Retinoic acid derived from the fetal ovary initiates meiosis in mouse germ cells

Meiotic initiation of germ cells at 13.5 dpc (days post‐coitus) indicates female sex determination in mice. Recent studies reveal that mesonephroi‐derived retinoic acid (RA) is the key signal for induction of meiosis. However, whether the mesonephroi is dispensable for meiosis is unclear and the role of the ovary in this meiotic process remains to be clarified. This study provides data that RA derived from fetal ovaries is sufficient to induce germ cell meiosis in a fetal ovary culture system. When fetal ovaries were collected from 11.5 to 13.5 dpc fetuses, isolated and cultured in vitro, germ cells enter meiosis in the absence of mesonephroi. To exclude RA sourcing from mesonephroi, 11.5 dpc urogenital ridges (UGRs; mesonephroi and ovary complexes) were treated with diethylaminobenzaldehyde (DEAB) to block retinaldehyde dehydrogenase (RALDH) activity in the mesonephros and the ovary. Meiosis occurred when DEAB was withdrawn and the mesonephros was removed 2 days later. Furthermore, RALDH1, rather than RALDH2, serves as the major RA synthetase in UGRs from 12.5 to 15.5 dpc. DEAB treatment to the ovary alone was able to block germ cell meiotic entry. We also found that exogenously supplied RA dose‐dependently reduced germ cell numbers in ovaries by accelerating the entry into meiosis. These results suggest that ovary‐derived RA is responsible for meiosis initiation. J. Cell. Physiol. 228: 627–639, 2013. © 2012 Wiley Periodicals, Inc.     

Retinoic acid prevents germ cell mitotic arrest in mouse fetal testes

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, p63γ 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.     

Somatic and germ cell interactions during histogenetic aggregation of mouse fetal testes.

In the present study we examined the capacity of somatic and germ cells dissociated from fetal mouse testes at various stages to reform seminiferous cords in culture. We found that after 12 h in culture, seminiferous cords became segregated from stromal cells. Although Sertoli cells were incorporated into seminiferous cords at all stages studied, the germ cells dramatically changed their histogenetic behavior with age. Most germ cells which had been dissociated at 12.5 days postcoitum (dpc) were incorporated into the seminiferous cords, whereas at 14.5 dpc or later the majority remained among the stromal cells or as clusters on the surface of the aggregates. We considered three possible causes for this change in behavior of germ cells: (i) Failure to deposit some extracellular matrix components in the aggregates. (ii) Decrease in adhesiveness of prospermatogonia to either extracellular matrix components or Sertoli cells. (iii) A change in adhesiveness of Sertoli cells to germ cells with age. We found that laminin and fibronectin were similarly deposited in aggregates at 12.5 and 15.5 dpc. When prospermatogonia at 15.5 dpc labeled with colloidal gold were reaggregated with somatic cells at 12.5 dpc, 50% were incorporated into seminiferous cords. Moreover, [3H]thymidine-labeled Sertoli cells at 15.5 dpc formed heterochronic seminiferous cords with Sertoli cells at 12.5 dpc. These results suggest that mouse Sertoli cells change their surface property which is essential for binding to germ cells when they enter the mitotic resting stage (T-prospermatogonia).     

PAR6, A Potential Marker for the Germ Cells Selected to Form Primordial Follicles in Mouse Ovary

Partitioning-defective proteins (PAR) are detected to express mainly in the cytoplast, and play an important role in cell polarity. However, we showed here that PAR6, one kind of PAR protein, was localized in the nuclei of mouse oocytes that formed primordial follicles during the perinatal period, suggesting a new role of PAR protein. It is the first time we found that, in mouse fetal ovaries, PAR6 appeared in somatic cell cytoplasm and fell weak when somatic cells invaded germ cell cysts at 17.5 days post coitus (dpc). Meanwhile, the expression of PAR6 was observed in cysts, and became strong in the nuclei of some germ cells at 19.5 dpc and all primordial follicular oocytes at 3 day post parturition (dpp), and then obviously declined when the primordial follicles entered the folliculogenic growth phase. During the primordial follicle pool foundation, the number of PAR6 positive germ cells remained steady and was consistent with that of formed follicles at 3 dpp. There were no TUNEL (apoptosis examination) positive germ cells stained with PAR6 at any time studied. The number of follicles significantly declined when 15.5 dpc ovaries were treated with the anti-PAR6 antibody and PAR6 RNA interference. Carbenoxolone (CBX, a known blocker of gap junctions) inhibited the expression of PAR6 in germ cells and the formation of follicles. Our results suggest that PAR6 could be used as a potential marker of germ cells for the primordial follicle formation, and the expression of PAR6 by a gap junction-dependent process may contribute to the formation of primordial follicles and the maintenance of oocytes at the diplotene stage.     

Apoptosis of fetal testicular cells is regulated by both p53-dependent and independent mechanisms

A portion of fetal germ cells undergoes apoptosis in the physiological context, but the molecular mechanisms of their apoptosis are largely unknown. Because p53 tumor suppressor gene product promotes apoptosis in various types of cells, we have investigated the expression of p53 in fetal gonads and examined the influence of loss of p53 function in fetal gonad cells using mice deficient in the p53 gene. We found that the expression of p53 protein in fetal testis was induced after 15.5 dpc (days post coitum), while the expression was not detected in fetal ovary. The number of apoptotic cells found in the seminiferous tubules of fetal testes was not significantly different between p53-deficient and wild-type mice until 16.5 dpc. At 17.5 dpc, however, more apoptotic cells were observed in wild-type testes than in the p53-deficient mice. In contrast, a similar number of apoptotic cells was found in fetal ovaries throughout these developmental stages. These observations indicate that p53 promotes apoptosis of fetal testicular cells after 16.5 dpc.     

G2/M checkpoint gene expression in developing germ cells

Cell cycle progression is prevented by signal transduction pathways known as checkpoints which are activated in response to replication interference and DNA damage. We cloned a G2/M cell cycle phase-related checkpoint gene from a neonatal mouse testis cDNA library which was identified as mouse claspin, a proposed adaptor protein for Chk1. As part of a study on germ cell differentiation we examined the expression of the checkpoint gene, Chk1, and claspin at 12.5 and 14.5 days post coitum (dpc) and in the post-natal phase. Chk1 mRNA expression increased from 12.5 to 14.5 dpc in female gonads and was strong in males at both time points. Claspin however, was not detected until 14.5 dpc. This suggests there may be some dissociation of claspin expression from Chk1 in fetal germ cell development. Chk1 and claspin expression was also studied in testis over the first 3 days following birth, when apoptosis regulates germ stem cell number. We modulated checkpoint-related gene expression in testis using the anti-metabolite, 5-fluorouracil, resulting in increased apoptosis and upregulation of Chk1 (P<0.0001) and Cdc2 (P<0.02) mRNA. Although we do not fully understand the role checkpoint gene expression has during mammalian germ cell development this report is the first to show the expression of checkpoint-related genes in early mammalian germ cells.     

Male differentiation of germ cells induced by embryonic age-specific Sertoli cells in mice

Retinoic acid (RA) is a meiosis-inducing factor. Primordial germ cells (PGCs) in the developing ovary are exposed to RA, resulting in entry into meiosis. In contrast, PGCs in the developing testis enter mitotic arrest to differentiate into prospermatogonia. Sertoli cells express CYP26B1, an RA-metabolizing enzyme, providing a simple explanation for why XY PGCs do not initiate meios/is. However, regulation of entry into mitotic arrest is likely more complex. To investigate the mechanisms that regulate male germ cell differentiation, we cultured XX and XY germ cells at 11.5 and 12.5 days postcoitus (dpc) with an RA receptor inhibitor. Expression of Stra8, a meiosis initiation gene, was suppressed in all groups. However, expression of Dnmt3l, a male-specific gene, during embryogenesis was elevated but only in 12.5-dpc XY germ cells. This suggests that inhibiting RA signaling is not sufficient for male germ cell differentiation but that the male gonadal environment also contributes to this pathway. To define the influence of Sertoli cells on male germ cell differentiation, Sertoli cells at 12.5, 15.5, and 18.5 dpc were aggregated with 11.5 dpc PGCs, respectively. After culture, PGCs aggregated with 12.5 dpc Sertoli cells increased Nanos2 and Dnmt3l expression. Furthermore, these PGCs established male-specific methylation imprints of the H19 differentially methylated domains. In contrast, PGCs aggregated with Sertoli cells at late embryonic ages did not commit to the male pathway. These findings suggest that male germ cell differentiation is induced both by inhibition of RA signaling and by molecule(s) production by embryonic age-specific Sertoli cells.     

The matricellular protein SPARC is internalized in Sertoli, Leydig, and germ cells during testis differentiation

The gene encoding the matricellular protein secreted protein, acidic and rich in cysteine (SPARC) was identified in a screen for genes expressed sex-specifically during mouse gonad development, as being strongly upregulated in the male gonad from very early in testis development. We present here a detailed analysis of SPARC gene and protein expression during testis development, from 11.5 to 15.5 days post coitum (dpc). Section in situ hybridization analysis revealed that SPARC mRNA is expressed by the Sertoli cells in the testis cords and the fetal Leydig cells, found within the interstitial space between the testis cords. Immunodetection with anti-SPARC antibody showed that the protein was located inside the testis cords, within the cytoplasm of Sertoli and germ cells. In the interstitium, SPARC was present intracellularly within the Leydig cells. The internalization of SPARC in Sertoli, Leydig, and germ cells suggests that it plays an intracellular regulatory role in these cell types during fetal testis development.     

Sexual dimorphism in the regulation of meiotic process in the rabbit

Meiosis, mitosis, and apoptosis during fetal and postnatal periods were investigated in order to explore mechanisms of sexual dimorphism in initiation of germ cell meiosis. Gonads were obtained from Japanese white rabbits from 23 to 51 days postcoitum (dpc). Gonadal thin sections were stained with hematoxylin and eosin. Germ cell alkaline phosphatase and apoptosis were detected with histochemical and immunohistochemical methods, respectively. In the ovary, meiotic germ cells were initially recognized at 29 dpc and arrested after enclosure within follicles. Similarly, meiotic germ cells were recognized outside seminiferous tubules at 29 dpc, but no meiotic figures were identified in intratubular spaces. Apoptotic germ cells were not recognized in the intratubular spaces before 35 dpc, and no apoptotic figures were recognized in the ovary during the period studied. In conclusion, the initiation of meiosis in testicular interstitial tissue at the time comparable to that in the ovary indicates that germ cells of both sexes have the ability to enter meiosis during the same stage of fetal development; and it appears most likely that delayed initiation of meiosis in the intratubular space is attributable to meiosis-inhibiting substance(s) present in seminiferous tubules.     

In vitro adhesion of mouse fetal germ cells to extracellular matrix components

Mouse primordial germ cells (PGCs) isolated from the dorsal mesentery and gonadal ridges of 10.5–12.5 days post coitum (dpc) embryos showed a progressively increasing adhesiveness to laminin and fibronectin coated substrates, whereas type I collagen and various glycosaminoglycans (hyaluronic acid, heparin and chondroitinsulphates) were poor adhesive substrates. At later stages germ cells appeared to lose their adhesiveness to fibronectin and laminin substrates; the ability to adhere to laminin decreased very rapidly in male and slowly in female germ cells. Oocytes and prospermatogonia from 15.5 dpc fetal gonads showed poor adhesiveness to all substrates tested. PGC adhesion to laminin and fibronectin substrates did not require calcium but was markedly trypsin sensitive. Antibodies against the fibronectin receptor of CHO fibroblasts and short peptides containing the Arg-Gly-Asp sequence greatly reduced PGC adhesion to fibronectin. Following adhesion to laminin or fibronectin, most PGCs did not exhibit a morphology typical of motile cells, but remained spherical. A significant proportion (about 30%) of oocytes from 13.5–14.5 dpc embryos appeared, however, able to spread and elongate following attachment to laminin. The results support the hypothesis that mouse PGCs may utilize laminin and/or fibronectin as adhesive substrates during migration and gonad colonization, but indicate that additional factors are probably required to promote PGC motility. In addition, our data provide indirect evidence that binding sites for specific components of extracellular matrix are present in PGCs, and that their expression may be developmentally regulated.     

A study of meiosis in chimeric mouse fetal gonads

The influence of somatic environment on the onset and progression of meiosis in fetal germ cells was studied in chimeric gonads produced in vitro by dissociation-reaggregation experiments. Germ cells isolated from testes or ovaries of 11.5-13.5 days post coitum (dpc) CD-1 mouse embryos were loaded with the fluorescent supravital dye 5-6 carboxyfluorescein diacetate succinimyl ester (CFSE) and mixed with a cell suspension obtained by trypsin-EDTA treatment of gonads of various ages and of the same or opposite sex. Whereas 11.5 dpc donor germ cells appeared unable to survive in the chimeric gonads obtained, about 76% of the CFSE-labeled female germ cells obtained from 12.5 dpc donor embryos (premeiotic germ cells) found viable within host ovarian tissues showed a meiotic nucleus. In contrast, a smaller number (about 19%) were in meiosis in chimeric testes. None or very few of donor male germ cells entered meiosis in testes or ovarian host tissues. Aggregation of meiotic 13.5 dpc female germ cells with testis tissues from 13.5 to 14.5 dpc embryos resulted in inhibition of meiotic progression and pyknosis in most donor germ cells. These results support the existence of a meiosis-preventing substance or a factor causing oocyte degeneration in the fetal mouse testis, but not of a meiosis-inducing substance in the fetal ovary.     

Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells that originate from primordial germ cells (PGCs) in the early embryo. Although spermatogonial stem cells arise from PGCs, it is not clear whether fetal male germ cells function as spermatogonial stem cells able to produce functional sperm. In the present study, we examined the timing and mechanisms of the commitment of fetal germ cells to differentiate into spermatogonial stem cells by transplantation techniques. Transplantation of fetal germ cells into the seminiferous tubules of adult testis showed that donor germ cells, at 14.5 days postcoitum (dpc), were able to initiate spermatogenesis in the adult recipient seminiferous tubules, whereas no germ cell differentiation was observed in the transplantation of 12.5-dpc germ cells. These results indicate that the commitment of fetal germ cells to differentiate into spermatogonial stem cells initiates between embryonic days 12.5 and 14.5. Furthermore, the results suggest the importance of the interaction between germ cells and somatic cells in the determination of fetal germ cell differentiation into spermatogonial stem cells, as normal spermatogenesis was observed when a 12.5-dpc whole gonad was transplanted into adult recipient testis. In addition, sperm obtained from the 12.5- dpc male gonadal explant had the ability to develop normally if injected into the cytoplasm of oocytes, indicating that normal development of fetal germ cells in fetal gonadal explant occurred in the adult testicular environment.     

TGFβ signaling in male germ cells regulates gonocyte quiescence and fertility in mice

During testis development, proliferation and death of gonocytes are highly regulated to establish a standard population of adult stem spermatogonia that maintain normal spermatogenesis. As Transforming Growth Factor beta (TGFbeta) can regulate proliferation and apoptosis, we investigated its expression and functions during testis development. We show that TGFbeta2 is only expressed in quiescent gonocytes and decreases gonocyte proliferation in vitro. To study the functions of TGFbeta2, we developed conditional mice that invalidate the TGFbeta receptor type II in germ cells. Most of the knock-out animals die during fetal life, but the surviving adults show a reduced pool of spermatogonial stem/progenitor cells and become sterile with time. Using an organ culture system mimicking in vivo development, we show higher proportions of proliferating and apoptotic gonocytes from 13.5 dpc until 1 dpp, suggesting a reduction of germinal quiescence in these animals. Conversely, a 24-hour TGFbeta2-treatment of explanted wild-type testes, isolated every day from 13.5 dpc until 1 dpp, increased the duration of quiescence.These data show that the TGFbeta signaling pathway plays a physiological role during testis development by acting directly as a negative regulator of the fetal and neonatal germ cell proliferation, and indicate that the TGFbeta signaling pathway might regulate the duration of germ cell quiescence and is necessary to maintain adult spermatogenesis.     

Pluripotential stem cells derived from migrating primordial germ cells

Pluripotent stem cells termed embryonic germ cells (EGCs) have earlier been derived from pre- and post-migrating mouse primordial germ cells (PGCs). We have recently obtained four EGC lines from migrating PGCs of 9.5 days post coitum (dpc) embryos. All lines were male with normal karyotype and showed properties that are similar to previously established EGC lines, including colony morphology, expression of alkaline phosphatase (AP), and expression of SSEA-1 antigen. The developmental potency of two of these lines was tested in vivo. They contributed to a range of tissues in fetal chimeras including heart, lung, kidney, intestine, muscle, brain and skin. We also examined the methylation status of the imprinted genes: Igf2r, p57Kip2, Lit1, H19 and Igf2. Igf2r, p57Kip2 and Lit1 were unmethylated in all analysed EGC lines, whereas H19 and Igf2 showed significant hypo-methylation in the 9.5 dpc EGC-1 line when compared to previously derived 11.5 dpc male EGC lines. This suggests that imprint erasure in the male germ line occurs prior to 9.5 dpc for all imprinted genes examined.     

FGF9 promotes survival of germ cells in the fetal testis

In addition to its role in somatic cell development in the testis, our data have revealed a role for Fgf9 in XY germ cell survival. In Fgf9-null mice, germ cells in the XY gonad decline in numbers after 11.5 days post coitum (dpc), while germ cell numbers in XX gonads are unaffected. We present evidence that germ cells resident in the XY gonad become dependent on FGF9 signaling between 10.5 dpc and 11.5 dpc, and that FGF9 directly promotes XY gonocyte survival after 11.5 dpc, independently from Sertoli cell differentiation. Furthermore, XY Fgf9-null gonads undergo true male-to-female sex reversal as they initiate but fail to maintain the male pathway and subsequently express markers of ovarian differentiation (Fst and Bmp2). By 14.5 dpc, these gonads contain germ cells that enter meiosis synchronously with ovarian gonocytes. FGF9 is necessary for 11.5 dpc XY gonocyte survival and is the earliest reported factor with a sex-specific role in regulating germ cell survival.     

Expression of DNA methyltransferase (Dnmt1) in testicular germ cells during development of mouse embryo

The DNA methylation pattern is reprogrammed in embryonic germ cells. In female germ cells, the short-form DNA methyltransferase Dnmt1, which is an alternative isoform specifically expressed in growing oocytes, plays a crucial role in maintaining imprinted genes. To evaluate the contribution of Dnmt1 to the DNA methylation in male germ cells, the expression profiles of Dnmt1 in embryonic gonocytes were investigated. We detected a significant expression of Dnmt1 in primordial germ cells in 12.5-14.5 day postcoitum (dpc) embryos. The expression of Dnmt1 was downregulated after 14.5 dpc after which almost no Dnmt1 was detected in gonocytes prepared from 18.5 dpc embryos. The short-form Dnmt1 also was not detected in the 16.5-18.5 dpc gonocytes. On the other hand, Dnmt1 was constantly detected in Sertoli cells at 12.5-18.5 dpc. The expression profiles of Dnmt1 were similar to that of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, suggesting that Dnmt1 was specifically expressed in the proliferating male germ cells. Inversely, genome-wide DNA methylation occurred after germ cell proliferation was arrested, when the Dnmt1 expression was downregulated. The present results indicate that not Dnmt1 but some other type of DNA methyltransferase contributes to the creation of DNA methylation patterns in male germ cells.     

Synthesis of glycoconjugates in mouse primordial germ cells

The synthesis of protein-bound carbohydrates has been studied in primordial germ cells (PGCs) and in somatic cells of 12.5 to 13.5-days-postcoitum (dpc) fetal mouse gonads. Both cell types were shown to synthesize asparagine-linked glycopeptides and glycosaminoglycans (GAGs). In addition, PGCs also synthesize lactosaminoglycans (LAGs) although in different proportions in female and male germ cells. Female PGCs, which at 13.5 dpc are entering meiosis, synthesize mainly LAGs, and minor amounts of hyaluronic acid (HA) and chondroitin sulfate (CS). Male germ cells, on the other hand, synthesize mainly CS. Furthermore, somatic cells of fetal gonads synthesize HA as the major class of GAGs. It is suggested that the activation of LAG synthesis in developing germ cells might be related to the beginning of meiosis. Moreover, we propose that HA synthesis might be developmentally regulated in somatic cells of the gonad, in order to regulate the establishment of specific interactions with germ cells.