Evidence for a role for anti-Mullerian hormone in the suppression of follicle activation in mouse ovaries and bovine ovarian cortex grafted beneath the chick chorioallantoic membrane

The first critical transition in follicular development, the activation of primordial follicles to leave the pool of resting follicles and begin growth, is poorly understood, but it appears that the balance between inhibitory and stimulatory factors is important in regulating the exodus of follicles from the resting pool. There is evidence that anti-Mullerian hormone (AMH; also known as MIS) inhibits follicle activation in mice, but whether it plays a similar role in non rodent species is not known. When pieces of bovine ovarian cortex, rich in primordial follicles, are cultured in serum-free medium, most follicles initiate growth, but when cortical pieces are grafted beneath the chorioallantoic membrane (CAM) of chick embryos, follicle activation does not occur. Since embryonic chick gonads of both sexes produce and secrete high levels of AMH, the hypothesis that the AMH in the chick circulation inhibits follicle activation was tested. In Experiment 1, whole newborn mouse ovaries were grafted beneath the CAM (placed "in ovo") or cultured in vitro for 8 days. In vitro (or after 8 days in vivo) follicles activated and proceeded to the primary or secondary stage, but activation was suppressed in ovo. This inhibition was reversed if ovaries were removed from beneath the CAM and cultured in vitro. In contrast, when ovaries from mice null mutant for the AMH type II receptor were CAM-grafted in Experiment 2, follicle activation occurred in a similar fashion to activation in vitro. This finding strongly implicates AMH as the inhibitor of follicle activation in ovo. Since chick embryonic gonads are the source of circulating AMH, chicks were gonadectomized in Experiment 3, prior to grafting of pieces of bovine ovarian cortex beneath their CAMs. Bovine primordial follicles activated in the gonadectomized chicks, similar to the results for mice lacking the AMH type II receptor. Taken together these experiments provide strong evidence that AMH is the inhibitor of mouse follicle activation present in the circulation of embryonic chicks and provide indirect, and hence more tentative, evidence for AMH as an inhibitor of bovine follicle activation.     

Transforming Growth Factor-β Signaling Participates in the Maintenance of the Primordial Follicle Pool in the Mouse Ovary

Physiologically, only a few primordial follicles are activated to enter the growing follicle pool each wave. Recent studies in knock-out mice show that early follicular activation depends on signaling from the tuberous sclerosis complex, the mammalian target of rapamycin complex 1 (mTORC1), phosphatase and tensin homolog deleted on chromosome 10, and phosphatidylinositol 3-kinase (PI3K) pathways. However, the manner in which these pathways are normally regulated, and whether or not TGF-β acts on them are poorly understood. So, this study aims to identify whether or not TGF-β acts on the process. Ovary organ culture experiments showed that the culture of 18.5 days post-coitus (dpc) ovaries with TGF-β1 reduced the total population of oocytes and activated follicles, accelerated oocyte growth was observed in ovaries treated with TGF-βR1 inhibitor 2-(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-yl-amine (SD208) compared with control ovaries, the down-regulation of TGF-βR1 gene expression also activated early primordial follicle oocyte growth. We further showed that there was dramatically more proliferation of granulosa cells in SD208-treated ovaries and less proliferation in TGF-β1-treated ovaries. Western blot and morphological analyses indicated that TGF-β signaling manipulated primordial follicle growth through tuberous sclerosis complex/mTORC1 signaling in oocytes, and the mTORC1-specific inhibitor rapamycin could partially reverse the stimulated effect of SD208 on the oocyte growth and decreased the numbers of growing follicles. In conclusion, our results suggest that TGF-β signaling plays an important physiological role in the maintenance of the dormant pool of primordial follicles, which functions through activation of p70 S6 kinase 1 (S6K1)/ribosomal protein S6 (rpS6) signaling in mouse ovaries.     

Tumor necrosis factor (TNF) receptor type 2 is an important mediator of TNF alpha function in the mouse ovary

It is believed that a finite pool of primordial follicles is established during embryonic and neonatal life. At birth, the mouse ovary consists of clusters of interconnected oocytes surrounded by pregranulosa cells. Shortly after birth these structures, termed germ cell cysts or nests (GCN), break down to facilitate primordial follicle formation. Tumor necrosis factor alpha (TNF) is a widely expressed protein with myriad functions. TNF is expressed in the ovary and may regulate GCN breakdown in rats. We investigated whether it participates in GCN breakdown and follicle formation in mice by using an in vitro ovary culture system as well as mutant animal models. We found that TNF and both receptors (TNFRSF1A and TNFRSF1B) are expressed in neonatal mouse ovaries and that TNF promotes oocyte death in neonatal ovaries in vitro. However, deletion of either receptor did not affect follicle endowment, suggesting that TNF does not regulate GCN breakdown in vivo. Tnfrsf1b deletion led to an apparent acceleration of follicular growth and a concomitant expansion of the primordial follicle population. This expansion of the primordial follicle population does not appear to be due to decreased primordial follicle atresia, although this cannot be ruled out completely. This study demonstrates that mouse oocytes express both TNF receptors and are sensitive to TNF-induced death. Additionally, TNFRSF1B is demonstrated to be an important mediator of TNF function in the mouse ovary and an important regulator of folliculogenesis.     

The chemokine SDF1/CXCL12 and its receptor CXCR4 regulate mouse germ cell migration and survival

In mouse embryos, germ cells arise during gastrulation and migrate to the early gonad. First, they emerge from the primitive streak into the region of the endoderm that forms the hindgut. Later in development, a second phase of migration takes place in which they migrate out of the gut to the genital ridges. There, they co-assemble with somatic cells to form the gonad. In vitro studies in the mouse, and genetic studies in other organisms, suggest that at least part of this process is in response to secreted signals from other tissues. Recent genetic evidence in zebrafish has shown that the interaction between stromal cell-derived factor 1 (SDF1) and its G-protein-coupled receptor CXCR4, already known to control many types of normal and pathological cell migrations, is also required for the normal migration of primordial germ cells. We show that in the mouse, germ cell migration and survival requires the SDF1/CXCR4 interaction. First, migrating germ cells express CXCR4, whilst the body wall mesenchyme and genital ridges express the ligand SDF1. Second, the addition of exogenous SDF1 to living embryo cultures causes aberrant germ cell migration from the gut. Third, germ cells in embryos carrying targeted mutations in CXCR4 do not colonize the gonad normally. However, at earlier stages in the hindgut, germ cells are unaffected in CXCR4(-/-) embryos. Germ cell counts at different stages suggest that SDF1/CXCR4 interaction also mediates germ cell survival. These results show that the SDF1/CXCR4 interaction is specifically required for the colonization of the gonads by primordial germ cells, but not for earlier stages in germ cell migration. This demonstrates a high degree of evolutionary conservation of part of the mechanism, but also an area of evolutionary divergence.     

Effect of bcl-2 on the primordial follicle endowment in the mouse ovary

Little is known about the embryonic factors that regulate the size of the primordial follicle endowment at birth. A few studies suggest that members of the B-cell lymphoma/leukemia-2 (bcl-2) family of protooncogenes may be important determinants. Thus, the purpose of this study was to test whether bcl-2 regulates the size of the primordial follicle pool at birth. To test this hypothesis, three lines of transgenic mice (c-kit/bcl-2 mice) were generated that overexpress human bcl-2 in an effort to reduce prenatal oocyte loss. The overexpression was targeted to the ovary and appropriate embryonic time period with the use of a 4.8-kilobase c-kit promoter. This promoter provided two to three times more expression of bcl-2 in the ovaries with minimal or no overexpression in most nongonadal tissues. On Postnatal Days 8-60, ovaries were collected from homozygous c-kit/bcl-2 and nontransgenic littermates (controls) and processed for histological evaluation of follicle numbers. All lines of c-kit/bcl-2 mice were born with significantly more primordial follicles than control mice (P < or = 0.05). By Postnatal Days 30-60, however, there were no significant differences in follicle numbers between c-kit/bcl-2 and control mice. These results indicate that bcl-2 overexpression increases the number of primordial follicles at birth, but that the surfeit of primordial follicles is not maintained in postnatal life. These data suggest that it is possible that the ovary may contain a census mechanism by which excess numbers of primordial follicles at birth are detected and removed from the ovary by adulthood.     

Low expression of SEMA6C accelerates the primordial follicle activation in the neonatal mouse ovary

The primordial follicle assembly, activation and the subsequent development are critical processes for female reproduction. A limited number of primordial follicles are activated to enter the growing follicle pool each wave, and the primordial follicle pool progressively diminishes over a woman's life‐time. The number of remaining primordial follicles represents the ovarian reserve. Identification and functional investigation of the factors involved in follicular initial recruitment will be of great significance to the understanding of the female reproduction process and ovarian ageing. In this study, we aimed to study whether and how semaphorin 6C (Sema6c) regulated the primordial follicle activation in the neonatal mouse ovary. The attenuation of SEMA6C expression by SiRNA accelerated the primordial follicle activation in the in vitro ovary culture system. PI3K‐AKT‐rpS6 pathway was activated when SEMA6C expression was down‐regulated. And the LY294002 could reverse the effect of low SEMA6C expression on primordial follicle activation. Our findings revealed that Sema6c was involved in the activation of primordial follicles, and the down‐regulation of SEMA6C led to massive primordial follicle activation by interacting with the PI3K‐AKT‐rpS6 pathway, which might also provide valuable information for understanding premature ovarian failure and ovarian ageing.     

In vivo evidence of role of bone morphogenetic protein-4 in the mouse ovary

The transition of a primordial follicle to a primary follicle is an early step in folliculogenesis. All female mammals are born with a fixed stock of primordial follicles, and exhaustion of that stock leads to menopause or infertility. Recently, several in vitro studies have indicated that BMP-4, BMP-7, and several other growth factors affect the transition of primordial to primary follicles. The aim of our present study was to investigate role of BMP-4 in this process using passive immunization to investigate the role of BMP-4 in a prepubertal mouse model. After seven days of treatment, the weight of antiBMP-4 treated ovaries was significantly lower than the ovaries from mice treated with nonimmune Ig. The number of primary follicles was lower, and the numbers of primordial follicles were higher in antiBMP-4 treated ovaries compared to control ovaries. Treatment with equine chorionic gonadotrophin (eCG) showed no influence on the effects of antiBMP-4 in the mouse ovary. Thus, the results of our study indicate that in vivo BMP-4 acts as transition factor in transition of primordial to primary follicle.     

PAR6, A Potential Marker for the Germ Cells Selected to Form Primordial Follicles in Mouse Ovary

Partitioning-defective proteins (PAR) are detected to express mainly in the cytoplast, and play an important role in cell polarity. However, we showed here that PAR6, one kind of PAR protein, was localized in the nuclei of mouse oocytes that formed primordial follicles during the perinatal period, suggesting a new role of PAR protein. It is the first time we found that, in mouse fetal ovaries, PAR6 appeared in somatic cell cytoplasm and fell weak when somatic cells invaded germ cell cysts at 17.5 days post coitus (dpc). Meanwhile, the expression of PAR6 was observed in cysts, and became strong in the nuclei of some germ cells at 19.5 dpc and all primordial follicular oocytes at 3 day post parturition (dpp), and then obviously declined when the primordial follicles entered the folliculogenic growth phase. During the primordial follicle pool foundation, the number of PAR6 positive germ cells remained steady and was consistent with that of formed follicles at 3 dpp. There were no TUNEL (apoptosis examination) positive germ cells stained with PAR6 at any time studied. The number of follicles significantly declined when 15.5 dpc ovaries were treated with the anti-PAR6 antibody and PAR6 RNA interference. Carbenoxolone (CBX, a known blocker of gap junctions) inhibited the expression of PAR6 in germ cells and the formation of follicles. Our results suggest that PAR6 could be used as a potential marker of germ cells for the primordial follicle formation, and the expression of PAR6 by a gap junction-dependent process may contribute to the formation of primordial follicles and the maintenance of oocytes at the diplotene stage.     

Fate of the initial follicle pool: empirical and mathematical evidence supporting its sufficiency for adult fertility

The importance of the initial follicle pool in fertility in female adult mammals has recently been debated. Utilizing a mathematical model of the dynamics of follicle progression (primordial to primary to secondary), we examined whether the initial follicle pool is sufficient for adult fertility through reproductive senescence in CD1 mice. Follicles in each stage were counted from postnatal day 6 through 12 months and data were fit to a series of first-order differential equations representing two mechanisms: an initial pool of primordial follicles as the only follicle source (fixed pool model), or an initial primordial follicle pool supplemented by germline stem cells (stem cell model). The fixed pool model fit the experimental data, accurately representing the maximum observed primary follicle number reached by 4-6 months of age. Although no germline stem cells could be identified by SSEA-1 immunostaining, the stem cell model was tested using a range of de novo primordial follicle production rates. The stem cell model failed to describe the observed decreases in follicles over time and did not parallel the accumulation and subsequent reduction in primary follicles during the early fertile lifespan of the mouse. Our results agree with established dogma that the initial endowment of ovarian follicles is not supplemented by an appreciable number of stem cells; rather, it is sufficient to ensure the fertility needs of the adult mouse.     

Proliferating cell nuclear antigen (PCNA) regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries

Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA), a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFα and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer granulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV-induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries.     

Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development

The growth and development of follicles within the ovary are highly dependent on autocrine and paracrine signaling involving growth factors from granulosa cells, theca cells, stromal interstitial cells, and the oocytes. The growth factor bone morphogenetic protein-4 (BMP-4) and its receptor (BMPR-IB) have been detected in ovaries, and a mutation in BMPR-IB has been associated with abnormal ovulation rate. The objective of the current study was to examine the role that BMP-4 plays in the early stages of primordial follicle development. Ovaries from 4-day-old rats were placed into a whole-ovary organ culture system for 2 wk to investigate the effect that treatment with exogenous BMP-4 has on early follicle development. BMP-4-treated ovaries had a significantly higher proportion of developing primary follicles and fewer arrested primordial follicles than did untreated controls. This indicates that BMP-4 promotes primordial follicle development and the primordial-to-primary follicle transition. Ovaries were also treated with neutralizing antibody against BMP-4 to determine effects of removing endogenously produced BMP-4. Interestingly, ovaries treated with BMP-4 antibody were markedly smaller than controls. This was associated with a progressive loss of oocytes and primordial follicles, a progressive increase in cellular apoptosis, and an accompanying loss of normal ovarian tissue morphology over time. Immunocytochemistry localized BMP-4 protein to isolated stromal cell populations, selected stromal cells (i.e., pretheca cells) associated with developing primordial follicles, and the basement membrane of follicles. Ovaries were treated with BMP-4 and RNA collected after organ culture to determine whether BMP-4 signaling affects expression of other growth factors. Kit ligand and basic fibroblast growth factor expression was unchanged, but TGFalpha expression was decreased in whole ovaries. Taken together, these data suggest that BMP-4 plays an important role in promoting the survival and development of primordial follicles in the neonatal ovary.     

Cryopreservation of the mouse ovary inhibits the onset of primordial follicle development

The cryopreservation of ovarian tissue has been reported to affect the development of preantral follicles. However, the effect of cryopreservation of ovarian tissue on the development of primordial follicles remains to be elucidated. This study was conducted to evaluate the effect of cryopreservation on the development of frozen-thawed mouse primordial follicles. One-day-old mouse ovaries were cryopreserved by either slow-freezing or a vitrification method. The development of primordial follicles was evaluated histologically and also with markers for follicle development such as: GDF-9, inhibin-alpha subunit and ZP3 in fresh and frozen-thawed ovaries cultured for five days. The proportion of apoptotic and necrotic areas was analyzed in fresh and frozen-thawed ovaries at one and five days after culture, in order to examine the viability of ovarian cells that influence primordial follicle development. The development rate of primordial follicles was significantly lower in slow-frozen and vitrified ovaries than the fresh controls after five days of in vitro culture (P<0.05). The mRNA expression for all developmental markers was slightly decreased in the frozen-thawed ovaries; this difference was not significant. The proportion of apoptosis was significantly increased in the slow-frozen and vitrified ovaries compared to the fresh ovaries at one day (P<0.05); however, there was no difference at five days after culture. The proportion of the area of necrosis was significantly higher in slow-frozen and vitrified ovaries compared to the fresh ovaries at one and five days after culture (P<0.05). Our preliminary data suggest that ovarian tissue cryopreservation using slow-freezing and vitrification methods inhibits development of primordial follicles. This may be caused by the death of ovarian cells through apoptosis and necrosis after cryopreservation.     

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime’s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.     

HDAC6 regulates primordial follicle activation through mTOR signaling pathway

Primordial follicle pool established perinatally is a non-renewable resource which determines the female fecundity in mammals. While the majority of primordial follicles in the primordial follicle pool maintain dormant state, only a few of them are activated into growing follicles in adults in each cycle. Excessive activation of the primordial follicles accelerates follicle pool consumption and leads to premature ovarian failure. Although previous studies including ours have emphasized the importance of keeping the balance between primordial follicle activation and dormancy via molecules within the primordial follicles, such as TGF-β, E-Cadherin, mTOR, and AKT through different mechanisms, the homeostasis regulatory mechanisms of primordial follicle activation remain unclear. Here, we reported that HDAC6 acts as a key negative regulator of mTOR in dormant primordial follicles. In the cytoplasm of both oocytes and granulosa cells of primordial follicles, HDAC6 expressed strong, however in those activated primordial follicles, its expression level is relatively weaker. Inhibition or knockdown of HDAC6 significantly promoted the activation of limited primordial follicles while the size of follicle pool was not affected profoundly in vitro. Importantly, the expression level of mTOR in the follicle and the activity of PI3K in the oocyte of the follicle were simultaneously up-regulated after inhibiting of HDAC6. The up-regulated mTOR leads to not only the growth and differentiation of primordial follicles granulosa cells (pfGCs) into granulosa cells (GCs), but the increased secretion of KITL in these somatic cells. As a result, inhibition of HDAC6 awaked the dormant primordial follicles of mice in vitro. In conclusion, HDAC6 may play an indispensable role in balancing the maintenance and activation of primordial follicles through mTOR signaling in mice. These findings shed new lights on uncovering the epigenetic factors involved physiology of sustaining female reproduction.Subject terms: TOR signalling, Cell proliferation, Endocrine reproductive disorders     

The capacity of primordial follicles in fetal bovine ovaries to initiate growth in vitro develops during mid-gestation and is associated with meiotic arrest of oocytes

In cattle and other species in which the pool of resting, primordial follicles is formed during fetal life, little is known about the regulation of the early stages of ovarian follicular development. We used histological morphometry and a combination of observations in vivo and experiments in vitro to study the timing and regulation of follicle formation and the acquisition of the capacity of primordial follicles to initiate growth in cattle. In vivo, primordial, primary, and secondary follicles were first observed around Days 90, 140, and 210 of gestation, respectively. The long interval between the first appearance of primordial and primary follicles suggests that primordial follicles are not capable of activating when they are first formed, or they are inhibited from activating. This hypothesis was confirmed by the finding that most primordial follicles in pieces of ovarian cortex obtained from fetal ovaries older than 140 days activated (i.e., initiated growth) after 2 days in vitro, whereas follicles in cortical pieces from 90- to 140-day-old fetal ovaries did not. We tested the hypothesis that the oocytes of newly formed primordial follicles are not in meiotic arrest and found that before Day 141, most oocytes ( approximately 73%) were in prediplotene stages of prophase I, whereas after Day 140, the majority of oocytes ( approximately 85%) had arrested at the diplotene stage. This observation was further confirmed by the finding that levels of mRNA for YBX2, a protein associated with meiotic arrest, were 2.3 times higher in ovarian cortical pieces isolated after versus before Day 141. Primordial follicles in cortical pieces from 90- to 140-day-old fetal ovaries did activate during a longer, 10-day culture, but activation could be inhibited by adding estradiol or progesterone, but not dihydrotestosterone (all at 10(-6) M). Fetal ovaries secreted estradiol in vitro, and secretion by ovaries from 83 to 140-day-old fetuses declined precipitously ( approximately 30-fold) with age, consistent with the hypothesis that estradiol inhibits activation of newly formed primordial follicles in vivo. In summary, the results show that newly formed primordial follicles do not activate in vivo or within 2 days in vitro and that capacity to activate is correlated with achievement of meiotic arrest by the oocyte and can be inhibited by estradiol, which fetal ovaries actively produce around the time of follicle formation.     

Expression of ErbB3-Binding Protein-1 (EBP1) during Primordial Follicle Formation: Role of Estradiol-17?

The formation of primordial follicles involves the interaction between the oocytes and surrounding somatic cells, which differentiate into granulosa cells. Estradiol-17ß (E) promotes primordial follicle formation in vivo and in vitro; however, the underlying mechanisms are poorly understood. The expression of an ERBB3-binding protein 1 (EBP1) is downregulated in 8-day old hamster ovaries concurrent with the increase in serum estradiol levels and the formation of primordial follicles. The objectives of the present study were to determine the spatio-temporal expression and putative E regulation of EBP1 in ovarian cells during perinatal development with respect to primordial follicle formation. Hamster EBP1 nucleic acid and amino acid sequences were more than 93% and 98% similar, respectively, to those of mouse and human, and contained nucleolar localization signal, RNA-binding domain and several phosphorylation sites. EBP1 protein was present in somatic cells and oocytes from E15, and declined in oocytes by P1 and in somatic cells by P5. Thereafter, EBP1 expression increased through P7 with a transient decline on P8 primarily in interstitial cells. EBP1 mRNA levels mirrored protein expression pattern. E treatment on P1 and P4 upregulated EBP1 expression by P8 whereas E treatment on P4 downregulated it by 72 h suggesting a compensatory upregulation due to E pretreatment. Treatment with an FSH-antiserum, which suppressed primordial follicle formation, prevented the decline in EBP1 levels, and the effect was reversed by E treatment. Therefore, the results provide the first evidence that EBP1 may play an important role in mediating the effect of E in the differentiation of somatic cells into granulosa cells during primordial follicle formation.     

Follistatin288 Regulates Germ Cell Cyst Breakdown and Primordial Follicle Assembly in the Mouse Ovary

In mammals, the primordial follicle pool represents the entire reproductive potential of a female. The transforming growth factor-β (TGF-β) family member activin (ACT) contributes to folliculogenesis, although the exact mechanism is not known. The role of FST288, the strongest ACT-neutralizing isoform of follistatin (FST), during cyst breakdown and primordial follicle formation in the fetal mice ovary was assessed using an in vitro culture system. FST was continuously expressed in the oocytes as well as the cuboidal granulosa cells of growing follicles in perinatal mouse ovaries. Treatment with FST288 delayed germ cell nest breakdown, particularly near the periphery of the ovary, and dramatically decreased the percentage of primordial follicles. In addition, there was a dramatic decrease in proliferation of granulosa cells and somatic cell expression of Notch signaling was impaired. In conclusion, FST288 impacts germ cell nest breakdown and primordial follicle assembly by inhibiting somatic cell proliferation.