 were significantly elevated in the theca interna. PMID:25775029

  3. 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.

  4. Growing Teratoma Syndrome Secondary to Ovarian Giant Immature Teratoma in an Adolescent Girl: A Case Report and Literature Review.

    PubMed

    Li, Song; Liu, Zhenzhen; Dong, Chengyong; Long, Fei; Liu, Qinlong; Sun, Deguang; Gao, Zhenming; Wang, Liming

    2016-02-01

    Growing teratoma syndrome (GTS) is a rare clinical entity first described by Logothetis et al in 1982. Although it is unusual for GTS to be located in the ovary, this report is of a case of an adolescent girl who underwent a complete surgical resection of the mass. Histopathology confirmed only an immature teratoma had originated from the ovary and so she received adjuvant chemotherapy with blemycin, etopside, and cisplatin over 4 cycles. Results from an abdominal enhanced CT (computed tomography) 9 years later revealed a giant mass had compressed adjacent tissues and organs. Laparotomy was performed and a postoperative histopathology showed the presence of a mature teratoma, and so the diagnosis of ovarian GTS was made. One hundred one cases of ovarian GTS from English literature published between 1977 and 2015 were collected and respectively analyzed in large samples for the first time. The median age of diagnosis with primary immature teratoma was 22 years (range 4-48 years, n = 56). GTS originating from the right ovary accounted for 57% (27/47, n = 47) whereas the left contained 43% (20/47, n = 47). Median primary tumor size was 18.7 cm (range 6-45 cm, n = 28) and median subsequent tumor size was 8.6 cm (range 1-25 cm, n = 25). From the primary treatment to the diagnosis of ovarian GTS, median tumor growth speed was 0.94 cm/month (range 0.3-4.3 cm/month, n = 21). Median time interval was 26.6 months (range 1-264 months, n = 41). According to these findings, 5 patients did have a pregnancy during the time interval between primary disease and GTS, making our patient the first case of having a pregnancy following the diagnosis of ovarian GTS. Because of its high recurrence and insensitiveness to chemotherapy, complete surgical resection is the preferred treatment and fertility-sparing surgery should be considered for women of child-bearing age. Anyhow GTS of the ovary has an excellent prognosis. Patients with GTS had no evidence of

  5. Rosiglitazone stimulates peroxisome proliferator-activated receptor gamma expression and directly affects in vitro steroidogenesis in porcine ovarian follicles.

    PubMed

    Rak-Mardyła, Agnieszka; Karpeta, Anna

    2014-07-01

    Rosiglitazone is a peroxisome proliferator-activated receptor gamma (PPARγ) synthetic activator from the group of thiazolidinediones often used in the treatment of chronic diseases such as type 2 diabetes and other forms of insulin resistance. The present in vitro study assessed the direct effects of rosiglitazone at 25 and 50 μM doses on PPARγ gene expression, steroid secretion (progesterone [P4], androstenedione [A4], testosterone [T], and estradiol), and protein expression of PPARγ, 3βHSD, CYP17, 17βHSD, CYP19 by porcine ovarian follicles from prepubertal and cycling animals. We analyzed also steroid enzymatic activity by conversion of pregnen-3β-ol-20-one to P4, P4 to A4, and A4 to T. Our results indicated that rosiglitazone increased significantly PPARγ expression, P4 secretion, 3βHSD activity, and protein expression. Rosiglitazone decreased A4 and T secretion by reducing the expression and activity of CYP17 and 17βHSD and did not change estradiol secretion and CYP19. Similarly results was observed both in prepubertal and cycling pigs. Our results indicate that these direct effects of rosiglitazone on ovarian steroidogenesis provide a framework for testing several potential new mechanisms of PPAR-γ actions on porcine ovarian function. PMID:24681211

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

    PubMed Central

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

    2016-01-01

    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. PMID:26979400

  7. 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

    2016-01-01

    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. PMID:26979400

  8. 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.

  9. The potential of follicle-stimulating hormone peptide-modified triptolide-loaded nanoparticles to induce a mouse model of premature ovarian insufficiency

    PubMed Central

    Chen, Xiu-Ying; Chen, Wu-Lian; Ma, Min; Gu, Chao; Xiao, Xi-Rong; Li, Bin

    2015-01-01

    The use of triptolide (TP) is limited by its poor water solubility and severe toxicity. In this study, we developed an active drug delivery system (TP-loaded nanoparticles) that could help improve the water solubility of TP and decrease its toxicity. Then, we investigated whether TP-loaded nanoparticles could be used to establish a novel premature ovarian insufficiency mouse model. The mice treated with TP-loaded nanoparticles for 35 days displayed normal growth, decreased serum antimullerian hormone, prominent ovarian fibrosis and vacuolar changes, fewer follicles and corpus lutea, increased collapsed oocytes and follicle apoptosis, and sterility. In conclusion, this model appears to show the reproductive characteristics associated with premature ovarian insufficiency in women and will allow us to study the mechanism of the effects of traditional Chinese medicine on gonadal toxicity. PMID:25897221

  10. In vitro culture methods of preantral follicles.

    PubMed

    Green, Lisa J; Shikanov, Ariella

    2016-07-01

    Folliculogenesis is a highly regulated, dynamic process requiring various autocrine, paracrine, and endocrine factors that need to be reproduced in vitro. The mammalian ovary consists of a finite number of follicles at birth, with the majority in a quiescent state, awaiting recruitment. In vitro culture of preantral follicles presents a unique challenge of mimicking the dynamics of the ovarian environment, a methodical sequence of cell communication, and interaction with secretory, hormonal, and growth factors. In larger mammalian species, maintenance of follicular morphology is imperative to maintain communication between the cumulus-oocyte complexes. Three-dimensional culture systems help to maintain this architecture and have been used for the successful culture of follicles in various animal models, including humans and nonhuman primates, and have resulted in live birth in rodent models. Clinically, in vitro follicular development could revolutionize assisted reproductive technology by providing a means of using the more numerous immature ovarian follicles in patient populations that are unable to undergo ovarian stimulation. PMID:27173961

  11. 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

  12. Embryos of the Viviparous Dermapteran, Arixenia esau Develop Sequentially in Two Compartments: Terminal Ovarian Follicles and the Uterus

    PubMed Central

    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. PMID:23667700

  13. 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. PMID:23667700

  14. Expression of leptin and its receptor genes in the ovarian follicles of cycling and early pregnant pigs.

    PubMed

    Smolinska, N; Kaminski, T; Siawrys, G; Przala, J

    2013-01-01

    Leptin is a polypeptide hormone produced primarily by adipocytes. It has been implicated in the regulation of satiety and energy homeostasis. Leptin has been suggested to play a role in reproduction based on its involvement in the regulation of the hypothalamic-pituitary-gonadal axis via endocrine, paracrine and/or autocrine pathways. The aim of the present study was to localize the cellular distribution of leptin and the long isoform of leptin receptor (OB-Rb) genes in porcine ovarian antral follicles and to compare the expression levels of leptin and OB-Rb mRNAs in porcine granulosa cells (GC), theca interna (TIC) and theca externa (TEC) cells during the luteal phase of the estrous cycle and in early pregnancy. The expression of leptin and OB-Rb genes was detected in GC, TIC and TEC. Significantly higher levels of leptin gene expression in GC were observed during the mid- and late-luteal phases of the cycle than on days 30 to 32 of pregnancy. On days 14 to 16 of pregnancy, leptin mRNA expression was higher than that on days 14 to 16 of the cycle. The expression of the OB-Rb gene in GC and TEC increased during pregnancy in comparison with the analyzed luteal phases of the cycle. Our results validate the hypothesis that locally produced leptin plays a role in the regulation of porcine reproduction at the ovarian level and exerts a direct effect on porcine follicles. The differences in OB-Rb gene expression in porcine GC and theca cells also suggest that their sensitivity to leptin varies in the ovaries of pregnant and cyclic pigs. PMID:23031202

  15. 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

  16. Androgen receptor and miRNA-126* axis controls follicle-stimulating hormone receptor expression in porcine ovarian granulosa cells.

    PubMed

    Du, Xing; Li, Qiqi; Pan, Zengxiang; Li, Qifa

    2016-08-01

    Androgen, which acts via the androgen receptor (AR), plays crucial roles in mammalian ovarian function. Recent studies showed that androgen/AR signaling regulates follicle-stimulating hormone receptor (FSHR) expression in follicles; however, the detailed mechanism underlying this regulation remained unknown. Here, we demonstrate that AR and miR-126* cooperate to inhibit FSHR expression and function in pig follicular granulosa cells (pGCs). In pGCs, overexpression of AR decreased, whereas knockdown increased, FSHR mRNA and protein expression; however, neither manipulation affected FSHR promoter activity. Using a dual-luciferase reporter assay, we found that the FSHR gene is a direct target of miR-126*, which inhibits FSHR expression and increases the rate of AR-induced apoptosis in pGCs. Collectively, our data show for the first time that the AR/miR-126* axis exerts synergetic effects in the regulation of FSHR expression and apoptosis in pGCs. Our findings thus define a novel pathway, AR/miR-126*/FSHR, that regulates mammalian GC functions. PMID:27222597

  17. 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. PMID:26464260

  18. Increased incidence of DNA amplification in follicular than in uterine and blood samples indicates possible tropism of Neospora caninum to the ovarian follicle.

    PubMed

    Silva, Andressa F; Rangel, Lucia; Ortiz, Carlos Garcia; Morales, Elizabeth; Zanella, Eraldo L; Castillo-Velázquez, Uziel; Gutierrez, Carlos G

    2012-08-13

    This study evaluated the presence of Neospora caninum in ovarian follicle aspirates and uterine flushes obtained from N. caninum seropositive dairy cows. Ninety-two cows that aborted within the previous 90 days were sampled to determine the presence of antibodies against N. caninum. Thirteen seropositive cows were chosen for collection of blood leukocytes, uterine flushes (UF; n=12) and follicular aspirates (OPU; n=13). Samples were centrifuged and the cellular sediment from the follicular fluid, uterine flushes and blood leukocytes were used for DNA extraction and PCR. Follicular aspirates had the highest frequency of DNA amplification for N. caninum (p<0.05, 92.3%; 12/13). Whereas uterine (4/12) and blood leukocyte (5/13) samples had similar (p>0.05) rate of positive results. Nonetheless, there was no agreement between blood leukocytes and follicular samples taken from the same animal (Cohen Kappa=-0.16). Similarly, blood leukocytes and uterine results had moderate agreement between them (Cohen Kappa=0.47). This study indicates that N. caninum is present in the ovarian follicle and uterus of seropositive cows, suggesting a possible risk of neosporosis transmission between females during oocyte and embryo collection and transfer. Hence, precautions should be taken to minimize the risk of N. caninum transmission. Furthermore, the high incidence of positive results in follicle samples, that exceeded that of their paired blood leukocytes, suggests a possible tropism of N. caninum for the ovarian follicle. PMID:22440723

  19. Segmentation of ovarian follicles using geometric properties, texture descriptions, and boundary information

    NASA Astrophysics Data System (ADS)

    Robinson, Glynn P.; Chakraborty, Amit; Johnston, Michael; Reuss, M. Lynne; Duncan, James S.

    1996-04-01

    The size and number of follicles present within an ovary may be used as an indicator of fertility in women. Ultrasound is the imaging modality of choice for obtaining information on the follicles as it is inexpensive and readily available. A method of segmenting the follicles and ovary and producing accurate 2D and 3D representation would be of great benefit to a large segment of the population. However, the nature of ultrasound images means that standard approaches to segmentation based on image gradients or detecting regions of homogeneous gray-level alone are inadequate. A semi-automatic method of segmentation which combined a texture based classification for initial segmentation with deformable models to provide descriptions of individual objects is extended by imposing geometric constraints on the relationships between the individual objects present within an image. Since we are interested in segmenting the individual objects over a 3D spatial stack we use the results from one image in the sequence as the initial estimates for the next image. This reduces the need for operator intervention and provides representations of individual objects through the whole sequence. These representations can then be used for accurate measurement of area/volume and for three-dimensional visualization of the relationships between the individual follicles and the enclosing ovary.

  20. Molecular cloning, expression profile and transcriptional modulation of two splice variants of very low density lipoprotein receptor during ovarian follicle development in geese (Anser cygnoide).

    PubMed

    Hu, Shenqiang; Liu, Hehe; Pan, Zhixiong; Xia, Lu; Dong, Xia; Li, Liang; Xu, Feng; He, Hua; Wang, Jiwen

    2014-10-01

    Very low density lipoprotein receptor (VLDLR)-mediated endocytosis of plasma lipoproteins into the ovary is essential for ovarian follicle development. Two splice variants of VLDLR have been identified in several species, yet little is known about their distinctive roles in ovarian developing follicles. In the present study, the full-length cDNAs of two splice isoforms of VLDLR were obtained from geese (Anser cygnoide) ovaries using the RACE method. The longer isoform (TypeI VLDLR) is 3141bp and contains five conserved structural domains, while the other (TypeII VLDLR) lacks 90bp encoding for the O-linked sugar domain. TypeII VLDLR was predominantly expressed in the ovary, with greater amounts of mRNA in theca and granulosa cells from early stages of follicle development but decreased during vitellogenesis. However, there was minimal expression of the TypeI VLDLR gene in theca cells and expression was almost undetectable in granulosa cells throughout follicle development. Yolk VLDL concentrations decreased as stage of development advanced while yolk triglyceride and cholesterol concentrations increased in a follicular size-dependent manner. The significant correlations between transcripts of TypeII VLDLR and yolk lipids supported its important role on yolk lipid deposition. In addition, in vitro experiments suggested that exogenous cholesterol, 25-hydroxycholesterol and mevinolin (a highly potent competitive inhibitor of HMG-CoA) treatment could significantly alter TypeII VLDLR gene expression in granulosa cells from both pre-hierarchical and pre-ovulatory follicles. Collectively, data from the present study indicate that TypeII VLDLR is more important for the transport of plasma lipoproteins into developing follicles than TypeI VLDLR, and provide new evidence about the influence of steroids in modulating VLDLR gene expression in ovarian cells. PMID:25018046

  1. 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

  2. Reductions in the number of mid-sized antral follicles are associated with markers of premature ovarian senescence in dairy cows.

    PubMed

    Modina, Silvia C; Tessaro, Irene; Lodde, Valentina; Franciosi, Federica; Corbani, Davide; Luciano, Alberto M

    2014-01-01

    High-producing dairy cows are subfertile; however, the mechanisms responsible for the decreased fertility are unknown. The aim of the present study was to test the hypothesis that culled dairy cows (4-8 years old) characterised by 'Lo' ovaries (i.e. those with <10 mid-antral follicles) are affected by premature ovarian senescence. Cows in which both ovaries were 'Lo' ovaries represented 5% of the total population analysed, and exhibited reduced ovarian size (P<0.001) and increased perifollicular stroma (P<0.05) compared with age-matched controls (i.e. cows in which both ovaries had >10 mid-antral follicles; 'Hi' ovaries). The total number of follicles, including healthy and atretic primordial, primary, secondary and small antral follicles, was lower in Lo ovaries (P<0.01). Interestingly, the primordial follicle population in Lo ovaries was lower (P<0.05) than in the control. Finally, the follicular fluid of mid-antral follicles from Lo ovaries had reduced oestradiol and anti-Müllerian hormone levels (P<0.05), but increased progesterone concentrations (P<0.05). Together, these data account for the reduced fertility of cows with Lo ovaries and are in agreement with previous observations that oocytes isolated from Lo ovaries have reduced embryonic developmental competence. Cows with a specific Lo ovary condition may represent a suitable model to address the causes of low fertility in high-yielding dairy cows, as well as the condition of premature ovarian aging in single-ovulating species. PMID:23327793

  3. Use of transcutaneous ultrasonography to characterize ovarian status, size distribution, and hierarchical status of follicles in Japanese quail (Coturnix coturnix japonica).

    PubMed

    Sreesujatha, R M; Jeyakumar, S; Kundu, A; Balasundaram, Chellam

    2016-09-15

    The present study was undertaken to evaluate the ovarian status of matured breeding female Japanese quail by means of B Mode portable ultrasound scanning. Active ovaries in various stages of hierarchical development and different morphological structure were observed. The large yellow follicles appeared as a cluster of dark circles with various intensity of hyperechoic concentric ring. Sonographically, these follicles were classified into F1, F2, and F3 measuring 16.20 ± 0.06 mm, 12.48 ± 0.09 mm, and 7.95 ± 0.16 mm, respectively. A comparative study on preslaughter and postslaughter ovarian follicular status revealed accuracy of follicles less than 10 mm, 10-15 mm, 15-18 mm, and greater than 18 mm as 26.31%, 78.9%, 93.75%, and 100%, respectively, with an overall accuracy of 71%. This is the first report on the application of transcutaneous ultrasonographic technique to monitor ovarian status on Japanese quail reporting that the ovarian follicular activity and morphological structure can be monitored nonintrusively throughout quail life which can used as model for reproductive and biomedical research. PMID:27233352

  4. 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

  5. Mixed ovarian germ cell tumor composed of immature teratoma, yolk sac tumor and embryonal carcinoma.

    PubMed

    Wang, Ying; Zhou, Feng; Qian, Zhida; Qing, Jiale; Zhao, Mengdam; Huang, Lili

    2014-11-01

    We report the case of a 19-year-old woman experiencing lower abdominal distension and pain. Laboratory tests indicated elevated serum levels of Alpha-Fetoprotein (AFP) and human Chorionic Gonadotropin (hCG). A large mass was detected in the abdomen by physical examination and by transvaginal ultrasonography. Exploratory laparotomy was performed, and a smooth-surfaced, spherical, solid tumor was found on the left ovary, measuring 11.5 x 9.9 x 6.9 cm. Histological evaluation revealed that the tumor consisted of a combination of immature teratoma, Yolk Sac Tumor, and embryonal carcinoma; this is a very rare combination in mixed germ cell tumors. PMID:25518772

  6. Roles of thyroid hormones in follicular development in the ovary of neonatal and immature rats.

    PubMed

    Fedail, Jaafar Sulieman; Zheng, Kaizhi; Wei, Quanwei; Kong, Lingfa; Shi, Fangxiong

    2014-08-01

    Thyroid hormones (TH) play a critical role in ovarian follicular development, maturation and the maintenance of various endocrine functions. However, whether TH can affect ovarian follicular development in neonatal and immature rats remains unclear. Therefore, the aim of the present study was to elucidate the effect of TH on ovarian follicular development in neonatal and immature rats. Thirty female post-lactation mothers of Sprague-Dawley rat pups were randomly divided into three groups: control, hyperthyroid (hyper), and hypothyroid (hypo). On postnatal days (PND) 10 and 21, body weights, serum hormones, ovarian histologic changes, and immunohistochemistry of thyroid hormone receptor alpha 1 (TRα1) and nitric oxide synthase types (NOS), and NOS activities, were determined. The data showed that body weights significantly decreased in both hyper and hypo groups compared with the control group (P < 0.05). In addition, the hyper group had increased serum concentrations of T3, T4, and E2; whereas the hypo group manifested reduced serum concentrations of T3, T4, and E2 on PND 10 and 21. The hyper and hypo groups showed significantly reduced total number of primordial, primary and secondary follicles on PND 10 and 21 compared with the control group (P < 0.05). Similarly, antral follicle numbers in the hyper and hypo groups were significantly decreased on PND 21 compared with the control group (P < 0.05). Immunostaining indicated that TRα1 and NOS were expressed in ovarian surface epithelium and oocytes of growing and antral follicles, with strong staining of the granulosa and theca cells of follicles. NOS activities were significantly augmented in the hyper, but diminished in the hypo groups on PND 10 and 21. In summary, our findings suggest that TH play important roles in ovarian functions and in the regulation of NOS activity. Our results also indicate that a relationship exists between the TH and NO signaling pathways during the process of ovarian follicular

  7. The porcine ovarian follicle: I. Selected chemical analysis of follicular fluid at different developmental stages.

    PubMed

    Chang, S C; Jones, J D; Ellefson, R D; Ryan, R J

    1976-10-01

    Numerous parameters of the chemical composition of porcine follicular fluid, obtained at different stages of follicle development, were analyzed. The concentrations of electrolytes, glucose, uric acid, selected enzymes, lipids, amino acids, cyclic AMP, and steroid hormones were determined and compared with concentrations in serum. There were both quantitative and qualitative differences in the composition of follicular fluid and plasma. These differences are discussed with regard to follicular development. PMID:183842

  8. Causes and consequences of the variation in the number of ovarian follicles in cattle.

    PubMed

    Evans, A C O; Mossa, F; Fair, T; Lonergan, P; Butler, S T; Zielak-Steciwko, A E; Smith, G W; Jimenez-Krassel, F; Folger, J K; Ireland, J L H; Ireland, J J

    2010-01-01

    In cattle we have noted that the antral follicle count (AFC, follicles > or = 3 mm in diameter) varies greatly among animals (from 5 to 50), is repeatable within animals, and is highly correlated with the total number of healthy follicles in ovaries. Also, animals with low AFC have higher serum concentrations of FSH and LH, but lower concentrations of Anti-Mullerian Hormone, progesterone and androgens than animals with high AFC. We have investigated the effect of maternal environment during gestation on their offspring AFC by restricting maternal nutrition to 60% of maintenance requirements (compared with 100% in controls) during the first third of gestation. Calves born to nutritionally restricted mothers had 60% lower AFC compared with calves born to mothers fed control diets. In other studies we have evidence to indicate that fertility may be compromised in animals with low AFC due to effects on oocytes, progesterone and the endometrium compared with animals with high AFC. To examine this directly we assessed AFC in post-partum dairy cows and found that cows with a high AFC had higher pregnancy rates, shorter calving to conception intervals and received fewer services during the breeding season compared with cows with a low AFC. In addition, the high variation in follicle numbers in adults may not only be reflective of reproductive disorders and suboptimal fertility, but there is evidence to indicate that it may be associated with alterations in the function of other non-reproductive systems (e.g. cardiovascular) that may have profound effects on the animal's health and welfare. PMID:21755688

  9. Dynamics of extracellular matrix in ovarian follicles and corpora lutea of mice.

    PubMed

    Irving-Rodgers, Helen F; Hummitzsch, Katja; Murdiyarso, Lydia S; Bonner, Wendy M; Sado, Yoshikazu; Ninomiya, Yoshifumi; Couchman, John R; Sorokin, Lydia M; Rodgers, Raymond J

    2010-03-01

    Despite the mouse being an important laboratory species, little is known about changes in its extracellular matrix (ECM) during follicle and corpora lutea formation and regression. Follicle development was induced in mice (29 days of age/experimental day 0) by injections of pregnant mare's serum gonadotrophin on days 0 and 1 and ovulation was induced by injection of human chorionic gonadotrophin on day 2. Ovaries were collected for immunohistochemistry (n=10 per group) on days 0, 2 and 5. Another group was mated and ovaries were examined on day 11 (n=7). Collagen type IV alpha1 and alpha2, laminin alpha1, beta1 and gamma1 chains, nidogens 1 and 2 and perlecan were present in the follicular basal lamina of all developmental stages. Collagen type XVIII was only found in basal lamina of primordial, primary and some preantral follicles, whereas laminin alpha2 was only detected in some preantral and antral follicles. The focimatrix, a specialised matrix of the membrana granulosa, contained collagen type IV alpha1 and alpha2, laminin alpha1, beta1 and gamma1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. In the corpora lutea, staining was restricted to capillary sub-endothelial basal laminas containing collagen type IV alpha1 and alpha2, laminin alpha1, beta1 and gamma1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. Laminins alpha4 and alpha5 were not immunolocalised to any structure in the mouse ovary. The ECM composition of the mouse ovary has similarities to, but also major differences from, other species with respect to nidogens 1 and 2 and perlecan. PMID:20033213

  10. Cyclin E-dependent protein kinase activity regulates niche retention of Drosophila ovarian follicle stem cells

    PubMed Central

    Wang, Zhu A.; Kalderon, Daniel

    2009-01-01

    Whether stem cells have unique cell cycle machineries and how they integrate with niche interactions remains largely unknown. We identified a hypomorphic cyclin E allele WX that strongly impairs the maintenance of follicle stem cells (FSCs) in the Drosophila ovary but does not reduce follicle cell proliferation or germline stem cell maintenance. CycEWX protein can still bind to the cyclin-dependent kinase catalytic subunit Cdk2, but forms complexes with reduced protein kinase activity measured in vitro. By creating additional CycE variants with different degrees of kinase dysfunction and expressing these and CycEWX at different levels, we found that higher CycE-Cdk2 kinase activity is required for FSC maintenance than to support follicle cell proliferation. Surprisingly, cycEWX FSCs were lost from their niches rather than arresting proliferation. Furthermore, FSC function was substantially restored by expressing either excess DE-cadherin or excess E2F1/DP, the transcription factor normally activated by CycE-Cdk2 phosphorylation of retinoblastoma proteins. These results suggest that FSC maintenance through niche adhesion is regulated by inputs that normally control S phase entry, possibly as a quality control mechanism to ensure adequate stem cell proliferation. We speculate that a positive connection between central regulators of the cell cycle and niche retention may be a common feature of highly proliferative stem cells. PMID:19966222

  11. Profiling follicle stimulating hormone-induced gene expression changes in normal and malignant human ovarian surface epithelial cells.

    PubMed

    Ho, Shuk-Mei; Lau, Kin-Mang; Mok, Samuel Chi-Ho; Syed, Viqar

    2003-07-01

    Epidemiological data have implicated the pituitary gonadotropin follicle stimulating hormone (FSH) as both a risk factor for and a protective agent against epithelial ovarian cancer. Yet, little is known about how this hormone could play such opposing roles in ovarian carcinogenesis. Complementary DNA microarrays containing 2400 named genes were used to examine FSH-induced gene expression changes in ovarian cancer (OC) and immortalized normal human ovarian surface epithelial (HOSE) cell lines. Two-way t-statistics analyses of array data identified two distinct sets of FSH-regulated genes in HOSE and in established OC cell lines established from patients (OVCA cell lines). Among the HOSE cell lines, FSH increased expression of 57% of the 312 genes and downregulated 43%. In contrast, FSH diminished expression of 92% of the 177 genes in the OVCA cell lines. All but 18 of the genes affected by FSH in HOSE cell lines were different from those altered in OVCA cell lines. Among the 18 overlapping genes, nine genes exhibited the same direction of change following FSH challenge, while the other nine showed discordance in response between HOSE and OVCA cell lines. The FSH-induced differential expression of seven out of nine genes was confirmed by real-time RT-PCR. Gene-specific antisense oligonuleotides (ODNs) were used to inhibit the expression of genes encoding GTPase activating protein (rap1GAP), neogenin, and restin in HOSE and OVCA cells. Antisense ODNs to neogenin and restin, but not an antisense ODN to rap1GAP, were effective in inhibiting OVCA cell growth, diminishing proliferating cell nuclear antigen expression, and increasing caspase 3 activities. Furthermore, the ODN to rap1GAP was further shown to be ineffective in altering migration properties of OVCA cell lines. HOSE cell proliferation was not affected by treatment with any of the antisense ODNs. In summary, gene profiling data reveal for the first time that FSH may exert different biological actions on OVCA

  12. Expression pattern of glycoconjugates in the Bidderian and ovarian follicles of the Brazilian toad Bufo ictericus analyzed by lectin histochemistry.

    PubMed

    Farias, C F; Azevedo, R A; Brito-Gitirana, L

    2006-02-01

    The Bidder's organ and ovary of the Brazilian toad Bufo ictericus were studied by light microscopy, using hematoxylin-eosin (HE) and periodic acid Schiff (PAS) staining. The expression and distribution of carbohydrate moieties was analyzed by lectin histochemistry, using 8 lectins with different carbohydrate specificities: Ulex europaeus (UEA I), Lens culinaris (LCA), Erythrina cristagalli (ECA), Arachis hypogaea (PNA), Ricinus communis (RCA I), Aleuria aurantia (AAA), Triticum vulgaris (WGA), and Glycine maximum (SBA). The results showed that the Bidderian zona pellucida presented alpha-mannose, alpha-L-fucose, beta-D-galactose, N-acetyl-D-glucosamine, and alpha/beta-N-acetyl-galactosamine residues. The Bidderian follicular cells showed the presence of beta-D-galactose and N-acetyl-D-glucosamine. In the extracellular matrix, alpha-mannose and alpha/beta-N-acetyl-galactosamine residues were detected. The ovarian zona pellucida showed alpha-L-fucose, N-acetyl-D-glucosamine, alpha/beta-N-acetyl-galactosamine residues, and alpha-mannose and N-acetyl-D-glucosamine residues were detected in the follicular cells. Thus, the zona pellucida in both organs contains N-acetyl-D-glucosamine, and alpha/beta-N-acetyl-galactosamine residues. alpha-L-fucose residues were detected in the zona pellucida of both organs, using different lectins. Considering that beta-D-galactose residue was absent from ovary but present in the Bidder's organ, this sugar residue may play an important role in follicle development, blocking the Bidderian follicles and preventing further development of the Bidder's organ into a functional ovary. PMID:16680305

  13. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on secretion of steroids and STAR, HSD3B and CYP19A1 mRNA expression in chicken ovarian follicles.

    PubMed

    Sechman, Andrzej; Antos, Piotr; Katarzyńska, Dorota; Grzegorzewska, Agnieszka; Wojtysiak, Dorota; Hrabia, Anna

    2014-03-01

    The aim of the study was to investigate the in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid hormone secretion by chicken ovarian follicles and mRNA expression of genes involved in steroids synthesis. In the first in vitro experiment, white (WF) and yellowish (YF) follicles and fragments of the theca (TL) and granulosa (GL) layers of the 3 largest yellow preovulatory follicles (F3-F1) were incubated in a medium supplemented with TCDD (0.01-100nM). In the second experiment, they were incubated in a medium with TCDD (10nM), ovine LH (10ng/mL; oLH) or a combination of oLH (10ng/mL) and TCDD (10nM). It was found that TCDD decreased estradiol (E2) secretion by WF and the TL of all preovulatory follicles, testosterone (T) secretion by WF, YF, and the TL of F2 and F1 follicles, and progesterone (P4) secretion by the GL of the preovulatory follicles. It also reduced oLH-stimulated E2 and P4 secretion by all examined follicles and T by WF. Real-time qPCR revealed that TCDD affected basal and oLH-stimulated expression of STAR, HSD3B and CYP19A1 mRNAs in all investigated ovarian follicles. In conclusion, the data obtained indicate that TCDD inhibits sex steroids secretion from chicken ovarian follicles. The effects of TCDD depend on its concentration and the stage of follicle maturation, and are associated with modulation of STAR, HSD3B and CYP19A1 mRNAs expression. These results indicate that the exposure of the laying hen to TCDD by influence of ovarian steroidogenesis may impair the selection of white follicles to preovulatory hierarchy and disturb their growth and preovulatory maturation. PMID:24398026

  14. Cholesterol side-chain cleavage cytochrome P450 and 3beta-hydroxysteroid dehydrogenase expression and the concentrations of steroid hormones in the follicular fluids of different phenotypes of healthy and atretic bovine ovarian follicles.

    PubMed

    Irving-Rodgers, Helen F; Krupa, Malgorzata; Rodgers, Raymond J

    2003-12-01

    Bovine ovarian antral follicles exhibit either one or the other of two patterns of granulosa cell death in atresia. Death can commence either from the antrum and progress toward the basal lamina (antral atresia) or the converse (basal atresia). In basal atresia, the remaining live antrally situated cells appeared to continue maturing. Beyond that, little is known about these distinct patterns of atresia. Healthy (nonatretic) follicles also exhibit either one or the other of two patterns of granulosa cell shape, follicular basal lamina ultrastructure or location of younger cells within the membrana granulosa. To examine these different phenotypes, the expression of the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in granulosa cells and concentrations of steroid hormones in follicular fluid were measured in individual histologically classified bovine antral follicles. Healthy follicles first expressed SCC and 3beta-HSD in granulosa cells only when the follicles reached an approximate threshold of 10 mm in diameter. The pattern of expression in antral atretic follicles was the same as healthy follicles. Basal atretic follicles were all <5 mm. In these, the surviving antral granulosa cells expressed SCC and 3beta-HSD. In examining follicles of 3-5 mm, basal atretic follicles were found to have substantially elevated progesterone (P < 0.001) and decreased androstenedione and testosterone compared to healthy and antral atretic follicles. Estradiol was highest in the large healthy follicles, lower in the small healthy follicles, lower still in the antral atretic follicles, and lowest in the basal atretic follicles. Our findings have two major implications. First, the traditional method of identifying atretic follicles by measurement of steroid hormone concentrations may be less valid with small bovine follicles. Second, features of the two forms of follicular atresia are so different as to

  15. Human chorionic gonadotrophin in early gestation induces growth of estrogenic ovarian follicles and improves primiparous sow fertility during summer.

    PubMed

    Seyfang, Jemma; Langendijk, P; Chen, T Y; Bouwman, E; Kirkwood, R N

    2016-09-01

    Reduced summer farrowing rates may be due to inadequate corpora luteal (CL) support. Porcine CL become dependent on LH from 12 d of pregnancy and the embryonic estrogen signal for maternal recognition of pregnancy (MRP) is initiated at about 11-12 d after insemination. We hypothesised that injection of the LH analogue human chorionic gonadotropin (hCG) would induce growth of estrogenic follicles and, by mimicking the signal for MRP and stimulating progesterone secretion, increase primiparous sow fertility. In Experiment 1, during a 28 d lactation 53 mixed parity sows were full-fed either throughout lactation (n=16) or until 18 d and then feed restricted during the last 10 d of lactation (n=36). At 12 d after mating restrict-fed sows were injected with 1000IU hCG (n=17) or were not injected (n=19); the full-fed sows acted as non-treated positive controls. Transrectal ovarian ultrasound exams were performed on days 12, 16, 20, 24, and 28; blood samples were obtained on days 12, 14, and 15 for estradiol and progesterone assay. For Experiment 2, during the summer months primiparous sows received 1000IU hCG 12 d after mating (n=28) or were non-injected controls (n=27). Pregnancy status was determined at 28 d and sows allowed to go to term to determine farrowing rates and litter sizes. In Experiment 1, injection of hCG increased (P<0.001) follicle diameter and serum concentrations of estradiol (P<0.01) and progesterone (P<0.05). There were no effects of lactation feeding level on wean-estrus interval, farrowing rate or subsequent litter size. In Experiment 2, hCG injection was associated with a higher pregnancy rate (P<0.05) and farrowing rate (P<0.08). There was no effect on litter size. These data confirm that hCG stimulates growth of estrogenic follicles and CL function, and improves primiparous sow fertility during the summer months. PMID:27397793

  16. Catalase addition to vitrification solutions maintains goat ovarian preantral follicles stability.

    PubMed

    Carvalho, A A; Faustino, L R; Silva, C M G; Castro, S V; Lobo, C H; Santos, F W; Santos, R R; Campello, C C; Bordignon, V; Figueiredo, J R; Rodrigues, A P R

    2014-08-01

    The aim of this study was to verify whether the addition of catalase (20 IU/mL) at different steps of goat ovarian tissue vitrification affects ROS levels, follicular morphology and viability, stromal cell density, apoptosis and the expression of proteins related to DNA-damage signaling (γH2AX) and repair (53BP1). Goat ovarian tissues were analyzed fresh (control) or after vitrification: without catalase (VS-/WS-), with catalase in vitrification solutions (VS+/WS-), with catalase in washing solutions (VS-/WS+) or with catalase in both solutions (VS+/WS+). The vitrification without catalase had higher ROS levels than the control. The catalase, regardless the step of addition, maintained ROS levels similar to the control. There were no difference between treatments regarding follicular viability, stromal cell density and detection of γH2AX and 53BP1. There was no difference in follicular morphology and DNA fragmentation between groups vitrified. In conclusion, catalase addition to vitrification solutions prevents ROS formation in cryopreserved goat ovarian tissues. PMID:24972862

  17. 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

  18. Daidzein affects steroidogenesis and oestrogen receptor expression in medium ovarian follicles of pigs.

    PubMed

    Nynca, Anna; Słonina, Dominika; Jablońska, Olga; Kamińska, Barbara; Ciereszko, Renata E

    2013-03-01

    Daidzein, a phytoestrogen present in soybean products used in swine feed, has been demonstrated to affect both reproductive and endocrine functions. The aims of this study were to examine the in vitro effects of daidzein on (1) progesterone (P4) and oestradiol (E2) secretion by porcine luteinised granulosa cells harvested from medium follicles, and (2) the mRNA and protein expression of oestrogen receptors α and β (ERα and ERβ) in these cells. The influence of E2 on P4 secretion and ERα and ERβ expression in the granulosa cells of pigs was also investigated. It was found that daidzein inhibited progesterone secretion by luteinised granulosa cells isolated from medium follicles. In contrast, E2 did not affect progesterone production by these cells. Moreover, daidzein did not alter the granulosal secretion of E2. Both daidzein and E2 decreased mRNA expression of ERα in the cells examined. The expression of ERβ mRNA was not affected by daidzein but was inhibited by E2. ERα protein was not detected while ERβ protein was found in the nuclei of the cells. Daidzein and E2 upregulated the expression of ERβ protein in the cells. In summary, the phytoestrogen daidzein directly affected the porcine ovary by inhibiting progesterone production and increasing ERβ protein expression. Daidzein-induced changes in follicular steroidogenesis and granulosal sensitivity to oestrogens may disturb reproductive processes in pigs. PMID:23439294

  19. Forkhead box O member FOXO1 regulates the majority of follicle-stimulating hormone responsive genes in ovarian granulosa cells.

    PubMed

    Herndon, Maria K; Law, Nathan C; Donaubauer, Elyse M; Kyriss, Brandon; Hunzicker-Dunn, Mary

    2016-10-15

    FSH promotes maturation of ovarian follicles. One pathway activated by FSH in granulosa cells (GCs) is phosphatidylinositol-3 kinase/AKT. The AKT target FOXO1 is reported to function primarily as a repressor of FSH genes, including Ccnd2 and Inha. Based on its broad functions in other tissues, we hypothesized that FOXO1 may regulate many more GC genes. We transduced GCs with empty adenovirus or constitutively active FOXO1 followed by treatment with FSH for 24 h, and conducted RNA deep sequencing. Results show that FSH regulates 3772 genes ≥2.0-fold; 60% of these genes are activated or repressed by FOXO1. Pathway Studio Analysis revealed enrichment of genes repressed by FOXO1 in metabolism, signaling, transport, development, and activated by FOXO1 in signaling, cytoskeletal functions, and apoptosis. Gene regulation was verified by q-PCR (eight genes) and ChIP analysis (two genes). We conclude that FOXO1 regulates the majority of FSH target genes in GCs. PMID:27328024

  20. Gankyrin facilitates follicle-stimulating hormone-driven ovarian cancer cell proliferation through the PI3K/AKT/HIF-1α/cyclin D1 pathway.

    PubMed

    Chen, J; Bai, M; Ning, C; Xie, B; Zhang, J; Liao, H; Xiong, J; Tao, X; Yan, D; Xi, X; Chen, X; Yu, Y; Bast, R C; Zhang, Z; Feng, Y; Zheng, W

    2016-05-12

    Gankyrin is a regulatory subunit of the 26kD proteasome complex. As a novel oncoprotein, gankyrin is expressed aberrantly in cancers from several different sites and has been shown to contribute to oncogenesis in endometrial and cervical carcinomas. Neither gankyrin's contribution to the development of epithelial ovarian cancer nor its interaction with follicle-stimulating hormone (FSH)-driven proliferation in ovarian cancer has been studied. Here we have found that gankyrin is overexpressed in ovarian cancers compared with benign ovarian cystadenomas and that gankyrin regulates FSH upregulation of cyclin D1. Importantly, gankyrin regulates PI3K/AKT signaling by downregulating PTEN. Prolonged AKT activation by FSH stimulation of the FSH receptor (FSHR) promotes gankyrin expression, which, in turn, enhances AKT activation by inhibiting PTEN. Overexpression of gankyrin decreases hypoxia inducible factor-1α (HIF-1α) protein levels, but has little effect on HIF-1α mRNA levels, which could be attributed to gankyrin mediating HIF-1α protein stability via the ubiquitin-proteasome pathway. Reduction in HIF-1α protein stability led to attenuation of the binding with cyclin D1 promoter, resulted in abolishment of the negative regulation of cyclin D1 by HIF-1α, which promotes proliferation of ovarian cancer cells. Our results document that gankyrin regulates HIF-1α protein stability and cyclin D1 expression, ultimately mediating FSH-driven ovarian cancer cell proliferation. PMID:26364616

  1. Fetal Cyclophosphamide Exposure Induces Testicular Cancer and Reduced Spermatogenesis and Ovarian Follicle Numbers in Mice

    PubMed Central

    Comish, Paul B.; Drumond, Ana Luiza; Kinnell, Hazel L.; Anderson, Richard A.; Matin, Angabin; Meistrich, Marvin L.; Shetty, Gunapala

    2014-01-01

    Exposure to radiation during fetal development induces testicular germ cell tumors (TGCT) and reduces spermatogenesis in mice. However, whether DNA damaging chemotherapeutic agents elicit these effects in mice remains unclear. Among such agents, cyclophosphamide (CP) is currently used to treat breast cancer in pregnant women, and the effects of fetal exposure to this drug manifested in the offspring must be better understood to offer such patients suitable counseling. The present study was designed to determine whether fetal exposure to CP induces testicular cancer and/or gonadal toxicity in 129 and in 129.MOLF congenic (L1) mice. Exposure to CP on embryonic days 10.5 and 11.5 dramatically increased TGCT incidence to 28% in offspring of 129 mice (control value, 2%) and to 80% in the male offspring of L1 (control value 33%). These increases are similar to those observed in both lines of mice by radiation. In utero exposure to CP also significantly reduced testis weights at 4 weeks of age to ∼70% of control and induced atrophic seminiferous tubules in ∼30% of the testes. When the in utero CP-exposed 129 mice reached adulthood, there were significant reductions in testicular and epididymal sperm counts to 62% and 70%, respectively, of controls. In female offspring, CP caused the loss of 77% of primordial follicles and increased follicle growth activation. The results indicate that i) DNA damage is a common mechanism leading to induction of testicular cancer, ii) increased induction of testis cancer by external agents is proportional to the spontaneous incidence due to inherent genetic susceptibility, and iii) children exposed to radiation or DNA damaging chemotherapeutic agents in utero may have increased risks of developing testis cancer and having reduced spermatogenic potential or diminished reproductive lifespan. PMID:24691397

  2. Fetal cyclophosphamide exposure induces testicular cancer and reduced spermatogenesis and ovarian follicle numbers in mice.

    PubMed

    Comish, Paul B; Drumond, Ana Luiza; Kinnell, Hazel L; Anderson, Richard A; Matin, Angabin; Meistrich, Marvin L; Shetty, Gunapala

    2014-01-01

    Exposure to radiation during fetal development induces testicular germ cell tumors (TGCT) and reduces spermatogenesis in mice. However, whether DNA damaging chemotherapeutic agents elicit these effects in mice remains unclear. Among such agents, cyclophosphamide (CP) is currently used to treat breast cancer in pregnant women, and the effects of fetal exposure to this drug manifested in the offspring must be better understood to offer such patients suitable counseling. The present study was designed to determine whether fetal exposure to CP induces testicular cancer and/or gonadal toxicity in 129 and in 129.MOLF congenic (L1) mice. Exposure to CP on embryonic days 10.5 and 11.5 dramatically increased TGCT incidence to 28% in offspring of 129 mice (control value, 2%) and to 80% in the male offspring of L1 (control value 33%). These increases are similar to those observed in both lines of mice by radiation. In utero exposure to CP also significantly reduced testis weights at 4 weeks of age to ∼ 70% of control and induced atrophic seminiferous tubules in ∼ 30% of the testes. When the in utero CP-exposed 129 mice reached adulthood, there were significant reductions in testicular and epididymal sperm counts to 62% and 70%, respectively, of controls. In female offspring, CP caused the loss of 77% of primordial follicles and increased follicle growth activation. The results indicate that i) DNA damage is a common mechanism leading to induction of testicular cancer, ii) increased induction of testis cancer by external agents is proportional to the spontaneous incidence due to inherent genetic susceptibility, and iii) children exposed to radiation or DNA damaging chemotherapeutic agents in utero may have increased risks of developing testis cancer and having reduced spermatogenic potential or diminished reproductive lifespan. PMID:24691397

  3. 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

  4. 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. PMID:26667040

  5. Expression and localization of ghrelin and its receptor in ovarian follicles during different stages of development and the modulatory effect of ghrelin on granulosa cells function in buffalo.

    PubMed

    Gupta, M; Dangi, S S; Singh, G; Sarkar, M

    2015-01-01

    Ghrelin, a hormone predominantly found in the stomach, was recently described as a factor that controls female reproductive function. The aim of our study was to investigate the expression and localization of ghrelin and its active receptor, growth hormone secretagogue receptor type 1a (GHS-R1a) in buffalo ovarian follicles of different follicular size and to investigate role of ghrelin on estradiol (E2) secretion, aromatase (CYP19A1), proliferating cell nuclear antigen (PCNA) and apoptosis regulator Bax gene expression on granulosa cell culture. Using real time PCR and western blot, we measured gene and protein expression of examined factors. Localization was done with immunofluorescence method. Expression of ghrelin increased with follicle size with significantly highest in dominant or pre-ovulatory follicle (P<0.05). Expression of GHS-R1a was comparable in medium and large follicle but was higher than small follicles (P<0.05). Both the factors were localized in granulosa and theca cells. Pattern of intensity of immunofluorescence was similar with mRNA and protein expression. In the in vitro study granulosa cells (GCs) were cultured and treated with ghrelin each at 1, 10 and 100ng/ml concentrations for two days after obtaining 75-80 per cent confluence. Ghrelin treatment significantly (P<0.05) inhibited E2 secretion, CYP19A1 expression, apoptosis and promoted cell proliferation. In conclusion, this study provides novel evidence for the presence of ghrelin and receptor GHS-R1a in ovarian follilcles and modulatory role of ghrelin on granulosa cell function in buffalo. PMID:25275756

  6. Non-genomic estrogen/estrogen receptor α promotes cellular malignancy of immature ovarian teratoma in vitro.

    PubMed

    Hung, Yao-Ching; Chang, Wei-Chun; Chen, Lu-Min; Chang, Ying-Yi; Wu, Ling-Yu; Chung, Wei-Min; Lin, Tze-Yi; Chen, Liang-Chi; Ma, Wen-Lung

    2014-06-01

    Malignant immature ovarian teratomas (IOTs) most often occur in women of reproductive age. It is unclear, however, what roles estrogenic signaling plays in the development of IOT. In this study, we examined whether estrogen receptors (ERα and β) promote the cellular malignancy of IOT. Estradiol (E2), PPT (propylpyrazole), and DPN (diarylpropionitrile) (ERα- and β-specific agonists, respectively), as well as ERα- or ERβ-specific short hairpin (sh)RNA were applied to PA-1 cells, a well-characterized IOT cell line. Cellular tumorigenic characteristics, for example, cell migration/invasion, expression of the cancer stem/progenitor cell marker CD133, and evidence for epithelial-mesenchymal transition (EMT) were examined. In PA-1 cells that expressed ERα and ERβ, we found that ERα promoted cell migration and invasion. We also found that E2/ERα signaling altered cell behavior through non-classical transactivation function. Our data show non-genomic E2/ERα activations of focal adhesion kinase-Ras homolog gene family member A (FAK-RhoA) and ERK governed cell mobility capacity. Moreover, E2/ERα signaling induces EMT and overexpression of CD133 through upregulation micro-RNA 21 (miR21; IOT stem/progenitor promoter), and ERK phosphorylations. Furthermore, E2/ERα signaling triggers a positive feedback regulatory loop within miR21 and ERK. At last, expression levels of ERα, CD133, and EMT markers in IOT tissue samples were examined by immunohistochemistry. We found that cytosolic ERα was co-expressed with CD133 and mesenchymal cell markers but not epithelial cell markers. In conclusion, estrogenic signals exert malignant transformation capacity of cancer cells, exclusively through non-genomic regulation in female germ cell tumors. PMID:24142535

  7. Concentration of progesterone during the development of the ovulatory follicle: I. Ovarian and embryonic responses.

    PubMed

    Cerri, R L A; Chebel, R C; Rivera, F; Narciso, C D; Oliveira, R A; Thatcher, W W; Santos, J E P

    2011-07-01

    Objectives were to evaluate the effects of differing progesterone concentrations during follicle development on follicular dynamics, fertilization, and embryo quality. Lactating Holstein cows (n=154) were assigned randomly to 1 of 2 treatments. Cows underwent a presynchronization of the estrous cycle composed of an injection of GnRH concurrently with the placement of a progesterone insert, an injection of PGF(2α) and insert removal 7 d later, and a second injection of GnRH 48 h later (study d -16). All cows were then submitted to a hormonal protocol identical to the presynchronization program starting on d 7 of the estrous cycle (study d -9). Cows enrolled in the high progesterone (HP) treatment received no further treatment. Cows in the low progesterone (LP) treatment received additional PGF(2α) injections on study d -14, -13.5, and -13 and again on study d -9, -7, -6.5, and -6. Ovaries were evaluated by ultrasonography, and blood was sampled for concentrations of progesterone and estradiol throughout the study. Uteri were flushed 6 d after artificial insemination (AI) and recovered oocytes-embryos were evaluated. Concentrations of progesterone were less for LP cows from study d -7 to -2; concentrations of estradiol at PGF(2α) and at the last GnRH of synchronization were greater for LP than HP. The proportion of cows in estrus at AI was greater for LP than for HP (38.0 vs. 5.3%). Ovulatory follicles of LP cows had larger diameters at the injections of PGF(2α) (17.2 vs. 14.6mm) and final GnRH (19.4 vs. 16.9%) of the synchronization, which resulted in a larger diameter of the corpus luteum 6 d after AI (24.3 vs. 22.6mm). Double ovulation after the last GnRH of the synchronization was increased in LP (18.6%) compared with HP (4.5%). Fertilization rate was similar and averaged 82.7%. The proportion of embryos and oocytes-embryos classified as grades 1 and 2, proportion of degenerated embryos, and unfertilized-degenerated oocytes-embryos were not different between

  8. Effects of cyclic increase in gonadotropins on the in vitro development of primordial follicles to antral stage.

    PubMed

    Fabbri, Raffaella; Pasquinelli, Gianandrea; Parazza, Isabella; Macciocca, Maria; Magnani, Valentina; Battaglia, Cesare; Paradisi, Roberto; Venturoli, Stefano

    2012-10-01

    The aim of this study was to evaluate the effects of FSH and LH on follicle development during a long-term culture of cryopreserved human ovarian tissue, using morphological and ultrastructural examinations. Thawed ovarian tissue slices from a 4-year-old child with Wilms tumor were cultured for 32 weeks in two different culture conditions, without (medium A) and with (medium B) a monthly peaked increase in FSH and LH. At week 32, in the medium B cultured tissue, a cluster of preantral follicles associated with two oocytes prematurely ovulated was observed, suggesting that the cyclic increase of gonadotropins promoted thawed follicles to grow up to the antral stage. However, the integrity and coordinated follicle development were not maintained. Indeed, ultrastructural analysis showed a well-preserved "naked" oocyte with concomitant features of immaturity and maturity, as if this culture condition had led to an asynchronous maturation of oocyte cytoplasmic components. PMID:23025653

  9. Snail controls proliferation of Drosophila ovarian epithelial follicle stem cells, independently of E-cadherin.

    PubMed

    Tseng, Chen-Yuan; Kao, Shih-Han; Hsu, Hwei-Jan

    2016-06-15

    Epithelial stem cells undergo constant self-renewal and differentiation to maintain the homeostasis of epithelial tissues that undergo rapid turnover. Recent studies have shown that the epithelial-mesenchymal transition (EMT), which is primarily mediated by Snail via the suppression of E-cadherin, is able to generate cells with stem cell properties. However, the role of Snail in epithelial stem cells remains unclear. Here, we report that Snail directly controls proliferation of follicle stem cells (FSCs) in Drosophila females. Disruption of Snail expression in FSCs compromises their proliferation, but not their maintenance. Conversely, FSCs with excessive Snail expression display increased proliferation and lifespan, which is accompanied by a moderate decrease in the expression of E-cadherin (required for adhesion of FSCs to their niche) at the junction between their adjacent cells, indicating a conserved role of Snail in E-cadherin inhibition, which promote epithelial cell proliferation. Interestingly, a decrease in E-cadherin in snail-knock down FSCs does not restore the decreased proliferation of snail-knock down FSCs, suggesting that adhesion strength of FSCs to their niche is dispensable for Snail-mediated FSC division. Our results demonstrate that Snail controls epithelial stem cell division independently of its known role in the EMT, which contributes to induction of cancer stem cells. PMID:27141871

  10. 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.

  11. A novel two-step strategy for in vitro culture of early-stage ovarian follicles in the mouse

    PubMed Central

    Jin, Shiying; Lei, Lei; Shikanov, Ariella; Shea, Lonnie D.; Woodruff, Teresa K.

    2009-01-01

    Objective To develop an in vitro strategy to support the growth of early-stage follicles and produce mature oocytes competent for fertilization. Design Whole ovaries from 8-day-old mice were cultured for 4 days, then secondary follicles were isolated and cultured for 12 days in a 3-dimensional (3-D) alginate or fibrin-alginate hydrogel matrix. Setting University-affiliated laboratory. Animals Mice Intervention(s) None Main Outcome Measures Histologic evaluation of follicle development, steroid hormone production, and rates of oocyte maturation, oocyte fertilization, and embryo formation. Results Culture of 8-day-old mouse ovaries for 4 days resulted in transition of the follicle population from primordial and primary follicles to secondary follicles, similar to that seen in a 12-day-old ovary. Isolated secondary follicles cultured for 12 days showed larger increases in oocyte diameter and more frequent antrum formation and theca cell differentiation in the FA-hydrogel matrix compared with the alginate matrix (P<0.05). Steroid hormone secretion patterns were consistent with the changes in follicle morphology and cell differentiation observed in the cultured follicles. Compared with oocytes from alginate follicle cultures, a greater number of oocytes retrieved from the FA-based follicle cultures progressed to metaphase I (MI), reached metaphase II (MII) and could be fertilized and cleaved to two-cell embryos (P<0.05). The organ culture plus FA-hydrogel follicle culture strategy produced a very high rate of oocyte progression to MII (88 ± 8.7%) and formation of 2-cell embryos (54 ± 4%). Conclusion A strategy combining whole ovary culture of early-stage follicles and subsequent FA hydrogel in vitro follicle culture produced a high percentage of oocytes competent for fertilization and may provide new options for fertility preservation in women and prepubertal girls facing fertility-threatening diseases or treatments. PMID:20004373

  12. Ovarian and endocrine responses associated with the treatment of cystic ovarian follicles in dairy cows with gonadotropin releasing hormone and prostaglandin F2α, with or without exogenous progesterone

    PubMed Central

    2004-01-01

    Abstract The objectives of this observational study were to document ovarian and endocrine responses associated with the treatment of cystic ovarian follicles (COFs) in dairy cows, using gonadotropin releasing hormone (GnRH) and prostaglandin F2α(PGF) with or without exogenous progesterone. A secondary objective was to determine pregnancy establishment following synchronization of ovulation and timed insemination in cows diagnosed with COFs. In trial I, 18 Holstein cows diagnosed with COFs received 2 injections of 100 μg GnRH, 9 d apart, with 25 mg PGF given 7 d after the 1st GnRH. A new follicle developed in all 18 cows after the 1st GnRH, and 83% of cows ovulated following the 2nd GnRH. Cows were inseminated 16 h after the 2nd GnRH. Of the 17 cows available for pregnancy diagnosis, 7 were confirmed pregnant. In trial II, 8 cows with COFs received GnRH and an intravaginal progesterone device (CIDR) concurrently, then PGF 7 d later. The CIDR was removed 2 d after PGF administration. Plasma estradiol concentrations declined following CIDR insertion. In all cows, a new follicle developed following GnRH treatment; estradiol-surge and estrus occurred spontaneously after CIDR-removal. Seven of 8 cows ovulated the new follicle. In dairy cows diagnosed with COFs, treatment with GnRH followed by PGF 7 d later, with or without exogenous progesterone, resulted in the recruitment of a healthy new follicle; synchronization of ovulation and timed insemination resulted in a 41% pregnancy rate. PMID:15600159

  13. The follicle-deplete mouse ovary produces androgen.

    PubMed

    Mayer, Loretta P; Devine, Patrick J; Dyer, Cheryl A; Hoyer, Patricia B

    2004-07-01

    The follicle-depleted postmenopausal ovary is enriched in interstitial cells that produce androgens. This study was designed to cause follicle depletion in mice using the industrial chemical, 4-vinylcyclohexene diepoxide (VCD), and characterize the steroidogenic capacity of cells in the residual ovarian tissue. From a dose-finding study, the optimal daily concentration of VCD was determined to be 160 mg/kg. Female B6C3F(1) immature mice were treated daily with vehicle control or VCD (160 mg kg(-1) day(-1), 15 days, i.p.). Ovaries were removed and processed for histological evaluation. On Day 15 following onset of treatment, primordial follicles were depleted and primary follicles were reduced to about 10% of controls. On Day 46, primary follicles were depleted and secondary and antral follicles were reduced to 0.7% and 2.6% of control, respectively. Seventy-five percent of treated mice displayed disruptions in estrous cyclicity. All treated mice were in persistent diestrus (acyclic) by Day 58. Plasma FSH levels were increased (P < 0.05) relative to controls on Day 37 and had plateaued by Day 100. Relative to age-matched cyclic controls, by Day 127, the significant differences in VCD-treated mice included reduced ovarian and uterine weights, elevated plasma LH and FSH, and reduced plasma progesterone and androstenedione. Furthermore, plasma 17beta-estradiol levels were nondetectable. Unlike controls, immunostaining for LH receptor, and the high density lipoprotein receptor (SR-BI), was diffuse in ovarian sections from VCD-treated animals. Ovaries from Day 120 control and VCD-treated animals were dissociated and dispersed cells were placed in culture. Cultured cells from ovaries of VCD-treated animals produced less LH-stimulated progesterone than control cells. Androstenedione production was nondetectable in cells from cyclic control animals. Conversely, cells from VCD-treated animals produced androstenedione that was doubled in the presence of insulin and LH (1 and

  14. 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

  15. Bax, caspase-2, and caspase-3 are required for ovarian follicle loss caused by 4-vinylcyclohexene diepoxide exposure of female mice in vivo.

    PubMed

    Takai, Yasushi; Canning, Jacqueline; Perez, Gloria I; Pru, James K; Schlezinger, Jennifer J; Sherr, David H; Kolesnick, Richard N; Yuan, Junying; Flavell, Richard A; Korsmeyer, Stanley J; Tilly, Jonathan L

    2003-01-01

    The industrial chemical, 4-vinylcyclohexene diepoxide (VCD), kills oocytes within immature follicles in the ovaries of mice and rats and is considered a potential occupational health hazard. It has been reported that VCD-induced follicle loss occurs via a cell death process involving elevated expression of Bax, a proapoptotic Bcl-2 family member, and increased caspase-3-like activity. We have previously shown that oocytes lacking acid sphingomyelinase (ASMase; an enzyme that generates the proapoptotic stress sensor ceramide), the aromatic hydrocarbon receptor (Ahr), Bax, or caspase-2 are resistant to apoptosis induced by other chemical toxicants. Therefore, this study was designed to investigate the functional importance of ASMase, Ahr, Bax, and caspase-2 as well as the related executioner enzyme caspase-3 to VCD-induced ovotoxicity in mice using gene knockout technology. For each gene mutant mouse line, wild-type and homozygous-null female siblings derived from heterozygous matings were given once-daily ip injections of either vehicle (sesame oil) or VCD (80 mg/kg body weight) for 15 d (three or four mice per treatment group per genotype). Ovaries were collected 24 h after the final injection and analyzed for the total number of nonatretic primordial and primary follicles remaining per ovary. No differences in the extent of primordial or primary follicle destruction resulting from VCD exposure were observed in wild-type vs. ASMase- or Ahr-deficient mice. By contrast, the extent of VCD-induced primordial follicle depletion in Bax-deficient mice (45 +/- 11%) was significantly (P < 0.05) lower than that in wild-type females (85 +/- 2%). The extent of primary follicle loss in bax-null mice exposed to VCD (3 +/- 22%) was also significantly (P < 0.05) lower than that in their wild-type sisters (86 +/- 4%). In caspase-2-deficient mice, significantly (P < 0.05) fewer oocyte-containing primary follicles were destroyed by VCD (17 +/- 19%) vs. wild-type controls (71 +/- 6

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

    PubMed

    Thompson, Kary E; Bourguet, Shannon M; Christian, Patricia J; Benedict, Jamie C; Sipes, I Glenn; Flaws, Jodi A; Hoyer, Patricia B

    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. PMID:15710172

  17. 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.

  18. Evidence that severe acute stress and starvation induce rapid atresia of ovarian vitellogenic follicles in Atlantic bluefin tuna, Thunnus thynnus (L.) (Osteichthyes: Scombridae).

    PubMed

    Corriero, A; Zupa, R; Bello, G; Mylonas, C C; Deflorio, M; Genovese, S; Basilone, G; Buscaino, G; Buffa, G; Pousis, C; De Metrio, G; Santamaria, N

    2011-11-01

    The effects of different stressors on the atretic degeneration of ovarian vitellogenic follicles, as well as on the ovarian mass, were examined in female Atlantic bluefin tuna, Thunnus thynnus (L.), from the Mediterranean Sea. The stressors taken into consideration were short-term starvation (up to 14 days), long-term cage rearing (1 year) and crowding-induced severe panic frenzy. Wild-caught individuals were used as a control group. Fish subjected to either severe panic frenzy or starvation exhibited a decrease in gonad mass and had significantly higher intensity of α atresia in the vitellogenic follicles (means: 78% and 58%, respectively; range: 36-100%) than either wild or long-term caged individuals (means: 32% and 30%, respectively; range: 19-44%). The extensive atresia in fish stressed by severe panic frenzy was observed as early as 24 h after the stressing event. The present study represents the first evidence of the extreme susceptibility of Atlantic bluefin tuna to severe acute stress during vitellogenesis; it also shows that starvation is associated with progressive reabsorption of vitellogenic oocytes. PMID:21988357

  19. 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.

  20. 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. PMID:24100206

  1. 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

  2. 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

  3. Novel needle-in-straw vitrification can effectively preserve the follicle morphology, viability, and vascularization of ovarian tissue in Japanese quail (Coturnix japonica).

    PubMed

    Liu, Jianan; Cheng, Kimberly M; Silversides, Frederick G

    2012-10-01

    Cryopreservation of ovarian tissue has been the only effective way of ex situ conservation of female germplasm in avian species. A novel needle-in-straw (NIS) vitrification method was developed to store tissue in straws instead of cryovials. Fragments of ovarian tissue from one-week old Japanese quail were transfixed on an acupuncture needle. They were immersed in equilibration and vitrification solutions containing dimethyl sulphoxide, ethylene glycol and sucrose. A layer of tin foil was rolled over the tissue fragments and the tin foil package was plunged into liquid nitrogen and inserted into a pre-cooled, 0.5-ml straw which was stored in liquid nitrogen. Tissue was also preserved using a needle immersed vitrification (NIV) method, in which tissue fragments transfixed on needles without tin foil and were stored in cryovials filled with liquid nitrogen. Cryopreserved tissue was warmed at room temperature (RT) or 37°C and the ratio of normal follicles to total visible follicles was determined by histological methods. In addition, cryopreserved and warmed tissue was cultured on the chorioallantoic membranes of fertilized chicken eggs for 5-6 days. The viability and vascularization of the grafts were evaluated. The tissue cryopreserved by NIS and warmed at RT showed comparable follicle morphology to fresh tissue and to that preserved by NIV and warmed at RT. No significant impairment on the viability or vascularization of the grafted tissue was observed. The NIS method allows tissue to be stored and transported safely and efficiently and can be used instead of cryovials in tissue cryobanking. PMID:22921390

  4. Effect of plasma lipoproteins in gonadotropin stimulation of 17 beta-estradiol production in the ovarian follicle of rainbow trout (Salmo gairdneri).

    PubMed

    Babin, P J

    1986-12-01

    The effect of trout plasma lipoproteins on the production of 17 beta-estradiol by trout ovarian follicles is investigated in vitro. 17 beta-Estradiol secretion into the medium was assayed as a function of follicular diameter in the presence of lipoproteins with and without salmonid gonadotropin (SGA-GTH). The presence of very low-density lipoproteins (VLDL) + chylomicrons (Chy), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) amplified the SGA-GTH effect at the lowest concentrations tested (less than 50 micrograms protein/ml). HDL is the most effective for increasing hormone accumulation on a microgram lipoprotein sterol basis. Autoradiography of 125I-labeled LDL showed that they were preferentially bound by thecal cells. Kinetics of 17 beta-estradiol release indicated that lipoprotein amplification occurred especially after 15 hr and subsequent metabolism of 17 beta-estradiol by follicular layers also led to an equilibrium. At the end of vitellogenesis apoprotein B lipoproteins (VLDL + Chy, LDL) apparently inhibited SGA-GTH stimulation. N',O'-Dibutyryl cAMP (10 mM) considerably stimulated 17 beta-estradiol production but lipoprotein amplification did not occur. Chloroquine (30 microM) inhibition of LDL and HDL amplification indicates that this process requires lysosomal degradation. Plasma lipoproteins in trout modulate SGA-GTH stimulation of 17 beta-estradiol production during exogenous vitellogenesis. Due to the ease and frequency with which the experiments can be carried out, the ovarian follicle of salmonids is an excellent model for the study of the role of lipoproteins in the regulation of ovarian steroids biosynthesis. PMID:3026884

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Comparisons of mRNA expression for aromatase, FSH receptor, and IGF-I in the granulosa of small ovarian follicles between cattle selected and unselected for twin ovulations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Long-term selection of cattle for the production of twin ovulations and births has enhanced the development of preantral and antral ovarian follicles and increased the frequency of twin or triplet ovulations to greater than 60%. However, these differences have not been linked to differences in FSH s...

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

    PubMed

    Bhattacharya, Poulomi; Keating, Aileen F

    2012-06-15

    The mammalian ovary is a heterogeneous organ and contains oocyte-containing follicles at varying stages of development. The most immature follicular stage, the primordial follicle, comprises the ovarian reserve and is a finite number, defined at the time of birth. Depletion of all follicles within the ovary leads to reproductive senescence, known as menopause. A number of chemical classes can destroy follicles, thus hastening entry into the menopausal state. The ovarian response to chemical exposure can determine the extent of ovotoxicity that occurs. Enzymes capable of bioactivating as well as detoxifying xenobiotics are expressed in the ovary and their impact on ovotoxicity has been partially characterized for trichloroethylene, 7,12-dimethylbenz[a]anthracene, and 4-vinylcyclohexene. This review will discuss those studies, as well as illustrate where knowledge gaps remain for chemicals that have also been established as ovotoxicants. PMID:22531813

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

    PubMed Central

    Bhattacharya, Poulomi; Keating, Aileen F.

    2012-01-01

    The mammalian ovary is a heterogeneous organ, and contains oocyte-containing follicles at varying stages of development. The most immature follicular stage, the primordial follicle, comprises the ovarian reserve and is a finite number, defined at the time of birth. Depletion of all follicles within the ovary leads to reproductive senescence, known as menopause. A number of chemical classes can destroy follicles thus hastening entry into the menopausal state. The ovarian response to chemical exposure can determine the extent of ovotoxicity that occurs. Enzymes capable of bioactivating as well as detoxifying xenobiotics are expressed in the ovary and their impact on ovotoxicity have been partially characterized for trichloroethylene, 7,12-dimethylbenz[a]anthracene, and 4-vinylcyclohexene. This review will discuss those studies, as well as illustrate where knowledge gaps remain for chemicals that have also been established as ovotoxicants. PMID:22531813

  9. Methoxychlor induces atresia by altering Bcl2 factors and inducing caspase activity in mouse ovarian antral follicles in vitro

    PubMed Central

    Basavarajappa, Mallikarjuna S.; Karman, Bethany N.; Wang, Wei; Gupta, Rupesh K.; Flaws, Jodi A.

    2012-01-01

    Methoxychlor (MXC) is an organochlorine pesticide widely used in many countries against various species of insects that attack crops and domestic animals. MXC reduces fertility by increasing atresia (death) of antral follicles in vivo. MXC also induces atresia of antral follicles after 96 h in vitro. The current work tested the hypothesis that MXC induces morphological atresia at early time points (24 and 48 h) by altering pro-apoptotic (Bax, Bok, Casp3, and caspase activity) and anti-apoptotic (Bcl2 and Bcl-xL) factors in the follicles. The results indicate that at 24 h, MXC increased Bcl-xL and Bax mRNA levels and increased the ratio of Bax/Bcl2. At 48–96 h, MXC induced morphological atresia. At 24–96 h, MXC increased caspase activities. These data suggest that MXC may induce atresia by altering Bcl2 factors and inducing caspase activities in antral follicles. PMID:23000595

  10. Ovarian follicle apoptosis at the onset of standing estrus in virgin and repeat-breeder dairy heifers.

    PubMed

    Båge, R; Bosu, W T; Rodríguez-Martínez, H

    2001-09-15

    There is evidence that repeat breeding in dairy cattle can be caused by both extrinsic, environmental factors and intrinsic, animal factors. In repeat-breeder heifers (RBH), disturbed endocrine patterns and estrous events result in a subsequent decreased fertility associated with delayed ovulation. Whether infertility is also due to the presence of an unsuitable follicular environment impairing normal fertilization, remains to be determined. At the onset of standing estrus, ovaries were obtained from 7 strictly defined RBH and 5 virgin heifers (VH) of the Swedish Red and White breed. Detection of apoptosis in the preovulatory and three subordinate follicle walls was done by using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) technique at light microscopy level. The follicles were histologically assessed for degree of atresia. The ultrastructure of the follicle wall and recovered oocytes was studied using transmission electron microscopy. The overall degree of apoptosis in membrana granulosa and theca interna of preovulatory and subordinate follicles did not differ between RBH and VH, but the numbers of TUNEL-positive cells differed significantly between preovulatory and subordinate follicles in both RBH and VH. There was a strong relationship between density of apoptotic cells and degree of atresia. No differences in follicle wall apoptosis nor morphology were detectable, suggesting that repeat breeder heifers enter standing estrus with the same morphological prerequisites as normal animals, considering follicular structure. PMID:11665874