Quantification of healthy follicles in the neonatal and adult mouse ovary: evidence for maintenance of primordial follicle supply

Proliferation and partial meiotic maturation of germ cells in fetal ovaries is believed to establish a finite, non-renewable pool of primordial follicles at birth. The supply of primordial follicles in postnatal life should be depleted during folliculogenesis, either undergoing atresia or surviving to ovulation. Recent studies of mouse ovaries propose that intra- and extraovarian germline stem cells replenish oocytes and form new primordial follicles. We quantified all healthy follicles in C57BL/6 mouse ovaries from day 1 to 200 using unbiased stereological methods, immunolabelling of oocyte meiosis (germ cell nuclear antigen (GCNA)) and ovarian cell proliferation (proliferating cell nuclear antigen (PCNA)) and electronmicroscopy. Day 1 ovaries contained 7924+/-1564 (s.e.m.) oocytes or primordial follicles, declining on day 7 to 1987+/-203, with 200-800 oocytes ejected from individual ovaries on that day and day 12. Discarded oocytes and those subjacent to the surface epithelium were GCNA-positive indicating their incomplete meiotic maturation. From day 7 to 100 mean numbers of primordial follicles per ovary were not significantly depleted but declined at 200 days to 254+/-71. Mean numbers of all healthy follicles per ovary were not significantly different from day 7 to 100 (range 2332+/-349-3007+/-322). Primordial follicle oocytes were PCNA-negative. Occasional unidentified cells were PCNA-positive with mitotic figures observed in the cortex of day 1 and 12 ovaries. Although we found no evidence for ovarian germline stem cells, our data support the hypothesis of postnatal follicle renewal in postnatal and adult ovaries of C57BL/6 mice.     

Platelet-derived growth factor modulates the primordial to primary follicle transition

Primordial follicles steadily leave the arrested pool and undergo a primordial to primary follicle transition during the female reproductive lifespan. When the available pool of primordial follicles is depleted reproduction ceases and humans enter menopause. The present study was designed to investigate the actions of several growth factors previously identified as candidate regulatory factors for the primordial to primary follicle transition with a microarray analysis. Ovaries from 4-day-old rats were placed into culture and treated for 2 weeks with platelet-derived growth factor (PDGF), anti-PDGF neutralizing antibody, vascular endothelial growth factor (VEGF), neuregulin (NRG), or kit ligand (KITL) as a positive control. PDGF-treatment resulted in a significant decrease in the percentage of primordial follicles and a concomitant increase in the percentage of developing primary follicles compared to controls. In contrast, ovaries treated with an anti-PDGF neutralizing antibody had a significant increase in the percentage of primordial follicles demonstrating an inhibition of endogenous follicle development. Ovaries incubated in the presence of VEGF or NRG had no change in follicle development. Observations indicate that PDGF, but not VEGF or NRG, promotes the primordial to primary follicle transition. Immunohistochemical localization indicated that the PDGF protein was present in the oocytes of both primordial and developing follicles. PDGF-treatment of cultured ovaries resulted in an increase in KITL mRNA expression. KITL has been previously shown to promote the primordial to primary follicle transition. KITL-treatment of ovaries had no effect on expression of Pdgf or any PDGF homologs or receptors. Therefore, PDGF appears to be produced by the oocyte and acts as one of several extracellular signaling factors that regulate the primordial to primary follicle transition. These observations provide insight into the cell-cell interactions involved in the regulation of primordial follicle development and can be used in the future development of therapies for some forms of infertility.     

The effects of FSH and activin A on follicle development in vitro

Numerous studies have reported on the roles of activins in gonadal regulation; however, little is known about their specific roles in early folliculogenesis. Ovarian follicular growth was investigated in 10-day cultures of day 4 postnatal whole ovaries treated with activin A (ActA; 50?ng/ml), with or without FSH (100?ng/ml) in vitro. We hypothesized that treatment with ActA±FSH would affect rates of growth and atresia in follicles. None of the treatments affected primordial follicle activation, and antral follicles were not observed after 10 days in culture. Primordial follicle numbers from all treatment groups were ～20% of those in day 4 fresh ovaries, indicating that activation had occurred. In the presence of ActA, preantral follicle numbers increased significantly (P<0.0001). ActA alone decreased the proportion of atretic follicles in the primary and preantral classes, whereas the combined treatment of ActA+FSH increased the proportion of atretic preantral oocytes. Real-time PCR analysis revealed that follistatin, FSH receptor, and activin βA and βB subunits were all expressed at significantly higher levels in the ActA-only treated group but not in the ActA+FSH group. Here, we report novel findings supporting the role of FSH in primordial follicle survival through an action on apoptosis and a stimulatory role of ActA in the primordial to primary and preantral stages of follicle development, suggesting an inhibitory action of activin on oocyte apoptosis.     

Stage-specific and differential expression of gap junctions in the mouse ovary: connexin-specific roles in follicular regulation

Gap junction communication plays an essential role in follicle growth. Immunocytochemistry and confocal microscopy were used to examine the expression of gap junction connexins of the alpha and beta subfamilies in follicles from primordial to preovulatory stages in the ovaries of prepubertal and adult mice. Connexin-specific antibodies detected alpha(1), alpha(4), alpha(6), beta(1), beta(2) and beta(4) connexins within follicles. In adult ovaries connexin immunolabelling was stronger in larger (more mature) follicles than it was in smaller follicles, with comparatively reduced labelling detected in prepubertal ovaries. In healthy follicles, labelling for alpha subfamily connexins was detected between granulosa cells, whereas labelling for beta subfamily connexins was found in the theca. Labelling for beta subfamily connexins and alpha(4) connexin (preantral stage) was detected on the oocyte surface membrane. In atretic follicles, labelling for beta(4) connexin appeared between the granulosa cells. These results demonstrate that alpha and beta connexin subfamilies are segregated to separate cellular compartments in the mouse follicle. The results are discussed in the light of possible roles for differential gap junctional communication in the regulation of folliculogenesis, oocyte maturation and atresia.     

BAX regulates follicular endowment in mice

It is believed that the endowment of primordial follicles in mammalian ovaries is finite. Once follicles are depleted, infertility ensues. Thus, the size of the initial endowment has consequences for fertility and reproductive longevity. Follicular endowment is comprised of various processes that culminate with the incorporation of meiosis-arrested oocytes into primordial follicles. Apoptosis is prominent during follicular endowment, and apoptosis regulatory genes are involved in its regulation. Conflicting data exist with regard to the role of the proapoptotic Bcl-2 associated X protein (BAX) in follicular endowment. Therefore, we investigated the role of BAX during follicular endowment in embryonic and neonatal ovaries. We found that BAX is involved in regulating follicular endowment in mice. Deletion of Bax yields increased oocyte numbers in embryonic ovaries and increased follicle numbers in neonatal ovaries when compared with wild-type ovaries. Increased follicular endowment in Bax -/- ovaries is not due to enhanced germ cell viability. Further, it is not due to an increased primordial germ cell (PGC) allotment, a delay in the onset of meiosis, or altered proliferative activity of oogonia. Instead, our data suggest that the regulatory activity of BAX in follicular endowment likely occurs during PGC migration, prior to PGC colonization of the gonad.     

In vitro development of mouse fetal germ cells into mature oocytes

Little is known about the mechanisms underlying primordial follicular formation and the acquisition of competence to resume meiosis by growing oocytes. It is therefore important to establish an in vitro experimental model that allows one to study such mechanisms. Mouse follicular development has been studied in vitro over the past several years; however, no evidence has been presented showing that mature oocytes can be obtained from mouse fetal germ cells prior to the formation of primordial follicles. In this study, a method has been established to obtain mature oocytes from the mouse fetal germ cells at 16.5 days postcoitum (dpc). From the initiation of primordial follicular formation to the growth of early secondary follicles, ovarian tissues from 16.5 dpc fetal mice were cultured in vitro for 14 days. Subsequently, 678 intact secondary follicles were isolated from 182 mouse fetal ovaries and cultured for 12 days. A total of 141 oocytes inside antral follicles were matured in vitro, and 102 oocytes underwent germinal vesicle breakdown. We found that 97 oocytes were fertilized and 15 embryos were able to form morula-blastocysts. We also analyzed various genomic imprinting markers and showed that the erasure of genomic imprinting markers in the parental generation was also imposed on the oocytes that developed from fetal germ cells. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with ovarian cells, and that oocytes within the growing follicles are able to mature normally in vitro.     

Influence of donor age on development of gonadal tissue from pouch young of the tammar wallaby, Macropus eugenii, after cryopreservation and xenografting into mice

Ovaries from a marsupial, the tammar wallaby (Macropus eugenii), were grafted into a eutherian recipient at known stages of development to ascertain whether normal development would occur. Xenografted ovaries from pouch young < 20 days old, before the onset of meiosis, retained few germ cells and developed tubule-like structures reminiscent of seminiferous cords. Ovaries from 50-day-old pouch young, which contain primordial follicles, developed into antral follicles and corpora lutea within the eutherian host, and produced hormones that stimulated the reproductive tract of the host. The timing of onset of antrum formation and the progress of follicle development were advanced relative to the timing of events in ovaries in situ. Frozen-thawed ovaries from 50-day-old donors developed into preantral follicles, but at a reduced rate and number. This finding shows that gonads of a marsupial species can develop as xenografts in a eutherian, forming large antral follicles. Accelerated follicular development in xenografts provides a potentially valuable model for studying the factors that control follicle development. Assisted reproduction of endangered marsupials may also be feasible using follicles from pouch young grown as xenografts in a eutherian host.     

Developmental expression and cellular distribution of Mullerian inhibiting substance in the primate ovary

Ovarian follicle formation during development and follicle maturation in adulthood are crucial determinants of female fertility and disruptions in these processes may result in subfertility or infertility. Among the several factors that are involved in ovarian physiology, Müllerian inhibiting substance (MIS) also known as anti-Müllerian hormone has emerged as an important marker to predict the follicle reserve. However, the roles of MIS in human ovarian physiology are unknown. To gain an insight into the potential roles of MIS in human ovarian differentiation during development and its regulation in adulthood, the expression profiles of MIS mRNA in the developing and adult human and monkey ovaries was examined by in situ hybridization. The results revealed that in the fetal human ovaries, MIS is specifically expressed at low levels in the granulosa cells of the developing primordial follicles; a small subset (approximately 2-3%) of oocytes express high amounts of MIS. In the adult human and monkey ovary, MIS mRNA is expressed at low levels in the primordial follicles, maximally in the primary and secondary follicles, and the expression is downregulated in the antral and atetric follicles. MIS expression is extinguished in the granulosa cells only after ovulation. These observations strongly favor the regulatory roles of MIS in folliculogenesis. MIS in the primate ovary may exert its effect during the primordial follicle formation to the terminal granulosa cell differentiation. The presence of MIS in a small subset of oocytes in the fetal ovary further points towards its additional role during fetal oocyte development.     

Mammalian foetal ovarian development: consequences for health and disease

The development of a normal ovary during foetal life is essential for the production and ovulation of a high-quality oocyte in adult life. Early in embryogenesis, the primordial germ cells (PGCs) migrate to and colonise the genital ridges. Once the PGCs reach the bipotential gonad, the absence of the sex-determining region on the Y chromosome (SRY) gene and the presence of female-specific genes ensure that the indifferent gonad takes the female pathway and an ovary forms. PGCs enter into meiosis, transform into oogonia and ultimately give rise to oocytes that are later surrounded by granulosa cells to form primordial follicles. Various genes and signals are implicated in germ and somatic cell development, leading to successful follicle formation and normal ovarian development. This review focuses on the differentiation events, cellular processes and molecular mechanisms essential for foetal ovarian development in the mice and humans. A better understanding of these early cellular and morphological events will facilitate further study into the regulation of oocyte development, manifestation of ovarian disease and basis of female infertility.     

Oocyte growth and follicular development in KIT-deficient Fas-knockout mice

In mammals, oocyte growth and follicular development are known to be regulated by KIT, a tyrosine kinase receptor. Fas is a member of the death receptor family inducing apoptosis. Here, we investigated germ cell survival, oocyte growth and follicular development in KIT-deficient (Wv/Wv:Fas+/+), Fas-deficient (+/+:Fas-/-), and both KIT- and Fas-deficient (Wv/Wv:Fas-/-) mice during fetal and postnatal periods. Further, the ovaries of these mice were transplanted in immunodeficient mice to compare oocyte growth and follicular development under a condition isolated from the extraovarian effects of KIT- and Fas-deficiency. Higher numbers of germ cells were found in the fetal and postnatal ovaries of Fas-deficient mice than in the same-aged wild-type mice. In KIT-deficient mice, ovaries at 13 days postcoitum (dpc) contained 1106+/-72 (n=3) germ cells, but the ovaries contained no oocytes after birth. Twenty-one days after transplantation of the ovaries at 13 dpc, no oocytes/germ cells were found. A higher number of germ cells (3843+/-108; n=3) were observed in the Wv/Wv:Fas-/- genotypes than in Wv/Wv:Fas+/+ mice at 13 dpc. Furthermore, Wv/Wv:Fas-/- mice contained 528+/-91 (n=3) oocytes at 2 days, and follicles developed to the antral stage at 14 days of age. After transplantation of fetal and neonatal ovaries from Wv/Wv:Fas-/- mice, increased numbers of growing oocytes and developing follicles were obtained compared with those in 14-day old ovaries in vivo. These results show that oocytes grow and follicles develop without KIT signaling, although KIT might be essential for the survival of germ cells/oocytes in mice.     

Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents

Folliculogenesis is a complex process regulated by various paracrine and autocrine factors. In vitro growth systems of primordial and preantral follicles have been developed for future use of immature oocytes, as sources of fertilizable oocytes and for studying follicular growth and oocyte maturation mechanisms. Rodents were often chosen for in vitro follicular culture research and a lot of factors implicated in folliculogenesis have been identified using this model. To date, the mouse is the only species in which the whole process of follicular growth, oocyte maturation, fertilization and embryo transfer into recipient females was successfully performed. However, the efficiency of in vitro culture systems must still be considerably improved. Within the follicle, numerous events affect cell proliferation and the acquisition of oocyte developmental competency in vitro, including interactions between the follicular cells and the oocyte, and the composition of the culture medium. Effects of the acting factors depend on the stage of follicle development, the culture system used and the species. This paper reviews the action of endocrine, paracrine factors and other components of culture medium on in vitro growth of preantral follicles in rodents.     

BAX is involved in regulating follicular growth, but is dispensable for follicle atresia in adult mouse ovaries

Mammalian females are endowed with a finite number of primordial follicles at birth or shortly thereafter. Immediately following the formation of the primordial follicle pool, cohorts of these follicles are recruited to begin growth, and this recruitment continues until the primordial follicle population is depleted. Once recruited, a follicle will either grow and ovulate or undergo atresia. Follicle atresia results from the apoptotic death of follicular cells. Members of the BCL-2 family of proteins are important regulators of apoptosis in most cells including in the ovary. Here, we tested the hypothesis that the proapoptotic BAX is an important regulator of follicle survival. We used a variety of histological and biochemical techniques to investigate the impact of Bax deletion on follicle growth and death. We observed that the Bax deletion results in delayed vaginal opening and altered follicular growth. Young adult Bax-deficient ovaries contained increased numbers of primordial follicles and a trend towards reduced numbers of growing follicles. Bax deficiency led to a reduction in average litter size, and also a reduction in the number of oocytes ovulated in response to exogenous gonadotropins. In contrast, Bax deficiency did not alter follicle atresia. In conclusion, BAX appears to be an important regulator of follicle growth, but is dispensable for follicle atresia in mice.     

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

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

Effects of IGF-I bioavailability on bovine preantral follicular development in vitro

The aim of this study was to determine the effect of regulation of IGF-I bioavailability on preantral follicle development in vitro. Bovine preantral follicles were cultured for 6 days in serum-free medium with increasing doses of Long R3 (LR3) IGF-I (an analog with low affinity for IGF-binding proteins (IGFBPs)), or human recombinant IGF-I (hrIGF-I). Follicle diameter and estradiol production were measured every second day. On day 6, ratios of oocyte/follicle diameter and oocyte morphology were assessed by histological examination, and IGFBP-2 and -3 were detected by immunocytochemistry and in situ hybridization respectively. Both types of IGF-I increased follicle diameter in a dose-dependent manner (P < 0.05) and increased estradiol production over control levels (P < 0.05). However, follicles treated with LR3 IGF-I and the highest concentration of hrIGF-I (1000 ng/ml) had smaller oocyte/follicle ratios, and increased oocyte degeneration, compared with controls or follicles treated with physiological concentrations of hrIGF-I (P < 0.05). IGFBPs were detected in cultured preantral follicles, indicating a requirement for regulation of IGF bioavailability during the early stages of follicular development. Specifically, IGFBP-3 mRNA was found to be expressed in oocytes, and IGFBP-2 immunoreactivity was detected in oocytes and granulosa cells of cultured follicles. In summary, the regulation of IGF-I bioavailability by IGFBPs is necessary for the co-ordination of oocyte and follicle development in vitro.