Search

Study Question: How can pre-meiotic germ cells persist in the human foetal ovary?, Summary Answer: 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., What Is Known Already: 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., Study Design Size, Duration: 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., Participants/materials Setting, Methods: 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., Main Results and the Role of Chance: 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., Large Scale Data: N/A., Limitations Reason for Caution: Our conclusions relied partly on in vitro experiments. Germ cells were not systematically identified with immunostaining and some may have thus escaped analysis., Wider Implications of the Findings: 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., Study Funding/competing Interest(s): 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 Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Uranium (U) is found in the environment and its use in industrial or military activities has led to concerns about its potential toxicity. The reprotoxicity of this heavy metal has been established in adult animals; however, no studies have examined its effect on human fetal gonads. Using an organ culture system, we investigated the effects of uranyl acetate on human gonads during the first trimester of gestation (7-12 weeks), which is a critical step in the development of a functional reproductive system. In human fetal ovaries, 0.05 mM U significantly decreased germ cell density by increasing their apoptosis rate. In human fetal testes, 0.1mM U similarly reduced the number of germ cells. The human fetal germ cells are more sensitive to U than mouse germ cells in the same experimental conditions. This is the first evidence that U may impair the development of the human gonads., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In mammals, early fetal germ cells are unique in their ability to initiate the spermatogenesis or oogenesis programs dependent of their somatic environment. In mice, female germ cells enter into meiosis at 13.5 dpc whereas in the male, germ cells undergo mitotic arrest. Recent findings indicate that Cyp26b1, a RA-degrading enzyme, is a key factor preventing initiation of meiosis in the fetal testis. Here, we report evidence for additional testicular pathways involved in the prevention of fetal meiosis. Using a co-culture model in which an undifferentiated XX gonad is cultured with a fetal or neonatal testis, we demonstrated that the testis prevented the initiation of meiosis and induced male germ cell differentiation in the XX gonad. This testicular effect disappeared when male meiosis starts in the neonatal testis and was not directly due to Cyp26b1 expression. Moreover, neither RA nor ketoconazole, an inhibitor of Cyp26b1, completely prevented testicular inhibition of meiosis in co-cultured ovary. We found that secreted factor(s), with molecular weight greater than 10 kDa contained in conditioned media from cultured fetal testes, inhibited meiosis in the XX gonad. Lastly, although both Sertoli and interstitial cells inhibited meiosis in XX germ cells, only interstitial cells induced mitotic arrest in germ cell. In conclusion, our results demonstrate that male germ cell determination is supported by additional non-retinoid secreted factors inhibiting both meiosis and mitosis and produced by the testicular somatic cells during fetal and neonatal life., (Copyright © 2010 Elsevier Inc. All rights reserved.)

During mouse fetal development, meiosis is initiated in female germ cells only, with male germ cells undergoing mitotic arrest. Retinoic acid (RA) is degraded by Cyp26b1 in the embryonic testis but not in the ovary where it initiates the mitosis/meiosis transition. However the role of RA status in fetal germ cell proliferation has not been elucidated. As expected, using organ cultures, we observed that addition of RA in 11.5 days post-conception (dpc) testes induced Stra8 expression and meiosis. Surprisingly, in 13.5 dpc testes although RA induced Stra8 expression it did not promote meiosis. On 11.5 and 13.5 dpc, RA prevented male germ cell mitotic arrest through PI3K signaling. Therefore 13.5 dpc testes appeared as an interesting model to investigate RA effects on germ cell proliferation/differentiation independently of RA effect on the meiosis induction. At this stage, RA delayed SSEA-1 extinction, p63gamma expression and DNA hypermethylation which normally occur in male mitotic arrested germ cells. In vivo, in the fetal male gonad, germ cells cease their proliferation and loose SSEA-1 earlier than in female gonad and RA administration maintained male germ cell proliferation. Lastly, inhibition of endogenous Cyp26 activity in 13.5 dpc cultured testes also prevented male germ cell mitotic arrest. Our data demonstrate that the reduction of RA levels, which occurs specifically in the male fetal gonad and was known to block meiosis initiation, is also necessary to allow the establishment of the germ cell mitotic arrest and the correct further differentiation of the fetal germ cells along the male pathway.

There is now compelling evidence that inappropriate exposure to estrogen during fetal or neonatal life could affect adult reproductive functions because the testis is sensitive to estrogens during specific periods of its development. Therefore, we investigated the effects of exogenous estrogens on gametogenesis and steroidogenesis during fetal and neonatal testicular development in the rat. We used in vitro systems, organ cultures, and dispersed testicular cell cultures, which allow the development of fetal and neonatal germ cells (gonocytes) and Leydig cells. Exogenous estrogens inhibited testosterone production in dispersed testicular cell cultures throughout fetal life, but this inhibition was observed only in the early fetal stages in organ culture. By using an aromatase inhibitor (letrozole, Novartis Pharma AG), we showed that the inhibitory effect of exogenous estrogens on testosterone production is masked in the whole testis at later stages (20.5 days postconception) due essentially to local production of estrogens. In both systems, additions of high concentrations (10(-6) M) of 17beta-estradiol or diethylstilbestrol decreased the number of gonocytes during the first fetal proliferative period but not during the neonatal period. Letrozole was without effect, suggesting that the aging-related loss of responsiveness of gonocytes is not due to any aromatase activity in the gonocytes.

The fetal and neonatal development of male germ cells (gonocytes) is a poorly understood but crucial process for establishing fertility. In rodents, gonocytes go through two phases of proliferation accompanied by apoptosis and separated by a quiescent period during the end of fetal development. P63 is a member of the P53 gene family that yields six isoforms. We detected only the p63 protein and no p53 and p73 in the nucleus of the gonocytes of mouse testes. We report for the first time the ontogeny of each p63 mRNA isoform during testis development. We observed a strong expression of p63gamma mRNA and protein when gonocytes are in the quiescent period. In vitro treatment with retinoic acid prevented gonocytes from entering the quiescent period and was correlated with a reduced production of p63gamma isoform mRNA. We investigated the function of p63 by studying the testicular phenotype of P63-null mice. P63 invalidation slightly, but significantly increased the number of gonocytes counted during the quiescent period. As P63-null animals die at birth we used an original organ culture that mimicked neonatal in vivo development to study further the testicular development. P63 invalidation resulted in a sharply increased number of gonocytes during the culture period due to a decrease in spontaneous apoptosis with no change in proliferation. P63 invalidation also caused abnormal morphologies in the germ cells that were also found in P63(+/-) adult male mice. Thus, p63 appears as an important regulator of germ cell development.

Estrogens are classically known to play a major role in female reproduction but there is now compelling evidence that they may also be involved in the regulation of male reproductive function. In humans, a decrease in sperm count and an increase in the incidences of testicular cancer, cryptorchidism and hypospadia have been observed in many countries over the last 50 years. Male reproductive alterations were also observed in wildlife. Such male reproductive disorders have been attributed to the increase in concentration of xenobiotics, and of xenoestrogens in particular, in the environment and in food. Epidemiological, clinical and experimental studies have suggested that excessive exposure to estrogens during fetal/neonatal life can lead to reproductive disorders in adulthood. Using an in vitro model we showed that estrogens directly affected the development of the fetal testis and we evidenced the existence of periods of sensitivity throughout development. Lastly, we clearly demonstrated that the fetal and neonatal testis is very sensitive to estrogens since the invalidation of estrogen receptor alpha leads to an increase of steroidogenesis and the invalidation of estrogen receptor beta enhances the development of the germ cell lineage in the male.

Estrogens are classically known to play a major role in female reproduction, but there is now compelling evidence that they may also be involved in the regulation of male reproductive function. In humans, a decrease in sperm count and an increase in the incidences of testicular cancer, cryptorchidism and hypospadia have been observed in many countries over the last 50 years. Male reproductive alterations were also observed in wildlife. Such male reproductive disorders have been attributed to the increase in concentration of xenobiotics, and of xenoestrogens in particular, in the environment and in food. Epidemiological, clinical and experimental studies have suggested that excessive exposure to estrogens during fetal/neonatal life can lead to reproductive disorders in adulthood. Using an in vitro model, we showed that estrogens directly affected the development of the fetal testis. Lastly, we clearly demonstrated that the fetal and neonatal testis is very sensitive to estrogens since the invalidation of estrogen receptor alpha leads to an increase of steroidogenesis and the invalidation of estrogen receptor beta enhances the development of the germ cell lineage in the male.

Epidemiological, clinical, and experimental studies have suggested that excessive exposure to estrogens during fetal/neonatal life can lead to reproductive disorders and sperm abnormalities in adulthood. However, it is unknown whether endogenous concentrations of estrogens affect the establishment of the male fetal germ cell lineage. We addressed this question by studying the testicular development of mice in which the estrogen receptor (ER) beta or the ERalpha gene was inactivated. The homozygous inactivation of ERbeta (ERbeta-/-) increased the number of gonocytes by 50% in 2- and 6-d-old neonates. The numbers of Sertoli and Leydig cells and the level of testicular testosterone production were unaffected, suggesting that estrogens act directly on the gonocytes. The increase in the number of gonocytes did not occur during fetal life but instead occurred just after birth, when gonocytes resumed mitosis and apoptosis. It seems to result from a decrease in the apoptosis rate evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method and cleaved caspase-3 immunohistochemical detection. Last, mice heterozygous for the ERbeta gene inactivation behaved similarly to their ERbeta-/- littermates in terms of the number of gonocytes, apoptosis, and mitosis, suggesting that these cells are highly sensitive to the binding of estrogens to ERbeta. ERalpha inactivation had no effect on the number of neonatal gonocytes and Sertoli cells. In conclusion, this study provides the first demonstration that endogenous estrogens can physiologically inhibit germ cell growth in the male. This finding may have important implications concerning the potential action of environmental estrogens.

A human sperm-oocyte binding protein, SOB1, was purified by two dimensional gel electrophoresis and sequenced. This protein was selected because it was recognized by a monoclonal antibody that inhibited the binding of human sperm to zona-free hamster oocytes. The sequences of the tryptic peptides were used to design degenerate primers. These were used to amplify a specific fragment from human testis cDNA by the polymerase chain reaction. This 1233 bp fragment was extended in 3' and 5' by RACE to obtain the 3 kb full length SOB1 cDNA. Sequence analysis indicated that the deduced open reading frame encodes a 853 amino acid protein, with a molecular mass of 94. 7 kDa. This is a new testis-specific cDNA. It is 27, 32.8 and 34.4% homologous to three sperm proteins, HI, Fsc1 and AKAP82 respectively. A single 3kb transcript was demonstrated only in the testis by northern blot analysis. It is a single copy gene, well conserved among mammals and located on human chromosome 12 at band p13., (Copyright 1999 Academic Press.)