The human ovarian reserve: the narrative and the science.

Role of environmental factors in timing the onset and progression of puberty

Author response: Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression

Decision letter: Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression

The number of germ cells in human embryonic and fetal ovaries in relation to age is currently based on volumetric estimations from one study including a total of 12 ovaries. Six recent publications present stereological estimations of the number of germ cells in ovaries and testes for the first two trimesters. Germ cell numbers from 103 human first and second trimester gonads aged 37-133 days post-conception (p.c.), obtained after legal termination of pregnancy, were collected from six independent studies that all used similar validated stereological methods for estimating germ cell numbers as well as somatic cell numbers. Statistically, the six studies estimated similar number of germ cells (P > 0.05) and no interaction between the studies and age was found (P > 0.05), indicating that the increase in cell numbers in relation to age was of comparable magnitude in each study. The number of germ cells increased from a mean of 7200 to 4,933,000 in fetal ovaries and from 3700 to 1,417,000 in fetal testes, from week 5 to week 19 p.c. A higher rate of increase was found for female germ cells as compared with males (P = 0.004). During the same period, the number of somatic cells increased from a mean of 158,000 to 1,017,000 in ovaries and from 154,000 to 2,035,000 in testes, respectively. CONCLUSIONS By the use of validated stereological methods, this study provides more accurate and improved information on human germ and somatic cell numbers in ovaries and testes during the first two trimesters of pregnancy.

Germline development from human pluripotent stem cells toward disease modeling of infertility

Regulation of cell death in human fetal and adult ovaries—Role of Bok and Bcl-X L

The development of mammalian fetal germ cells along oogenic or spermatogenic fate trajectories is dictated by signals from the surrounding gonadal environment. Germ cells in the fetal testis enter mitotic arrest, whilst those in the fetal ovary undergo sex-specific entry into meiosis, the initiation of which is thought to be mediated by selective exposure of fetal ovarian germ cells to mesonephros-derived retinoic acid (RA). Aspects of this model are hard to reconcile with the spatiotemporal pattern of germ cell differentiation in the human fetal ovary, however. We have therefore examined the expression of components of the RA synthesis, metabolism and signalling pathways, and their downstream effectors and inhibitors in germ cells around the time of the initiation of meiosis in the human fetal gonad. Expression of the three RA-synthesising enzymes, ALDH1A1, 2 and 3 in the fetal ovary and testis was equal to or greater than that in the mesonephros at 8–9 weeks gestation, indicating an intrinsic capacity within the gonad to synthesise RA. Using immunohistochemistry to detect RA receptors RARα, β and RXRα, we find germ cells to be the predominant target of RA signalling in the fetal human ovary, but also reveal widespread receptor nuclear localization indicative of signalling in the testis, suggesting that human fetal testicular germ cells are not efficiently shielded from RA by the action of the RA-metabolising enzyme CYP26B1. Consistent with this, expression of CYP26B1 was greater in the human fetal ovary than testis, although the sexually-dimorphic expression patterns of the germ cell-intrinsic regulators of meiotic initiation, STRA8 and NANOS2, appear conserved. Finally, we demonstrate that RA induces a two-fold increase in STRA8 expression in cultures of human fetal testis, but is not sufficient to cause widespread meiosis-associated gene expression. Together, these data indicate that while local production of RA within the fetal ovary may be important in regulating the onset of meiosis in the human fetal ovary, mechanisms other than CYP26B1-mediated metabolism of RA may exist to inhibit the entry of germ cells into meiosis in the human fetal testis.

The polycyclic aromatic hydrocarbon pollutant benzo[a]pyrene (BaP) is a known developmental gonadotoxicant. However, the mechanism of BaP-induced germ cell death is unclear. We investigated whether exposure to BaP induces apoptotic germ cell death in the mouse fetal ovary or testis. Mouse fetal gonads were dissected at embryonic day 13.5 days postcoitum (dpc) and fixed immediately or cultured for 6, 24, 48, or 72 h with various concentrations of BaP (1-1000 ng/ml). Germ cells numbers, apoptosis, and proliferation were evaluated by immunostaining. Treatment of fetal ovaries with BaP for 72 h concentration-dependently depleted germ cells. Treatment with BaP elevated the expression of BAX protein at 6 h and activated downstream caspases-9 and -3 at 24 h in a concentration-dependent manner in germ cells of fetal ovaries. As a consequence, ovarian germ cell numbers were significantly and concentration-dependently decreased at 48 h. Pretreatment with z-VAD-fmk, a pan-caspase inhibitor, prior to exposure to 1000 ng/ml BaP prevented BaP-mediated ovarian germ cell death; there were no effects of BaP or z-VAD-fmk on germ cell proliferation. No significant effects of BaP exposure on caspase 3 activation or germ cell numbers were observed in fetal testes after 48 h of culture. Our findings show that BaP exposure increases caspase-dependent and BAX-associated germ cell apoptosis in the mouse fetal ovary, leading to germ cell depletion. In contrast, the cultured 13.5 dpc fetal testis is relatively resistant to BaP-induced germ cell death. This study provides a novel insight into molecular mechanisms by which BaP has direct gonadotoxicity in the mouse fetal ovary.

Does PCOS have developmental origins?

The number and quality of oocytes stored within the ovary as primordial follicles influence the length of the fertile lifespan, the age at which menopause begins, and the health of offspring. Primordial germ cells and oogonia, which are the embryonic precursors of oocytes, multiply dramatically during the early stages of fetal development. Germ cell numbers then undergo a dramatic decline as a consequence of the permanent cessation of mitosis and the loss of approximately two-thirds of the newly made oogonia and oocytes. The loss of germ cells is largely due to apoptosis and this leaves a reduced number of oocytes within the ovary at birth, and, because new germ cells cannot be made after this point, it limits female fertility and reproductive lifespan. Despite the critical role of apoptosis in regulating the number of available oocytes, and hence in determining the length of a women's fertile lifespan and the timing of menopause, the proteins that regulate the death of germ cells are largely unknown and the reasons underlying their attrition are unclear. The overall aim of this project was to investigate the role of the Bci2-Modifying Factor (BMF), a pro-apoptotic protein belonging to the BH3-only sub-group of the Bcl-2 superfamily, in the death of germ cells during oogenesis in mice. BMF protein was immunohistochemically localised to germ cells at embryonic days (E) 15.5, 17.5 and PN1, coincident with entry into the meiotic prophase and the beginning of germ cell nest breakdown, but was undetectable at E13.5 and postnatal days {PN) 3 and 5. Targeted deletion of the Bmf gene in female mice resulted in a transient increase in germ cell number compared to wild type (WT) at (E) 15.5, and between PN1 and PN5. However, germ cell numbers were comparable in WT and Bmf1- ovaries at E13.5, E17.5 and PN10. Loss of BMF was also associated with a decrease in apoptosis, as determined by Terminal deoxynucleotidyl-transferase dUTP nick-end labelling (TUNEL) at ElS.S and E17.5. lmmunostaining for yH2AX, a meiotic prophase maker, and TAp63, a marker of diplotene arrest, indicated that germs cells cease proliferation and enter meiotic arrest at a similar time in ovaries from WT and Bmf1- mice. Additionally, immunostaining for PH3 (a marker of mitotic cells) and MVH (a marker of germ cells) showed no differences in the percentage of germ cells labelled with PH3 between Bm[1- and WT ovaries at E13.5. Thus, altered proliferation could not explain the observed increases in germ cell number in ovaries from Bmf1- compared to WT mice. Notably, BMF was dispensable for the massive germ cell loss that occurs during nest breakdown, which was compared in ovaries from WT and Bm[1- mice using morphological analyses as well as by immunostaining for MVH and the basal laminar marker laminin. Collectively, these data indicate that BMF is expressed in oocytes during the meiotic prophase and likely mediates their apoptosis at that time, but does not influence the number of primordial follicles initially making up the ovarian reserve at PNlO.

Does ibuprofen use during the first trimester of pregnancy interfere with the development of the human fetal ovary? In human fetuses, ibuprofen exposure is deleterious for ovarian germ cells. In utero stages of ovarian development define the future reproductive capacity of a woman. In rodents, analgesics can impair the development of the fetal ovary leading to early onset of fertility failure. Ibuprofen, which is available over-the-counter, has been reported as a frequently consumed medication during pregnancy, especially during the first trimester when the ovarian germ cells undergo crucial steps of proliferation and differentiation. Organotypic cultures of human ovaries obtained from 7 to 12 developmental week (DW) fetuses were exposed to ibuprofen at 1-100 μM for 2, 4 or 7 days. For each individual, a control culture (vehicle) was included and compared to its treated counterpart. A total of 185 individual samples were included. Ovarian explants were analyzed by flow cytometry, immunohistochemistry and quantitative PCR. Endpoints focused on ovarian cell number, cell death, proliferation and germ cell complement. To analyze the possible range of exposure, ibuprofen was measured in the umbilical cord blood from the women exposed or not to ibuprofen prior to termination of pregnancy. Human ovarian explants exposed to 10 and 100 μM ibuprofen showed reduced cell number, less proliferating cells, increased apoptosis and a dramatic loss of germ cell number, regardless of the gestational age of the fetus. Significant effects were observed after 7 days of exposure to 10 μM ibuprofen. At this concentration, apoptosis was observed as early as 2 days of treatment, along with a decrease in M2A-positive germ cell number. These deleterious effects of ibuprofen were not fully rescued after 5 days of drug withdrawal. N/A. This study was performed in an experimental setting of human ovaries explants exposed to the drug in culture, which may not fully recapitulate the complexity of in vivo exposure and organ development. Inter-individual variability is also to be taken into account. Whereas ibuprofen is currently only contra-indicated after 24 weeks of pregnancy, our results points to a deleterious effect of this drug on first trimester fetal ovaries ex vivo. These findings deserve to be considered in light of the present recommendations about ibuprofen consumption pregnancy, and reveal the urgent need for further investigations on the cellular and molecular mechanisms that underlie the effect of ibuprofen on fetal ovary development.

Apoptosis is responsible for primordial germ cell (PGC) attrition in the developing fetal ovary. In monolayer cultures of murine PGC, stem cell factor (SCF) and leukemia inhibitory factor (LIF) independently promote survival in vitro; however, the relevance of these data to fetal ovarian oogonium and oocyte survival, as well as the intracellular events involved in transducing the antiapoptotic actions of these cytokines in germ cells, remain to be elucidated. In this report, we investigated the effects of SCF and LIF, alone and in combination, on the survival of oogonia and oocytes, and elaborated on components of the signal transduction pathway used by these molecules, after validating a method of culturing fetal mouse ovaries. We further employed this system to also test the hypothesis that insulin-like growth factor-I (IGF-I), a classic antiapoptotic molecule, and transforming growth factor-beta (TGF-beta), a classic pro-apoptotic molecule, interact with the SCF/LIF pathway and function in a reciprocal fashion to precisely regulate germ cell numbers during fetal oogenesis. Freshly isolated embryonic day 13.5 ovaries contained nonapoptotic germ cells, as determined by histologic analysis of cellular morphology and in situ 3'-end-labeling of DNA integrity. In vitro culture of fetal ovaries without tropic support for 24, 48, and 72 h resulted in a time-dependent induction of germ cell apoptosis, such that most oogonia and oocytes present after 72 h were apoptotic. Morphometric analysis of serially sectioned ovaries indicated that the numbers of nonapoptotic germ cells remaining after 24, 48, and 72 h of culture were 78%, 38%, and 10%, respectively, of the number present before culture (P < 0.05 for all time points vs. 0 h). Inclusion of SCF (100 ng/ml) together with LIF (100 ng/ml) in the culture medium significantly attenuated germ cell apoptosis, with the SCF/LIF-treated ovaries retaining 5.5-fold more oogonia and oocytes after 72 h of culture as compared with control ovaries deprived of tropic support (P < 0.05). However, SCF or LIF, when added separately, had no (SCF) or little (LIF) inhibitory effect on germ cell apoptosis. Provision of 50 ng/ml IGF-I maintained survival of approximately two-thirds of the germ cells in cultured ovaries (P < 0.05), whereas a combination of all three growth factors (SCF, LIF, IGF-I) completely preserved the fetal ovary in culture to that resembling a freshly-isolated gonad. Cotreatment with 25 ng/ml TGF-beta partially reversed the survival actions of IGF-I or SCF/LIF, such that only one-third of the starting number of oogonia/oocytes remained after 72 h of culture (P < 0.05). Lastly, the antiapoptotic effects of SCF/LIF or IGF-I were almost entirely eliminated by cotreatment of fetal ovaries with either one of two inhibitors of phosphatidylinositol-3'-kinase (PI3K), LY294002 (5 microM) or wortmannin (50 nM), whereas cotreatment with an inhibitor of p70 S6 kinase (rapamycin, 25 ng/ml) was without effect. These data indicate that the combined actions of SCF, LIF, and IGF-I are required for maximal inhibition of apoptosis in germ cells of fetal mouse ovaries, and that the PI3K signaling pathway is an essential component of cytokine-mediated female germ cell survival. Moreover, TGF-beta can partially override the antiapoptotic actions of SCF/LIF or IGF-I in oogonia and oocytes, suggesting the existence of a complex signaling network that ultimately determines fetal ovarian germ cell fate.

How can pre-meiotic germ cells persist in the human foetal ovary? Numerous oogonia escaping meiotic entry were retrieved throughout human ovarian development simultaneously with the expression of signalling pathways preventing meiosis, typically described in the rodent embryonic testis. The transition from mitosis to meiosis is a key event in female germ cells that remains poorly documented in research on the human ovary. Previous reports described a strikingly asynchronous differentiation in the human female germ line during development, with the persistence of oogonia among oocytes and follicles during the second and third trimesters. The possible mechanisms allowing some cells to escape meiosis remain elusive. In order to document the extent of this phenomenon, we detailed the expression profile of germ cell differentiation markers using 73 ovaries ranging from 6.4 to 35 weeks post-fertilization. Pre-meiotic markers were detected by immunohistochemistry or qRT-PCR. The expression of the main meiosis-preventing factors identified in mice was analysed, and their functionality assessed using organ cultures. Oogonia stained for AP2γ could be traced from the first trimester until the end of the third trimester. Female germ cell differentiation is organized both in time and space in a centripetal manner in the foetal human ovary. Unexpectedly, some features usually ascribed to rodent pre-spermatogonia could be observed in human foetal ovaries, such as NANOS2 expression and quiescence in some germ cells. The two main somatic signals known to inhibit meiosis in the mouse embryonic testis, CYP26B1 and FGF9, were detected in the human ovary and act simultaneously to repress STRA8 and meiosis in human foetal female germ cells. N/A. Our conclusions relied partly on in vitro experiments. Germ cells were not systematically identified with immunostaining and some may have thus escaped analysis. We found evidence that a robust repression of meiotic entry is taking place in the human foetal ovary, possibly explaining the exceptional long-lasting presence of pre-meiotic germ cells until late gestational age. This result calls for a redefinition of the markers known as classical male markers, which may in fact characterize mammalian developing gonads irrespectively of their sex. This research was supported by the Université Paris Diderot-Paris 7 and Université Paris-Sud, CEA, INSERM, and Agence de la Biomédecine. The authors declare no conflict of interest.

The ovaries of foetal and neonatal rats (14·5 days post coitum to 2 days post partum ) have been studied in order to correlate morphological changes in oogonia and developing oocytes with fluctuations in their numbers. The chromosomal configurations of germ cells at different developmental stages were examined in squash preparations. A volumetric method was devised for the separate estimation of the populations of normal and degenerating germ cells. Counts were made of the numbers of mitoses in germinal and somatic cells up to 19·5 days p. c . Oogonia are mitotically active up to 17·5 days p. c . The onset of the leptotene phase of meiosis in the majority of oocytes (17·5 to 18·5 days p. c .) coincides with a sharp decline in the number of normal oogonial divisions. The duration of the zygotene phase appears to be considerably shorter (19·5 to 20·5 days p. c .) than that of pachytene (range 20·5 days p. c . to 2 days p. p .). Oocytes in the diplotene phase are typically seen at 2 days p. p . The number of germ cells attaining the dictyate or resting phase is small on the first day after birth, but increases on the second. Four different ‘waves’ of degeneration of germ cells were observed. The first, affecting a small number of oogonia, occurs before the onset of meiosis, and is characterized by pyknosis. The second is characterized by degeneration of dividing oogonia which probably sets in before or at mitotic prophase. Such ‘atretic divisions’ reach their peak at 18·5 days p. c ., when the frequency of normal mitotic divisions is relatively low. The third affects germ cells after they have entered meiotic prophase (particularly at the pachytene stage); the chromosomes condense and the cytoplasm becomes eosinophil (referred to as ‘ Z ’ cells). The fourth affects germ cells at the diplotene phase. Oogonial mitoses are associated with an increase in the population of germ cells from 12000 on day 14·5 to 71000 on day 17·5 p. c . The appearance of ‘atretic divisions’ is largely responsible for the reduction of about 3000 in the number of normal germ cells between days 17·5 and 18·5. Successful mitotic divisions, on the other hand, account for the increase of about 4000 in the total number of germ cells during the same interval. From day 18·5 onwards, both the total number and the number of normal germ cells decreases steadily, first due to the occurrence of ‘atretic divisions’ and later to degeneration of ' Z ' cells and atretic cells at diplotene. Thus by 2 days after birth, the peak number of normal germ cells (64000 at 17·5 days p. c .) has fallen to 19000, the total population (peak 75000 at 18·5 days p. c .) being reduced to 27000. The majority of germ cells appear to be eliminated from the ovary within 24 h of the onset of degeneration.

SummaryIn vertebrates, estrogen receptors are essential for estrogen-associated early gonadal sex development. Our previous studies revealed sexual dimorphic expression of estrogen receptor β2 (ERβ2) during embryogenesis of medaka, and here we investigated the functional importance of ERβ2 in female gonad development and maintenance using a transgenerational ERβ2-knockdown (ERβ2-KD) line and ERβ2-null mutants. We found that ERβ2 reduction favored male-biased gene transcription, suppressed female-responsive gene expression, and affected SDF1a and CXCR4b co-assisted chemotactic primordial germ cell (PGC) migration. Co-overexpression of SDF1a and CXXR4b restored the ERβ2-KD/KO associated PGC mismigration. Further analysis confirmed that curtailment of ERβ2 increased intracellular Ca2+ concentration, disrupted intra- and extracellular calcium homeostasis, and instigated autophagic germ cell degradation and germ cell loss, which in some cases ultimately affected the XX female sexual development. This study is expected improve our understanding of germ cell maintenance and sex spectrum, and hence open new avenues for reproductive disorder management.