Cortisol induces follicle regression, while FSH prevents cortisol-induced follicle regression in pigs.

During follicular development, a few dominant follicles develop to large antral dominant follicles, whereas the remaining follicles undergo atretic degeneration. Because vascularization on the follicular surface is a morphological feature of dominant follicles, we previously classified these follicles as vascularized follicles (VFs) and non-VFs (NVFs). In NVFs, progesterone producing genes were expressed similarly to that in VFs; however, the progesterone concentration in follicular fluid was low in large NVFs. Therefore, we estimated that progesterone is converted to cortisol, which induces the loss of follicular functions. In this study, we comparative analyzed the expression of genes for progesterone converting enzymes (Cytochrome (CYP)11B1, CYP21A2, Hydroxysteroid (HSD)11B2) and cortisol receptor (NR3C1) in VF and NVF granulosa cells. In NVFs, expression of cortisol producing genes (CYP11B1 and CYP21A2) was higher than in VFs. Expression of the gene for the cortisol metabolizing enzyme HSD11B2 in NVFs was significantly lower than in VFs. In NVFs, accompanied by increasing cortisol concentration in follicular fluid, apoptosis of granulosa and cumulus cells was observed. Cultivation with FSH and metyrapone (a CYP11B1 inhibitor) of NVF cumulus-oocyte complexes inhibited apoptosis of cumulus cells and induced cumulus cell proliferation and oocyte maturation. Cortisol-induced CYP11B1 and CYP21A2 expression, whereas FSH-induced HSD11B2 mRNA expression in VF granulosa cells in the presence of cortisol. Furthermore, an addition of 18ß-glycyrrhetinic acid (18-GA; a HSD17B2 inhibitor) to cortisol and FSH-containing medium increased apoptosis of VF granulosa cells. These results suggested that cortisol is a stimulatory factor that induces follicular atresia; furthermore, inhibition of cortisol production by FSH might increase the number of healthy preovulatory follicles in pigs.

Gut microbial metabolism of dietary fibre protects against high energy feeding induced ovarian follicular atresia in a pig model.

To investigate the effects of dietary fibre on follicular atresia in pigs fed a high-fat diet, we fed thirty-two prepubescent gilts a basal diet (CON) or a CON diet supplemented with 300 g/d dietary fibre (fibre), 240 g/d soya oil (SO) or both (fibre + SO). At the 19th day of the 4th oestrus cycle, gilts fed the SO diet showed 112 % more atretic follicles and greater expression of the apoptotic markers, Bax and caspase-3, and these effects were reversed by the fibre diet. The abundance of SCFA-producing microbes was decreased by the SO diet, but this effect was reversed by fibre treatment. Concentrations of serotonin and melatonin in the serum and follicular fluid were increased by the fibre diet. Overall, dietary fibre protected against high fat feeding-induced follicular atresia at least partly via gut microbiota-related serotonin-melatonin synthesis. These results provide insight into preventing negative effects on fertility in humans consuming a high-energy diet.

Roles of vitamin D and its receptor in the proliferation and apoptosis of luteinised granulosa cells in the goat.

The objective of this study was to investigate the dose-dependent effect of 1α,25-(OH)2VD3 (Vit D3) on invitro proliferation of goat luteinised granulosa cells (LGCs) and to determine the underlying mechanisms of its action by overexpressing and silencing vitamin D receptor (VDR) in LGCs. Results showed that VDR was prominently localised in GCs and theca cells (TCs) and its expression increased with follicle diameter, but was lower in atretic follicles than in healthy follicles. The proliferation rate of LGCs was significantly higher in the Vit D3-treated groups than in the control group, with the highest proliferation rate observed in the 10nM group; this was accompanied by changes in the expression of cell cycle-related genes. These data indicate that Vit D3 affects LGC proliferation in a dose-dependent manner. Contrary to the VDR knockdown effects, its overexpression upregulated and downregulated cell cycle- and apoptosis-related genes respectively; moreover, supplementation with 10nM of Vit D3 significantly enhanced these effects. These results suggest that changes in VDR expression patterns in LGCs may be associated with follicular development by regulation of cell proliferation and apoptosis. These findings will enhance the understanding of the roles of Vit D3 and VDR in goat ovarian follicular development.

Doxorubicin obliterates mouse ovarian reserve through both primordial follicle atresia and overactivation.

Ovarian toxicity and infertility are major side effects of cancer therapy in young female cancer patients. We and others have previously demonstrated that doxorubicin (DOX), one of the most widely used chemotherapeutic chemicals, has a dose-dependent toxicity on growing follicles. However, it is not fully understood if the primordial follicles are the direct or indirect target of DOX. Using both prepubertal and young adult female mouse models, we comprehensively investigated the effect of DOX on all developmental stages of follicles, determined the impact of DOX on primordial follicle survival, activation, and development, as well as compared the impact of age on DOX-induced ovarian toxicity. Twenty-one-day-old CD-1 female mice were intraperitoneally injected with PBS or clinically relevant dose of DOX at 10 mg/kg once. Results indicated that DOX primarily damaged granulosa cells in growing follicles and oocytes in primordial follicles and DOX-induced growing follicle apoptosis was associated with the primordial follicle overactivation. Using the 5-day-old female mice with a more uniform primordial follicle population, our data revealed that DOX also directly promoted primordial follicle death and the DNA damage-TAp63α-C-CASP3 pathway was involved in DOX-induced primordial follicle oocyte apoptosis. Compared to 21-day- and 8-week-old female mice that were treated with the same dose of DOX, the 5-day-old mice had the most severe primordial follicle loss as well as the least degree of primordial follicle overactivation. Taken together, these results demonstrate that DOX obliterates mouse ovarian reserve through both primordial follicle atresia and overactivation and the DOX-induced ovarian toxicity is age dependent.

Detection of the effects and potential interactions of FSH, VEGFA, and 2-methoxyestradiol in follicular angiogenesis, growth, and atresia in mouse ovaries.

Ovarian follicular development is a complex process that requires codevelopment of the perifollicular vascular network, which is closely regulated by angiogenic factors, gonadotropins, sex steroids, and their metabolites. To detect the effects of vascular endothelial growth factor 120 (VEGF120), follicle-stimulating hormone (FSH), and 2-methoxyestradiol (2ME2) on follicular angiogenesis during development and atresia, we treated sexually immature and mature female mice with VEGF120, FSH, 2ME2, and FSH receptor (FSHR) antagonist singly or in combination via intraperitoneal injection. The number of follicles and their perifollicular angiogenesis and atresia rates at different developmental stages were examined in paraffin sections after hematoxylin and eosin staining. The results showed that the exogenous factors have specific and precise effects on developmental, angiogenesis, and atresia processes in follicles of different sizes in mature and immature mice. Perifollicular angiogenesis was regulated by VEGFA and closely related to follicular development and atresia. 2ME2 affected angiogenesis through VEGFA and might regulate atresia directly. FSH might control VEGFA function via both transcriptional and posttranscriptional mechanisms because FSHR was required for achieving VEGFA functions at all the follicular development stages. The present study presents insights into the mechanisms of FSH, 2ME2, and VEGFA in follicular development and disorders and provides a foundation for the development of new therapeutic strategies.

Examination of the ovotoxicity of 5-fluorouracil in mice.

PURPOSE: Undesirable side effects of cancer treatments are common and include damage to the ovary, and depletion of the follicle reserve, which if severe enough, can lead to infertility and early menopause. Antimetabolite drugs, such as 5-fluorouracil (5-FU), are not considered to be detrimental to the ovary, but the ovotoxicity of 5-FU has not been evaluated in any detail. The purpose of this study was to evaluate the effects of 5-FU on follicle number. METHODS: In this study, adult female C57Bl6 mice (n = 4-6 animals/group) received a single dose of saline or 5-FU (150 mg/kg) and markers of ovarian damage and follicle depletion were assessed 12 h and 7 days later. RESULTS: Exposure to 5-FU did not alter primordial and primary follicle numbers. Atresia of secondary and antral follicles was increased significantly 12 h after 5-FU treatment, but atresia rates returned to levels similar to that of saline treated controls at 7 days. The number of corpora lutea were reduced 7 days after exposure to 5-FU, possibly as a consequence of earlier follicular atresia. CONCLUSIONS: These findings suggest that a single dose of 5-FU is mildly ovotoxic, but any effects on ovarian function are likely transient because the primordial follicle population is not depleted. Collectively, these data support the notion that 5-FU is unlikely to impact on the long-term fertility of women.

Low-dose metronomic delivery of cyclophosphamide is less detrimental to granulosa cell viability, ovarian function, and fertility than maximum tolerated dose delivery in the mouse.

Chemotherapy can cause early menopause or infertility in women and have a profound negative impact on the quality of life of young female cancer survivors. Various factors are known to influence the risk of chemotherapy-induced ovarian failure, including the drug dose and treatment duration; however, the scheduling of dose administration has not yet been evaluated as an independent risk factor. We hypothesized that low-dose metronomic (LDM) chemotherapy scheduling would be less detrimental to ovarian function than the traditional maximum tolerated dose (MTD) strategy. In vitro, MTD cyclophosphamide exposure resulted in decreased proliferation and increased granulosa cell apoptosis, while cells treated with LDM cyclophosphamide were not different from untreated controls. Treatments of MTD cyclophosphamide induced high levels of follicle atresia and enhanced follicle recruitment in mice. In contrast, LDM delivery of an equivalent dose of cyclophosphamide reduced growing follicle numbers, but was not associated with higher levels of follicle atresia or recruitment. MTD cyclophosphamide induced significant vascular disruption and DNA damage in vivo, while LDM chemotherapy with equal cumulative amounts of cyclophosphamide was not different from controls. MTD chemotherapy also had a negative effect on mouse-fertility outcomes. Our findings suggest that LDM scheduling could potentially minimize the long-term effects of cyclophosphamide on female fertility by preventing follicle depletion from enhanced activation.

Nanosilver particles increase follicular atresia: Correlation with oxidative stress and aromatization.

Present study was performed in order to update the possible mechanism(s), involving in nanosilver particles (NSPs)-induced detrimental impacts in ovarian tissue. For this purpose, 24 mature female rats were divided into control and 0.5, 1, 5 mg/kg NSPs-received groups (intraperitoneally, for 35 days). Follicular growth and atresia, ovarian total antioxidant capacity (TAC), malondialdehyde (MDA), superoxide dismutase (SOD) contents, serum estrogen (E2 ) level and macrophages infiltration were investigated. Moreover, ovarian angiogenesis, cellular mRNA damage and cytochrome aromatase CYP19 expression were analyzed. The NSPs enhanced follicular atresia diminished E2, reduced TAC and SOD level, elevated MDA content and up-regulated macrophages infiltration. Cellular mRNA damage, impaired angiogenesis and diminished CYP19 expression were revealed in NSPs-received groups. Therefore NSPs by down-regulating aromatization, reduce E2 synthesis which then it leads to impaired angiogenesis. The impaired angiogenesis in turn down-regulates ovarian antioxidant status, which partially enhances follicular atresia by triggering lipid peroxidation and mRNA damage.

Resveratrol protects the ovary against chromium-toxicity by enhancing endogenous antioxidant enzymes and inhibiting metabolic clearance of estradiol.

Resveratrol (RVT), a polyphenolic component in grapes and red wine, has been known for its cytoprotective actions against several diseases. However, beneficial effects of RVT against early exposure to endocrine disrupting chemicals (EDCs) have not been understood. EDCs are linked to several ovarian diseases such as premature ovarian failure, polycystic ovary syndrome, early menopause and infertility in women. Hexavalent chromium (CrVI) is a heavy metal EDC, and widely used in >50 industries. Environmental contamination with CrVI in the US is rapidly increasing, predisposing the human to several illnesses including cancers and still birth. Our lab has been involved in determining the molecular mechanism of CrVI-induced female infertility and intervention strategies to mitigate CrVI effects. Lactating mother rats were exposed to CrVI (50ppm potassium dichromate) from postpartum days 1-21 through drinking water with or without RVT (10mg/kg body wt., through oral gavage daily). During this time, F1 females received respective treatments through mother's milk. On postnatal day (PND) 25, blood and the ovary, kidney and liver were collected from the F1 females for analyses. CrVI increased atresia of follicles by increasing cytochrome C and cleaved caspase-3; decreasing antiapoptotic proteins; decreasing estradiol (E2) biosynthesis and enhancing metabolic clearance of E2, increasing oxidative stress and decreasing endogenous antioxidants. RVT mitigated the effects of CrVI by upregulating cell survival proteins and AOXs; and restored E2 levels by inhibiting hydroxylation, glucuronidation and sulphation of E2. This is the first study to report the protective effects of RVT against any toxicant in the ovary.

Identifying a novel role for X-prolyl aminopeptidase (Xpnpep) 2 in CrVI-induced adverse effects on germ cell nest breakdown and follicle development in rats.

Environmental exposure to endocrine-disrupting chemicals (EDCs) is one cause of premature ovarian failure (POF). Hexavalent chromium (CrVI) is a heavy metal EDC widely used in more than 50 industries, including chrome plating, welding, wood processing, and tanneries. Recent data from U.S. Environmental Protection Agency indicate increased levels of Cr in drinking water from several American cities, which potentially predispose residents to various health problems. Recently, we demonstrated that gestational exposure to CrVI caused POF in F1 offspring. The current study was performed to identify the molecular mechanism behind CrVI-induced POF. Pregnant rats were treated with 25 ppm of potassium dichromate from Gestational Day (GD) 9.5 to GD 14.5 through drinking water, and the fetuses were exposed to CrVI through transplacental transfer. Ovaries were removed from the fetuses or pups on Embryonic Day (ED) 15.5, ED 17.5, Postnatal Day (PND) 1, PND 4, or PND 25, and various analyses were performed. Results showed that gestational exposure to CrVI: 1) increased germ cell/oocyte apoptosis and advanced germ cell nest (GCN) breakdown; 2) increased X-prolyl aminopeptidase (Xpnpep) 2, a POF marker in humans, during GCN breakdown; 3) decreased Xpnpep2 during postnatal follicle development; and 4) increased colocalization of Xpnpep2 with Col3 and Col4. We also found that Xpnpep2 inversely regulated the expression of Col1, Col3, and Col4 in all the developmental stages studied. Thus, CrVI advanced GCN breakdown and increased follicle atresia in F1 female progeny by targeting Xpnpep2.

Involvement of hyaluronan synthesis in ovarian follicle growth in rats.

Most of the previous studies on ovarian hyaluronan (HA) have focused on mature antral follicles or corpora lutea, but scarcely on small preantral follicles. Moreover, the origin of follicular HA is unknown. To clarify the localization of HA and its synthases in small growing follicles, involvement of HA in follicle growth, and gonadotropin regulation of HA synthase (Has) gene expression, in this study, perinatal, immature, and adult ovaries of Wistar-Imamichi rats were examined histologically and biochemically and by in vitro follicle culture. HA was detected in the extracellular matrix of granulosa and theca cell layers of primary follicles and more advanced follicles. Ovarian HA accumulation ontogenetically started in the sex cords of perinatal rats, and its primary site shifted to the intrafollicular region of primary follicles within 5 days of birth. The Has1-3 mRNAs were expressed in the ovaries of perinatal, prepubertal, and adult rats, and the expression levels of Has1 and Has2 genes were modulated during the estrous cycle in adult rats and following administration of exogenous gonadotropins in immature acyclic rats. The Has1 and Has2 mRNAs were predominantly localized in the theca and granulosa cell layers of growing follicles respectively. Treatments with chemicals known to reduce ovarian HA synthesis induced follicular atresia. More directly, the addition of Streptomyces hyaluronidase, which specifically degrades HA, induced the arrest of follicle growth in an in vitro culture system. These results indicate that gonadotropin-regulated HA synthesis is involved in normal follicle growth.

Reproductive biomarkers responses induced by xenoestrogens in the characid fish Astyanax fasciatus inhabiting a South American reservoir: an integrated field and laboratory approach.

Field studies evaluating the effects of endocrine disruption chemicals (EDCs) on the fish reproduction are scarce worldwide. The goal of this study was to assess hepatic levels of vitellogenin (Vtg), zona radiata proteins (Zrp) and insulin-like growth factors (IGF-I and IGF-II), and relating them to reproductive endpoints in a wild fish population habiting a reservoir that receive domestic sewage, agricultural and industrial residues. Adult fish Astyanax fasciatus were sampled during the reproductive season in five sites from the Furnas Reservoir, Grande River, and Paraguay-Paraná basin. As a control to field data, fish were experimentally exposed via dietary intake, to oestradiol benzoate (OB) for 7 days. Fish from site with little anthropogenic interference showed hepatic levels of Vtg, Zrp and IGF-I and IGF-II similar to those from the non-treated experimental group. In sites located immediately downstream from the municipal wastewater discharges, the water total oestrogen was >120 ng/l, and male fish displayed increased Vtg and Zrp and decreased IGF-I levels similar to OB treated fish. In females, levels of Vtg, Zrp, IGF-I and IGF-II suggest an impairment of final oocyte maturation and spawning, as also detected by frequency of over-ripening, follicular atresia and fecundity. At the sites that receive agricultural and industrial residues, the water total oestrogen was <50 ng/l and females showed decreased Zrp and increased IGF-II levels associated to reduced diameter of vitellogenic follicles, indicating an inhibition of oocyte growth. Overall, the current study reports oestrogenic contamination impairing the reproduction of a wild fish from a hydroeletric reservoir and, the data contribute to improving the current knowledge on relationship between hepatic Vtg, Zrp and IGF-I and IGF-II, and reproductive endpoints in a teleost fish. In addition, our data point out novel reproductive biomarkers (IGF-I, IGF-II and over-ripening) to assessing xenoestrogenic contamination in freshwater ecosystems.

Copper mediated follicular atresia: Implications for granulosa cell death.

3-nitropropanoic acid is a potent oxidative stress inducer that is conventionally regarded as a regulator of follicular atresia by regulating granulosa cells (GCs) death through the apoptosis pathway. There has been no research investigating the impact of copper metal overload induced Cuproptosis in ovarian GCs as a factor contributing to hindered follicular development.To elucidate whether 3-NP-induced oxidative stress plays a contributory role in promoting Cuproptosis, and discuss the role of Cuproptosis in the development of ovarian follicles.We conducted an analysis of cuproptosis occurrence in murine GCs and C57BL/6 J mice under the influence of 3-NP and 3-NP with added exogenous copper.The results revealed that 3-NP serving as a robust facilitator of exogenous copper uptake by upregulating the expression of copper transporter 1 (CTR1). In turn, culminated in the accumulation of intracellular copper within mouse granulosa cells (mGCs). Furthermore, 3-NP promoted mitochondrial permeability transition pore opening and concurrently reduced the stability of lipoic acid proteins. These actions collectively induced the oligomerization of Dihydrolipoamide S-Acetyltransferase (DLAT), ultimately leading to cuproptosis in GCs and consequent follicular atresia. Heavy metal copper and fungal decomposition product 3-NP, induce ovarian atresia via cuproptosis, modulating the reproductive performance of female animals.

Mouse strain does not influence the overall effects of bisphenol a-induced toxicity in adult antral follicles.

Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) widely used in common consumer products containing polycarbonate plastics and epoxy resins. Previous studies indicate that other EDCs have species-dependent effects. Furthermore, some EDCs are known to have different effects in different strains within the same species. Little information, however, is known about whether the effects of BPA on the ovary differ by strain. Previous studies have shown that BPA inhibits follicle growth, induces atresia, and inhibits steroidogenesis and expression of steroidogenic enzymes in antral follicles from adult FVB mice. Thus, this study was designed to expand previous work by testing the hypothesis that mouse strain may differentially affect the susceptibility of adult antral follicles to BPA-induced toxicity. To test this hypothesis, antral follicles were mechanically isolated from adult FVB, CD-1, and C57BL/6 mice, individually cultured for 6-120 h and treated with either vehicle control (dimethylsulfoxide) or various concentrations of BPA (1.0 µg/ml, 10 µg/ml, or 100 µg/ml). After culture, media were subjected to measurements of hormone production via ELISA, and follicles were subjected to real-time PCR for analysis of genes known to regulate steroidogenesis, the cell cycle, and atresia. Overall, BPA inhibited follicle growth and steroidogenesis in all tested strains, but CD-1 follicles were slightly more sensitive to BPA at early time points than FVB and C57BL/6 follicles. These data suggest that CD-1, FVB, and C57BL/6 mice can all be used to investigate the effects of BPA on ovarian follicles.

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

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

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.

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 (P4) 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 P4, androstenedione (A), testosterone (T), estrone (E1), and 17ß-estradiol (E2) in media were determined, and follicles were processed for histological evaluation of atresia. Pregnenolone treatment alone stimulated production of all steroid hormones except E2. Mono-OH MXC-treated follicles had decreased sex steroids, but when given pregnenolone, produced levels of P4, A, T, and E1 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.

Lithium induces follicular atresia in rat ovary through a GSK-3ß/ß-catenin dependent mechanism.

Lithium chloride (LiCl) is a drug used to treat bipolar disorder, but has side effects in the female reproductive system. Although lithium is known to decrease folliculogenesis and induce follicular atresia in rodent ovaries, its cellular and molecular effects in the ovary have not yet been addressed. To investigate these effects, 23-day-old immature female rats were injected with 10 IU pregnant mare serum gonadotropin (PMSG), followed by injections of 250 mg/kg LiCl every 12 hr for four doses. Ovaries were removed 40 and 48 hr after PMSG administration and prepared for histology, immunohistochemistry, Western blotting, and DNA laddering analysis. Our results showed that in the ovaries of LiCl-treated rats, few antral but more atretic follicles were present compared to those of the control rats. The induction of atresia by LiCl was further confirmed by the presence of DNA fragmentation, accompanied by a reduced level of 17ß-estradiol in the serum. At the cellular level, lithium significantly decreased the number of proliferating cell nuclear antigen (PCNA)-positive cells and conversely increased the number of TUNEL-positive cells in the granulosa layer of the antral follicles. At the molecular level, lithium increased the level of phosphorylated glycogen synthase kinase-3ß, and unexpectedly decreased the expression of active (stabilized) ß-catenin. Altogether, our results indicate that lithium disrupts the balance between proliferation and apoptosis in granulosa cells, leading to follicular atresia possibly through the reduction in both the stabilized ß-catenin and 17ß-estradiol synthesis.

Rescue from dominant follicle atresia by follicle-stimulating hormone in mice.

We investigated the effects of follicle-stimulating hormone (FSH) on atresia of the dominant follicle and changes in relevant apoptosis genes in granulosa cells of dominant follicles regulated by FSH in vivo. Four-week-old mice were administered FSH by intraperitoneal injection to induce follicular maturation. Granulosa cells of dominant follicles were collected at 48, 72, and 96 h after the first FSH injection. Phosphate-buffered saline was injected as a control. The mRNA levels of relevant granulosa cell apoptosis genes were examined by real-time quantitative polymerase chain reaction, and apoptosis of granulosa cells in dominant ovarian follicles was determined by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Apoptosis in granulosa cells of dominant follicles was almost TUNEL-negative at 48, 72-66, 72, and 96-90 h after the first FSH injection, but granulosa cell apoptosis in dominant follicles was clearly detected at 96, 102, and 102-96 h by TUNEL. The BIM, caspase-3, and caspase-9 mRNA expression levels were significantly lower after FSH treatment at 72-66 and 96-90 h, compared with that at 72 and 96 h (P < 0.05). Caspase-8 and FasL mRNA expressions did not respond to FSH. FSH rescued granulosa cells from apoptosis when the relevant apoptosis genes were upregulated in early atretic follicles. FSH did not rescue granulosa cells from apoptosis if the DNA was cut into fragments by endonucleases. Thus, the rescue by FSH of granulosa cells from apoptosis and dominant follicle atresia may be accomplished by inhibition of apoptosis in mitochondria.

Inhibition of follicular development induced by chronic unpredictable stress is associated with growth and differentiation factor 9 and gonadotropin in mice.

Chronic psychosocial stress negatively affects ovarian function. Ovarian follicular development is regulated by both pituitary-derived gonadotropins and intraovarian regulatory factors. To date, the suppressive effects of chronic stress on the ovary have been observed to be manifested mainly as an inhibition of gonadotropin release. It is not clear whether there are any other intraovarian regulatory mechanisms involved in this process. Growth and differentiation factor 9 (GDF9) is an important, oocyte-specific paracrine regulator required for follicular development. In this study, the chronic unpredictable mild stress model was used to produce psychosocial stress in mice. The number of different developmental stages of follicles was counted on ovarian sections stained with hematoxylin and eosin. Real-time PCR and Western blotting were used to detect the mRNA and protein levels, respectively, of GDF9. The results show that chronic unpredictable stress inhibits follicular development, increases follicular atresia, and suppresses GDF9 expression. Exogenous gonadotropin treatment partly restores the repressed antral follicular development, but has no effect on the repressed secondary follicular development associated with chronic stress. Treatment with recombinant GDF9 restores secondary follicular development. Cotreatments with GDF9 and gonadotropins restore both secondary and antral follicular development in stressed mice. These findings demonstrate that inhibition of follicular development induced by chronic unpredictable stress is associated with GDF9 and gonadotropin.

Bisphenol A inhibits follicle growth and induces atresia in cultured mouse antral follicles independently of the genomic estrogenic pathway.

Bisphenol A (BPA) is an estrogenic chemical used to manufacture many commonly used plastic and epoxy resin-based products. BPA ubiquitously binds to estrogen receptors throughout the body, including estrogen receptor alpha (ESR1) in the ovary. Few studies have investigated the effects of BPA on ovarian antral follicles. Thus, we tested the hypothesis that BPA alters cell cycle regulators and induces atresia in antral follicles via the genomic estrogenic pathway, inhibiting follicle growth. To test this hypothesis, we isolated antral follicles from 32- to 35-day-old control and Esr1-overexpressing mice and cultured them with vehicle control (dimethylsulfoxide [DMSO]) or BPA (1-100 µg/ml). Additionally, antral follicles were isolated from 32- to 35-day-old FVB mice and cultured with DMSO, BPA (1-100 µg/ml), estradiol (10 nM), ICI 182,780 (ICI; 1 µM), BPA plus ICI, or BPA plus estradiol. Follicles were measured for growth every 24 h for 96-120 h and processed either for analysis of estrogen receptor, cell cycle, and/or atresia factor mRNA expression, or for histological evaluation of atresia. Results indicate that estradiol and ICI do not protect follicles from BPA-induced growth inhibition and that estradiol does not protect follicles from BPA-induced atresia. Furthermore, overexpressing Esr1 does not increase susceptibility of follicles to BPA-induced growth inhibition. Additionally, BPA up-regulates Cdk4, Ccne1, and Trp53 expression, whereas it down-regulates Ccnd2 expression. BPA also up-regulates Bax and Bcl2 expression while inducing atresia in antral follicles. These data indicate that BPA abnormally regulates cell cycle and atresia factors, and this may lead to atresia and inhibited follicle growth independently of the genomic estrogenic pathway.