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.     

Survival and normal function of glycolysis-deficient mouse oocytes

Mouse embryos homozygous for a null allele of Gpi1 which encodes the glycolytic enzyme glucose phosphate isomerase fail to complete gastrulation and die at about embryonic day 7.5, but mutant cells can survive in fetal chimaeras in which they are mixed with wild-type cells. An adult female mouse chimaera, composed of wild-type cells and homozygous Gpi1(-/-) null mutant cells, was produced to test whether the presence of wild-type cells in the ovary allowed mutant oocytes to survive and function. This mouse produced 28 offspring, eight of which were derived from homozygous Gpi1(-/-) null oocytes. DNA in situ hybridization also showed that some Gpi1(-/-) follicle cells were able to survive in chimaeric ovarian follicles. It is likely that the survival of mutant follicle cells and fully functional mutant oocytes was mediated by the presence of wild-type cells that could provide metabolic intermediates and so bypass the block in the glycolytic pathway. Wild-type cumulus cells probably supported the growing GPI-deficient oocytes via metabolic co-operation, by passing ATP and other glycolytic products through gap junctions. It was concluded that female mouse germ cells and ovarian follicle cells do not need an intact endogenous glycolytic pathway if they can obtain appropriate metabolites from an exogenous source.     

Distinct roles for the mammalian A-type cyclins during oogenesis

There are two A-type cyclins in higher vertebrates, cyclin A1 and A2. Targeted mutagenesis has shown that cyclin A2 is essential for early embryonic development while cyclin A1 is required only for male meiosis. The embryonic lethality of cyclin A2 knockout mice has obviated understanding its role in other aspects of mammalian development, including the germ line. We reported previously that cyclin A2 expression in the male germ line is consistent with a role in both mitotic and meiotic cell cycles. Using in situ hybridization and immunohistochemistry, we now observe high levels of cyclin A2 in granulosa cells and less-abundant but readily detectable expression in ovarian and ovulated oocytes. A decrease in cyclin A2 protein was observed in oocytes from embryonic stages to post-natal and adult ovaries. Interestingly, cyclin A2 protein was nuclear in oocytes from embryonic day 13.5 to 15.5, changing to largely cytoplasmic in oocytes from embryonic day 16.5 to post-natal and adults. Readily detectable expression of the cyclin-dependent kinases Cdk1 and Cdk2, two common partners for the A-type cyclins, was observed in granulosa cells and oocytes at all stages of folliculogenesis. Cdk1 was predominantly cytoplasmic, whereas Cdk2 was both cytoplasmic and nuclear in oocytes. No cyclin A1 expression, at either the mRNA level or the protein level was detected in either embryonic or adult ovaries, consistent with the full fertility observed in female cyclin A1-deficient mice. These results suggest that in the female germ line, cyclin A2 but not cyclin A1 has distinct roles in both mitosis and meiosis.     

Ovarian development in intrauterine growth-retarded and normally developed piglets originating from the same litter

Epidemiological studies in humans linking adult disease to growth in utero indicate that prenatal life is a critical period for the appropriate development of the reproductive axis. The aim of this study was to compare ovarian development in intrauterine growth-retarded and normally grown piglets originating from the same litter. Intrauterine growth-retarded piglets (runts) were identified on the basis of statistical analysis of the birth weight distribution within each litter. At birth, ovaries were collected from runt piglets (n=14) and their respective mean weight (normal, n=14) littermates. Ovaries were weighed and fixed, and development of ovarian germ cells was quantified in haematoxylin-eosin-stained paraffin wax sections using an image analysis system. Germ cell loss, using an in situ TdT-mediated dUTP nick-end labelling (TUNEL) assay for DNA fragmentation, and follicle cell activity, using immunohistochemistry to demonstrate vimentin, were studied in ovarian sections. At birth, body weight and absolute ovarian mass were significantly lower in runt piglets compared with their respective normally grown littermates (body weight: 733+/-38.5 versus 1530+/-39.7 g; ovarian mass: 51+/-3.0 versus 108+/-9.6 mg; P<0.001 for both). In the ovary, the proportion of nests of oogonia, the number of oocytes and TUNEL-positive cells, and the localization and intensity of vimentin immunoreactivity were not different between runt and normal littermates. However, runt piglets had more primordial follicles (268+/-18.6 versus 235+/-20.1 per mm(2) of cortex; P<0.05), fewer primary follicles (11+/-2.0 versus 20+/-3.0 per mm(2) of cortex; P<0.001) and no secondary follicles compared with normal piglets. These findings indicate that intrauterine growth retardation delayed follicular development in pig ovaries at birth.     

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.     

Autophagy is a cell survival program for female germ cells in the murine ovary

It is estimated that infertility affects 15-20% of couples and can arise from female or male reproductive defects. Mouse models have ascribed roles to over 100 genes in the maintenance of female fertility. Although previous models have determined roles for apoptosis in male and female fertility, we find that compromised autophagy within the perinatal ovary, through the loss of Becn1 or Atg7, results in the premature loss of female germ cells. Becn1(+/-) ovaries have a 56% reduction of germ cells compared with control ovaries at post-natal day 1, whereas Atg7(-/-) ovaries lack discernable germ cells at this stage. Thus autophagy appears to be a cell survival mechanism to maintain the endowment of female germ cells prior to establishing primordial follicle pools in the ovary.     

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.     

Murine homologues of the Drosophila gustavus gene are expressed in ovarian granulosa cells

Mammalian homologues of genes that control oogenesis in other organisms may play similar roles in mammalian ovarian development. In Drosophila melanogaster, GUSTAVUS (GUS) protein physically interacts with and is necessary for the proper posterior localization of VASA protein, and thus is required for specification of germ cells. We identified two mouse genes, SSB-1 and SSB-4 (SPRY domain SOCS box protein), whose protein products share 75% identity and are each approximately 70% identical to Drosophila GUS. Both SSB-1 and SSB-4 mRNA were detectable in mouse ovaries by Northern blotting of total and poly(A) + RNA, but were expressed in few other tissues. SSB-1 was detectable in testes, although the 3'-untranslated region of the mRNA was considerably shorter than the ovarian mRNA. In situ hybridization and RT-PCR analysis of ovaries revealed that both genes were expressed in granulosa cells at all stages of follicular development. In contrast, expression was barely detectable in in oocytes. Immunoblotting analysis revealed that SSB-1 protein was present in follicles at different stages of growth, and immunocytochemistry confirmed that SSB-1 and SSB-4 were detectable in granulosa cells of primary and subsequent stage follicles and that they were present in both mural and cumulus granulosa cells of antral follicles. These results establish that GUS-related proteins, which in Drosophila are restricted to the germ cells, are in the mouse instead expressed in the granulosa cells and are present throughout folliculogenesis. Based on their tissue-restricted pattern of expression and apparent abundance in granulosa cells, we propose that SSB-1 and SSB-4 play key roles in regulating granulosa cell physiology.     

Caspase-mediated apoptosis in the vertebrate ovary

In the vertebrate ovary, apoptosis is the process by which excess or non-viable germ and granulosa cells are eliminated early in ontogeny (often beginning before birth), and thereafter continuously throughout reproductive life. Accordingly, an excessively high rate or abnormal triggering of such cell death (and, by implication, follicle atresia) can negatively affect fertility. Programmed cell death involves the integration of many pathways and intracellular proteins, and central among these at almost every stage are members of the caspase family. Relatively little attention has been focused upon the ovary with regards to elucidating initiator and effector members of the caspase family, and pathways by which they are activated and inactivated. The present review briefly describes vertebrate caspases and the regulation of their function in non-ovarian tissues. Subsequently, the status of caspase expression and function in orchestrating apoptotic cell death in ovarian germ and follicle somatic cells is considered. The most compelling results implicating specific caspases in ovarian function have been derived from mouse single and double knockout model systems. The final outcome of continued studies, in addition to providing information regarding understanding and management of infertility, will influence the development of strategies to treat ovarian cancers and ameliorate the adverse effects of their therapy (for example, chemotherapy).     

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.     

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.     

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.     

Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth factor receptor-3c and -4, in bovine antral follicles

Paracrine cell signaling is believed to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family has been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR3c and FGFR4) are expressed in bovine antral follicles. RT-PCR was used to analyze bovine Fgf8, Fgfr3c and Fgfr4 mRNA levels in oocytes, and granulosa and theca cells. Fgf8 expression was detected in oocytes and in granulosa and theca cells; this expression pattern differs from that reported in rodents. Granulosa and theca cells, but not oocytes, expressed Fgfr3c, and expression in granulosa cells increased significantly with follicle estradiol content, a major indicator of follicle health. Fgfr4 expression was restricted to theca cells in the follicle, and decreased significantly with increasing follicle size. To investigate the potential regulation of Fgfr3c expression in the bovine granulosa, cells were cultured in serum-free medium with FSH or IGF-I; gene expression was upregulated by FSH but not by IGF-I. The FSH-responsive and developmentally regulated patterns of Fgfr3c mRNA expression suggest that this receptor is a potential mediator of paracrine signaling to granulosa cells during antral follicle growth in cattle.     

The developing ovary of the South American plains vizcacha, Lagostomus maximus (Mammalia, Rodentia): massive proliferation with no sign of apoptosis-mediated germ cell attrition

Apoptosis-dependent massive germ cell death is considered a constitutive trait of the developing mammalian ovary that eliminates 65-85% of the germinal tissue depending on the species. After birth and during adult lifetime, apoptotic activity moves from the germ cell proper to the somatic compartment, decimating germ cells through follicular atresia until the oocyte reserve is exhausted. In contrast, the South American rodent Lagostomus maximus shows suppressed apoptosis-dependent follicular atresia in the adult ovary, with continuous folliculogenesis and massive polyovulation, which finally exhausts the oocyte pool. The absence of follicular atresia in adult L. maximus might arise from a failure to move apoptosis from the germinal stratum to the somatic compartment after birth or being a constitutive trait of the ovarian tissue with no massive germ cell degeneration in the developing ovary. We tested these possibilities by analysing oogenesis, expression of germ cell-specific VASA protein, apoptotic proteins BCL2 and BAX, and DNA fragmentation by TUNEL assay in the developing ovary of L. maximus. Immunolabelling for VASA revealed a massive and widespread colonisation of the ovary and proliferation of germ cells organised in nests that disappeared at late development when folliculogenesis began. No sign of germ cell attrition was found at any time point. BCL2 remained positive throughout oogenesis, whereas BAX was slightly detected in early development. TUNEL assay was conspicuously negative throughout the development. These results advocate for an unrestricted proliferation of germ cells, without apoptosis-driven elimination, as a constitutive trait of L. maximus ovary as opposed to what is normally found in the developing mammalian ovary.