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.     

TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.     

Recent achievements in in vitro culture and preservation of ovarian follicles in mammals

The mammalian ovary contains a large number of follicles that are in various developmental stages. The largest portion of them are primordial follicles. However, throughout the female reproductive lifespan only a small proportion of these follicles will produce oocytes competent to undergo successful maturation and ovulation. The rest of the ovarian oocytes (>99.9%) undergo atresia. It would be of great practical benefit to rescue some of these follicles by growing them in culture in order to provide an extra source of gametes. There is considerable interest in developing technologies that aim to produce fully-grown, developmentally competent oocytes from a pool of early developmental stages of follicles. Two methods have been used: 1/ long-term in vitro culture of either follicles or oocytes, and 2/ transplantation of ovarian tissue grafts. The development of efficient technologies may provide an additional source of oocytes for livestock production and reproduction in humans and rare or endangered species. The aim of this paper is to present a comprehensive review of recent achievements in the utilization of small ovarian follicles (primordial, preantral and early antral) by long-term in vitro culture and/or transplantation of ovarian tissue grafts (fresh and cryopreserved) in mammals including humans.     

Inactivation of Retinoblastoma Protein (Rb1) in the Oocyte: Evidence That Dysregulated Follicle Growth Drives Ovarian Teratoma Formation in Mice

The origin of most ovarian tumors is undefined. Here, we report development of a novel mouse model in which conditional inactivation of the tumor suppressor gene Rb1 in oocytes leads to the formation of ovarian teratomas (OTs). While parthenogenetically activated ooctyes are a known source of OT in some mutant mouse models, enhanced parthenogenetic propensity in vitro was not observed for Rb1-deficient oocytes. Further analyses revealed that follicle recruitment and growth is disrupted in ovaries of mice with conditional inactivation of Rb1, leading to abnormal accumulation of secondary/preantral follicles. These findings underpin the concept that miscues between the germ cell and somatic compartments cause premature oocyte activation and the formation of OTs. Furthermore, these results suggest that defects in folliculogenesis and a permissive genetic background are sufficient to drive OT development, even in the absence of enhanced parthenogenetic activation. Thus, we have discovered a novel role of Rb1 in regulating the entry of primordial oocytes into the pool of growing follicles and signaling between the oocyte and granulosa cells during the protracted process of oocyte growth. Our findings, coupled with data from studies of other OT models, suggest that defects in the coordinated regulation between growth of the oocyte and somatic components in follicles are an underlying cause of OT formation.     

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.     

Follicular expression of c-Kit/SCF and inhibin-alpha in mouse ovary during development.

The mechanism of development of the ovarian follicles has been largely unknown. We performed an immunohistochemical (IHC) study to determine the follicular expressions of c-kit, SCF, and inhibin-alpha at different developmental stages in mouse ovary. Ovaries were obtained from 14 and 16 days post coitum and 2, 7, and 21 days post partum (dpp) mice. IHC for c-kit, SCF, and inhibin-alpha was carried out. c-Kit and SCF were expressed on oogonia regardless of the developmental stage. Immunoreactive c-kit and SCF antigens were expressed on oocytes of primordial and primary follicles of neonate mouse ovaries. In 21 dpp mouse ovary, the expression of c-kit/SCF in oocytes gradually decreased as the follicles developed. c-Kit/SCF was expressed strongly in oocytes of preantral follicles and weakly in granulosa and thecal cells. Inhibin-alpha was mainly expressed on granulosa cells of preantral and early antral follicles of the 21 dpp mouse ovaries. These findings suggest that the IHC expression of c-kit/SCF proteins is specific in all developmental stages of ovarian follicles and is decreased after the follicle starts to grow. The expression of inhibin-alpha is negatively correlated with the expression of c-kit/SCF in the ovarian follicles in mice.     

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.     

Development of primordial and prenatal follicles from undifferentiated somatic cells and oocytes in the hamster prenatal ovary in vitro: effect of insulin

Fetal hamster ovaries were cultured for up to 16 days in the presence or absence of various dosages of insulin to evaluate the induction of folliculogenesis in vitro. In the absence of insulin, a few primordial follicle-like structures appeared by the 4th day, and distinct primary follicles (stage 1) appeared by the 12th day of culture. The organelles in the oocytes and adjacent granulosa cells developed along with follicular growth. Moreover, gap junctions between the oocyte and somatic cell plasma membrane also developed as early as 8 days in culture. In the presence of 0.2 microg/ml insulin, primary follicles developed after 8 days, and approximately 4% secondary follicles with 2-3 layers of granulosa cells appeared after 16 days of culture. However, higher dosages (> 0.2 microg/ml) of insulin retarded primary follicle formation and induced the formation of primordial follicles with larger oocytes. An increased number of larger oocytes with a few granulosa cells accumulated at the periphery of the ovary. The results indicate that although primordial and primary follicles can develop after 12 days in vitro in the absence of exogenous insulin, the latter is required for timely progression of follicular development through primary and secondary stages.     

Characterization of integrin expression in the mouse ovary

Integrin alpha:beta heterodimers mediate cell contacts to the extracellular matrix and initiate intracellular signaling cascades in response to a variety of factors. Integrins interact with many determinants of cellular phenotypes and play roles in controlling the development, structural integrity, and function of every type of tissue. Despite their importance, little is known about the regulation of integrin subunits in the mammalian ovary and how they function in folliculogenesis. To determine their relevance to ovarian physiology, we have studied the expression of integrin subunit mRNAs by Northern blot analysis and in situ hybridization in ovaries of wild-type, growth differentiation factor 9 (Gdf 9) knockout, FSHbeta (Fshb) knockout, and inhibin alpha (Inha) knockout mice. Integrin alpha6 mRNA is expressed in oocytes and granulosa cells of single-layer follicles and in oocytes and theca cells of multilayer follicles. Integrin alpha6 is highly expressed in Gdf 9 knockout ovaries, which are enriched in oocytes and primary (single layer) follicles because of a block at this stage of follicular development. Integrin alpha(v) mRNA is most highly expressed in the granulosa cells of multilayer growing follicles, and therefore only low levels of expression are detectable in the Gdf 9 knockout ovaries. Integrin beta1 mRNA exhibits a broad expression pattern in ovaries, including oocytes, granulosa cells, theca cells, and corpora lutea. Integrin beta3 mRNA is expressed in theca and interstitial cells and is upregulated in corpora lutea. It is nearly undetectable in ovaries of Fshb knockout mice, which develop preantral follicles but have no luteal cells. Integrin beta5 mRNA is predominantly expressed in granulosa cells of multilayer follicles. It is expressed at high levels in the Fshb knockout mice and in a compartmentalized manner in the granulosa cell/Sertoli cell tumors that develop in the Inha knockout mice. Specific integrins are associated with ovarian cellular phenotypes in mice, which raises intriguing possibilities as to integrin functions in oocyte competence, follicular development, luteinization, and granulosa cell proliferation.     

Localization of a carbohydrate antigen associated with growing oocytes and ovarian surface epithelium.

We used a monoclonal antibody (PS1) to a carbohydrate antigen to study the development of the oocyte and follicle during early stages of differentiation in several mammalian species. This antigen has been shown to localize within the cytoplasm of oocytes in primordial follicles as well as in growing oocytes. It is also localized within distinct layers of the zona pellucida (ZP) of developing follicles. Although this antibody was made against a specific ZP glycoprotein, the antigen also appears to be abundant in cells of the ovarian surface epithelium (OSE). The localization of this carbohydrate moiety has been observed in ovaries of rabbits of different ages as well as in the ovarian surface epithelium of other mammalian species including cat, cynomolgus monkey, baboon, and human. These studies demonstrate that there is an abundant carbohydrate antigenic determinant which is associated with both the mammalian oocyte and the ovarian surface epithelium but which is not apparent in other ovarian cell types or in non-ovarian secretory epithelium. This antibody probe should provide a valuable tool for studying the development and differentiation of the ovary, since this antigen is associated with two highly differentiated but distinct cell types.     

Unilaminar follicular cells transiently express galectin-3 during ovarian folliculogenesis in pigs

The localization of galectin-3, a β-galactoside-binding animal lectin, was immunohistochemically studied in the ovaries of pigs to determine its expression in ovarian folliculogenesis. Various stages of ovarian follicles were identified in the ovaries of adult pigs. Galectin-3 was immunostained in the squamous follicular cells surrounding oocytes in primordial follicles and in the unilaminar granulosa cells of primary follicles, but not in oocytes of multilaminar follicles (including primary, secondary, and tertiary Graafian follicles). As in adult ovaries, galectin-3 immunoreactivity was prominent in the unilaminar follicles in neonatal ovaries. Galectin-3 was also immunolocalized in the luteal cells in the corpus luteum and granulosa cells of atretic follicles as well as in interstitial macrophages in porcine ovaries. Collectively, these results suggest that galectin-3 is transiently expressed in follicular cells in the unilaminar ovarian follicles (primordial and primary) but not in multilaminar ovarian follicles (primary to tertiary), implying that galectin-3 is embryologically involved in ovum generation.     

Activation of bovine and baboon primordial follicles in vitro

Mammalian ovaries contain a large pool of non-growing, primordial follicles. The ability to initiate growth of this pool of resting follicles in vitro and to maintain follicular growth to a stage when the oocyte could be matured and fertilized would increase the reproductive potential of valuable domestic animals, endangered species and infertile women. This paper summarizes our progress to date in activating primordial follicles of cattle and baboons. Pieces of ovarian cortex, rich in primordial follicles, were obtained from fetal bovine and baboon ovaries during late gestation. Pieces were maintained in organ culture in serum-free medium containing ITS+ (insulin-transferrin-selenium-linoleic acid-BSA) for up to 20 days and at various times during culture some pieces were fixed for histological morphometry. As early as 2 days of culture, the number of primordial follicles had decreased by 88% or 55%, whereas the number of primary follicles had increased 2.5- or 5-fold, compared to tissue freshly isolated from bovine or baboon ovaries, respectively (P < 0.01). In baboon cortical pieces a small number of secondary follicles developed during a 20-day culture period. The development of primary and secondary follicles was accompanied by an increase in diameter of both the granulosa cell layer and the oocyte. The addition of FSH (1, 10, or 100 ng/ml) had no effect on the development of follicles in bovine cortical pieces after 7 or 14 days of culture, relative to control cultures without FSH. These results show that a high percentage of primordial follicles from cattle and baboons can be activated to grow in serum-free medium in the absence of gonadotropins. Conditions that will support further growth in vitro of follicles from these species remain to be elucidated. The culture system we have developed could be used to develop such conditions and to explore factors that regulate the movement of primordial follicles into the pool of growing follicles.     

Up-regulation of alpha-inhibin expression in the fetal ovary of estrogen-suppressed baboons is associated with impaired fetal ovarian folliculogenesis

We recently demonstrated that the number of primordial follicles was significantly reduced in the ovaries of near-term baboon fetuses deprived of estrogen in utero and restored to normal in animals administered estradiol. Although the baboon fetal ovary expressed estrogen receptors alpha and beta, the mechanism(s) of estrogen action remains to be determined. It is well established that inhibin and activins function as autocrine/paracrine factors that impact adult ovarian function. However, our understanding of the expression of these factors in the primate fetal ovary is incomplete. Therefore, we determined the expression of alpha-inhibin, activin beta(A), activin beta(B), and activin receptors in fetal ovaries obtained at mid and late gestation from untreated baboons and at late gestation from animals in which fetal estrogen levels were reduced by >95% by maternal administration of the aromatase inhibitor CGS 20267 or restored to 30% of normal by treatment with CGS 20267 and estradiol benzoate. Immunocytochemical expression of alpha-inhibin was minimal to nondetectable in fetal ovaries from untreated baboons. In contrast, in baboons depleted of estrogen, alpha-inhibin was abundantly expressed in pregranulosa cells of interfollicular nests and granulosa cells of primordial follicles. Thus, the number (mean +/- SEM) per 0.08 mm2 of fetal ovarian cells expressing alpha-inhibin, determined by image analysis, was similar at mid and late gestation and increased approximately 8-fold (P < 0.01) near term in baboons treated with CGS 20267 and was restored (P < 0.01) to normal in baboons treated with CGS 20267 plus estradiol. Activin beta(A) was detected in oocytes and pregranulosa cells at midgestation and in oocytes and granulosa cells of primordial follicles at late gestation. Activin beta(B) was also expressed in pregranulosa cells and granulosa cells at mid and late gestation, respectively, but was not detected in oocytes. Neither the pattern nor the apparent level of expression of activin beta(A) or beta(B) were altered in fetal ovaries of baboons treated with CGS 20267 or CGS 20267 and estrogen. Activin receptors IA, IB, IIA, and IIB were detected by Western blot analysis in fetal ovaries at mid and late gestation, and expression was not altered by treatment with CGS 20267 or CGS 20267 and estrogen. Activin receptors IB and IIA were localized to oocytes and pregranulosa cells at midgestation and to granulosa cells and oocytes of primordial follicles at late gestation. Thus, the decrease in the number of follicles in the primate fetal ovary of baboons deprived of estrogen in utero was associated with increased expression of alpha-inhibin. Therefore, we propose that estrogen regulates fetal ovarian follicular development by controlling alpha-inhibin expression and, thus, the intraovarian inhibin:activin ratio.     

Activation of follicle development: the primordial follicle

Investigations of primordial follicle formation and growth are fundamental to our understanding of female gamete production. In all mammalian females the full complement of oocytes is established during fetal development. This store of primordial follicles is not renewable and serves the entire reproductive life span of the adult. The correct programming of fetal ovarian development and the number of primordial follicles formed will therefore limit the fecundity of the ovary. Primordial follicles are characterized by the presence of a single oocyte surrounded by a varying number of pregranulosa cells. The relatively small size, undifferentiated status and large numbers of primordial follicles make them prime candidates for use in basic and applied research in animal production, gene transfer and cloning. Furthermore, the development of cell culture systems that use primordial follicles as a source of oocytes for in vitro growth and maturation will enable us to maximize the potential of high genetic merit females and to shorten generation intervals. Despite these possibilities, primordial follicles are the least understood of all stages of follicle development. The factor(s) responsible for maintaining the primordial pool or, conversely, for activating primordial follicle growth remain elusive.     

KIT/KIT ligand in mammalian oogenesis and folliculogenesis: roles in rabbit and murine ovarian follicle activation and oocyte growth

In rodent ovaries Kit ligand (KITL) and its receptor KIT have diverse roles, including the promotion of primordial follicle activation, oocyte growth, and follicle survival. Studies were undertaken to determine whether KITL and KIT carry out similar activities in rabbits.KitlandKitmRNA and protein were localized to oocytes and granulosa cells, respectively, in the rabbit ovary. Ovarian cortical explants from juvenile rabbits and neonatal mouse ovaries were subsequently cultured with recombinant mouse KITL and/or KITL neutralizing antibody. Indices of follicle growth initiation were compared with controls and between treatment groups for each species. Recombinant mouse KITL had no stimulatory effect on primordial follicle recruitment in cultured rabbit ovarian explants. However, the mean diameter of oocytes from primordial, early primary, primary, and growing primary follicles increased significantly in recombinant mouse KITL-treated explants compared with untreated tissues. In contrast, recombinant mouse KITL promoted both primordial follicle activation and an increase in the diameter of oocytes from primordial and early primary follicles in the mouse, and these effects were inhibited by coculture with KITL-neutralizing antibody. Recombinant mouse KITL had no effect on follicle survival for either species. These data demonstrate that KITL promotes the growth of rabbit and mouse oocytes and stimulates primordial follicle activation in the mouse but not in the rabbit. We propose that the physiologic roles of KITL and KIT may differ between species, and this has important implications for the design of in vitro culture systems for folliculogenesis in mammals, including the human.     

A quantitative cytological study of polyovular follicles in mammalian ovaries with particular reference to the domestic bitch (Canis familiaris)

The incidence of polyovular types in the growing follicle population was estimated using quantitative cytology. Of 15 species studied, polyovular follicles were recorded in the following species and in ascending order of abundance: rabbits, rhesus monkeys, humans, cats, dogs. The incidence in bitches was 14% in animals aged 1-2 years but only 5% at 7-11 years old. The frequency of the various types of polyovular preantral follicle varied inversely with the numbers of oocytes per follicle and the probability of finding a follicle with more than 5 oocytes was remote. In young ovaries the frequency was constant in the early stages of growth but decreased in the largest preantral stage. The pattern in ageing ovaries was, by contrast, one of declining frequency such that few if any polyovular types completed development. The ovary of the ageing bitch was also characterized by a higher incidence of degenerating follicles and a much smaller pool of primordial stages. Polyovular follicles were larger than uniovular types at comparable stages which were defined by the number of granulosa cell layers. Their oocytes were smaller but the overall ooplasmic mass was increased with a corresponding increase in the mass of granulosa cells.     

Consequences of fetal irradiation on follicle histogenesis and early follicle development in rat ovaries

Follicle histogenesis, in which follicles arise from fragmenting ovigerous cords, is a poorly understood mechanism that is strictly dependent upon the presence of germ cells. Our previous studies have shown that severely germ cell-depleted rat ovaries after fetal gamma-irradiation display modifications of follicular endowment and dynamics during the immature period. The primordial follicle stock was absent and the follicles with primary appearance remained quiescent longer than in control ovaries during the neonatal period. The aim of the present work was to analyze the initial steps of follicle histogenesis, and to investigate the etiology of the alterations observed in the development of irradiated ovaries. Just after birth, we observed, in addition to sterile ovigerous cords, the emergence of the first follicles which exhibited several abnormal features as compared to those of control ovaries. Most of the follicles appeared as primary follicles, as they were composed of a layer of cuboidal-shaped granulosa cells surrounding an enlarged oocyte. Interestingly, the granulosa cells of these primary-like follicles did not proliferate and did not express the genes for anti-Müllerian hormone (Amh) or bone morphogenetic protein receptor type II (Bmpr2), both of which are normally expressed from the primary stage onwards. In contrast, the oocytes strongly expressed the gene for growth and differentiation factor 9 (Gdf9), which is normally upregulated from the primary follicle stage onwards, which suggests an uncoupling of granulosa cell development from oocyte development. In addition, irradiated ovaries displayed a higher frequency of follicles that contained 2 or 3 oocytes, which are also referred to as multi-oocyte follicles (MOFs). Examination at the time of follicle histogenesis indicated that MOFs arise from incomplete ovigerous cord breakdown. Taken together, the results of this study indicate that severe perturbations of follicular histogenesis take place following irradiation and massive germ cell depletion during fetal life. In addition to the classically described sterile cords, we have pointed out the differentiation of MOFs and primary-like quiescent follicles, which finally evolve into growing follicles and participate in ovarian function. We propose that these phenotypes are closely correlated to the proportion of granulosa cells to oocytes at the time of neonatal follicle histogenesis.