XY Germ Cells Research Articles

Delayed male germ cell sex-specification permits transition into embryonal carcinoma cells with features of primed pluripotency.

Testicular teratomas result from anomalies in embryonic germ cell development. In 129 inbred mice, teratoma initiation coincides with germ cell sex-specific differentiation and the mitotic-meiotic switch: XX and XY germ cells repress pluripotency, XX germ cells initiate meiosis, and XY germ cells activate male-specific differentiation and mitotic arrest. Here, we report that expression of Nanos2, a gene that is crucial to male sex specification, is delayed in teratoma-susceptible germ cells. Decreased expression of Nanos2 was found to be due, in part, to the Nanos2 allele present in 129 mice. In teratoma-susceptible germ cells, diminished expression of genes downstream of Nanos2 disrupted processes that were crucial to male germ cell differentiation. Deficiency for Nanos2 increased teratoma incidence in 129 mice and induced developmental abnormalities associated with tumor initiation in teratoma-resistant germ cells. Finally, in the absence of commitment to the male germ cell fate, we discovered that a subpopulation of teratoma-susceptible germ cells transition into embryonal carcinoma (EC) cells with primed pluripotent features. We conclude that delayed male germ cell sex-specification facilitates the transformation of germ cells with naïve pluripotent features into primed pluripotent EC cells.

Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice.

The mechanism for sex determination in mammalian germ cells remains unclear. Here, we reconstitute the female sex determination in mouse germ cells invitro under a defined condition without the use of gonadal somatic cells. We show that retinoic acid (RA) and its key effector, STRA8, are not sufficient to induce the female germ-cell fate. In contrast, bone morphogenetic protein (BMP) and RA synergistically induce primordial germ cells (PGCs)/PGC-like cells (PGCLCs) derived from embryonic stem cells (ESCs) into fetal primary oocytes. The induction is characterized by entry into the meiotic prophase, occurs synchronously and recapitulates cytological and transcriptome progression invivo faithfully. Importantly, the female germ-cell induction necessitates a proper cellular competence-most typically, DNA demethylation of relevant genes-which is observed in appropriately propagated PGCs/PGCLCs, but not in PGCs/PGCLCs immediately after induction. This provides an explanation for the differential function of BMP signaling between PGC specification and female germ-cell induction. Our findings represent a framework for a comprehensive delineation of the sex-determination pathway in mammalian germ cells, including humans.

Cell adhesion molecules expression pattern indicates that somatic cells arbitrate gonadal sex of differentiating bipotential fetal mouse gonad

Unlike other organ anlagens, the primordial gonad is sexually bipotential in all animals. In mouse, the bipotential gonad differentiates into testis or ovary depending on the genetic sex (XY or XX) of the fetus. During gonad development cells segregate, depending on genetic sex, into distinct compartments: testis cords and interstitium form in XY gonad, and germ cell cysts and stroma in XX gonad. However, our knowledge of mechanisms governing gonadal sex differentiation remains very vague. Because it is known that adhesion molecules (CAMs) play a key role in organogenesis, we suspected that diversified expression of CAMs should also play a crucial role in gonad development. Using microarray analysis we identified 129 CAMs and factors regulating cell adhesion during sexual differentiation of mouse gonad. To identify genes expressed differentially in three cell lines in XY and XX gonads: i) supporting (Sertoli or follicular cells), ii) interstitial or stromal cells, and iii) germ cells, we used transgenic mice expressing EGFP reporter gene and FACS cell sorting. Although a large number of CAMs expressed ubiquitously, expression of certain genes was cell line- and genetic sex-specific. The sets of CAMs differentially expressed in supporting versus interstitial/stromal cells may be responsible for segregation of these two cell lines during gonadal development. There was also a significant difference in CAMs expression pattern between XY supporting (Sertoli) and XX supporting (follicular) cells but not between XY and XX germ cells. This indicates that differential CAMs expression pattern in the somatic cells but not in the germ line arbitrates structural organization of gonadal anlagen into testis or ovary.

Dazl is a target RNA suppressed by mammalian NANOS2 in sexually differentiating male germ cells.

Evolutionally conserved Nanos RNA-binding proteins play crucial roles in germ cell development. While a mammalian Nanos family protein, NANOS2, is required for sexual differentiation of male (XY) germ cells in mice, the underlying mechanisms and the identities of its target RNAs in vivo remain elusive. Using comprehensive microarray analysis and a bacterial artificial chromosome transgenic system, here we identify Dazl, a germ cell-specific gene encoding an RNA-binding protein implicated in translation, as a crucial target of NANOS2. Importantly, removal of the Dazl 3′-untranslated region in XY germ cells stabilizes the Dazl mRNA, resulting in elevated meiotic gene expression, abnormal resumption of the cell cycle and impaired processing-body formation, reminiscent of Nanos2-knockout phenotypes. Furthermore, our data suggest that NANOS2 acts as an antagonist of the DAZL protein. We propose a dual system of NANOS2-mediated suppression of Dazl expression as a pivotal molecular mechanism promoting sexual differentiation of XY germ cells.

Testicular oocytes in MRL/MpJ mice possess similar morphological, genetic, and functional characteristics to ovarian oocytes

In general, mammalian males produce only spermatozoa in their testes and females produce only oocytes in their ovaries. However, newborn MRL/MpJ male mice produce oocytes within their testes. In this study, we examined the initiation and progression of oogenesis in fetal and neonatal MRL/MpJ mouse testes and evaluated the characteristics of testicular oocytes. Germ cells with positive reactions to oogenesis markers such as NOBOX oogenesis homeobox and synaptonemal complex protein 3 were observed in the MRL/MpJ fetal testes on embryonic day 18.5. These fetal testicular oocytes possessed maternal-specific methylation patterns of histone and DNA. The level of DNA methylation was still low in postnatal testicular oocytes at day 14 after birth. Additionally, the postnatal testicular oocytes contained both X and Y chromosomes and had the ability to fuse with sperm. These results suggest that some XY germ cells in fetal testes of MRL/MpJ mice enter meiosis prematurely, undergo oogenesis, and differentiate into oocytes. In addition, MRL/MpJ testicular oocytes have the ability to carry on oogenesis before and shortly after birth until they obtain some of the morphological, epigenetic, and functional characteristics of oocytes.

SMAD2 and p38 signaling pathways act in concert to determine XY primordial germ cell fate in mice.

The sex of primordial germ cells (PGCs) is determined in developing gonads on the basis of cues from somatic cells. In XY gonads, sex-determining region Y (SRY) triggers fibroblast growth factor 9 (FGF9) expression in somatic cells. FGF signaling, together with downstream nodal/activin signaling, promotes male differentiation in XY germ cells by suppressing retinoic acid (RA)-dependent meiotic entry and inducing male-specific genes. However, the mechanism by which nodal/activin signaling regulates XY PGC fate is unknown. We uncovered the roles of SMAD2/3 and p38 MAPK, the putative downstream factors of nodal/activin signaling, in PGC sexual fate decision. We found that conditional deletion of Smad2, but not Smad3, from XY PGCs led to a loss of male-specific gene expression. Moreover, suppression of RA signaling did not rescue male-specific gene expression in Smad2-mutant testes, indicating that SMAD2 signaling promotes male differentiation in a RA-independent manner. By contrast, we found that p38 signaling has an important role in the suppression of RA signaling. The Smad2 deletion did not disrupt the p38 signaling pathway even though Nodal expression was significantly reduced, suggesting that p38 was not regulated by nodal signaling in XY PGCs. Additionally, the inhibition of p38 signaling in the Smad2-mutant testes severely impeded XY PGC differentiation and induced meiosis. In conclusion, we propose a model in which p38 and SMAD2 signaling coordinate to determine the sexual fate of XY PGCs.

Analysis of a novel gene, Sdgc, reveals sex chromosome-dependent differences of medaka germ cells prior to gonad formation.

In vertebrates that have been examined to date, the sexual identity of germ cells is determined by the sex of gonadal somatic cells. In the teleost fish medaka, a sex-determination gene on the Y chromosome, DMY/dmrt1bY, is expressed in gonadal somatic cells and regulates the sexual identity of germ cells. Here, we report a novel mechanism by which sex chromosomes cell-autonomously confer sexually different characters upon germ cells prior to gonad formation in a genetically sex-determined species. We have identified a novel gene, Sdgc (sex chromosome-dependent differential expression in germ cells), whose transcripts are highly enriched in early XY germ cells. Chimeric analysis revealed that sexually different expression of Sdgc is controlled in a germ cell-autonomous manner by the number of Y chromosomes. Unexpectedly, DMY/dmrt1bY was expressed in germ cells prior to gonad formation, but knockdown and overexpression of DMY/dmrt1bY did not affect Sdgc expression. We also found that XX and XY germ cells isolated before the onset of DMY/dmrt1bY expression in gonadal somatic cells behaved differently in vitro and were affected by Sdgc. Sdgc maps close to the sex-determination locus, and recombination around the two loci appears to be repressed. Our results provide important insights into the acquisition and plasticity of sexual differences at the cellular level even prior to the developmental stage of sex determination.

CYP26B1 promotes male germ cell differentiation by suppressing STRA8-dependent meiotic and STRA8-independent mitotic pathways

Germ cell sex is defined by factors derived from somatic cells. CYP26B1 is known to be a male sex-promoting factor that inactivates retinoic acid (RA) in somatic cells. In CYP26B1-null XY gonads, germ cells are exposed to a higher level of RA than in normal XY gonads and this activates Stra8 to induce meiosis while male-specific gene expression is suppressed. However, it is unknown whether meiotic entry by an elevated level of RA is responsible for the suppression of male-type gene expression. To address this question, we have generated Cyp26b1/Stra8 double knockout (dKO) embryos. We successfully suppressed the induction of meiosis in CYP26B1-null XY germ cells by removing the Stra8 gene. Concomitantly, we found that the male genetic program represented by the expression of NANOS2 and DNMT3L was totally rescued in about half of dKO germ cells, indicating that meiotic entry causes the suppression of male differentiation. However, half of the germ cells still failed to enter the appropriate male pathway in the dKO condition. Using microarray analyses together with immunohistochemistry, we found that KIT expression was accompanied by mitotic activation, but was canceled by inhibition of the RA signaling pathway. Taken together, we conclude that inhibition of RA is one of the essential factors to promote male germ cell differentiation, and that CYP26B1 suppresses two distinct genetic programs induced by RA: a Stra8-dependent meiotic pathway, and a Stra8-independent mitotic pathway.

NANOS2 promotes male germ cell development independent of meiosis suppression

NANOS2 is an RNA-binding protein essential for fetal male germ cell development. While we have shown that the function of NANOS2 is vital for suppressing meiosis in embryonic XY germ cells, it is still unknown whether NANOS2 plays other roles in the sexual differentiation of male germ cells. In this study, we addressed the issue by generating Nanos2/Stra8 double knockout (dKO) mice, whereby meiosis was prohibited in the double-mutant male germ cells. We found that the expression of male-specific genes, which was decreased in the Nanos2 mutant, was hardly recovered in the dKO embryo, suggesting that NANOS2 plays a role in male gene expression other than suppression of meiosis. To investigate the molecular events that may be controlled by NANOS2, we conducted a series of microarray analyses to search putative targets of NANOS2 that fulfilled 2 criteria: (1) increased expression in the Nanos2 mutant and (2) the mRNA associated with NANOS2. Interestingly, the genes predominantly expressed in undifferentiated primordial germ cells (PGCs) were significantly selected, implying the involvement of NANOS2 in the termination of the characteristics of PGCs. Furthermore, we showed that NANOS2 is required for the maintenance of mitotic quiescence, but not for the initiation of the quiescence in fetal male germ cells. These results suggest that NANOS2 is not merely a suppressor of meiosis, but instead plays pivotal roles in the sexual differentiation of male germ cells.

Identification of Novel Markers of Mouse Fetal Ovary Development

In contrast to the developing testis, molecular pathways driving fetal ovarian development have been difficult to characterise. To date no single master regulator of ovarian development has been identified that would be considered the female equivalent of Sry. Using a genomic approach we identified a number of novel protein-coding as well as non-coding genes that were detectable at higher levels in the ovary compared to testis during early mouse gonad development. We were able to cluster these ovarian genes into different temporal expression categories. Of note, Lrrc34 and AK015184 were detected in XX but not XY germ cells before the onset of sex-specific germ cell differentiation marked by entry into meiosis in an ovary and mitotic arrest in a testis. We also defined distinct spatial expression domains of somatic cell genes in the developing ovary. Our data expands the set of markers of early mouse ovary differentiation and identifies a classification of early ovarian genes, thus providing additional avenues with which to dissect this process.

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.

Germ cell pluripotency, premature differentiation and susceptibility to testicular teratomas in mice

Testicular teratomas result from anomalies in germ cell development during embryogenesis. In the 129 family of inbred strains of mice, teratomas initiate around embryonic day (E) 13.5 during the same developmental period in which female germ cells initiate meiosis and male germ cells enter mitotic arrest. Here, we report that three germ cell developmental abnormalities, namely continued proliferation, retention of pluripotency, and premature induction of differentiation, associate with teratoma susceptibility. Using mouse strains with low versus high teratoma incidence (129 versus 129-Chr19(MOLF/Ei)), and resistant to teratoma formation (FVB), we found that germ cell proliferation and expression of the pluripotency factor Nanog at a specific time point, E15.5, were directly related with increased tumor risk. Additionally, we discovered that genes expressed in pre-meiotic embryonic female and adult male germ cells, including cyclin D1 (Ccnd1) and stimulated by retinoic acid 8 (Stra8), were prematurely expressed in teratoma-susceptible germ cells and, in rare instances, induced entry into meiosis. As with Nanog, expression of differentiation-associated factors at a specific time point, E15.5, increased with tumor risk. Furthermore, Nanog and Ccnd1, genes with known roles in testicular cancer risk and tumorigenesis, respectively, were co-expressed in teratoma-susceptible germ cells and tumor stem cells, suggesting that retention of pluripotency and premature germ cell differentiation both contribute to tumorigenesis. Importantly, Stra8-deficient mice had an 88% decrease in teratoma incidence, providing direct evidence that premature initiation of the meiotic program contributes to tumorigenesis. These results show that deregulation of the mitotic-meiotic switch in XY germ cells contributes to teratoma initiation.

What a difference a day makes! The contribution of intrinsic FGF9 signalling to germline masculinisation

Although the key discovery of sex-determining region Y (SRY) 20 years ago as the essential sex-determining gene emerged from studies of sex-reversed humans,1, 2 most of our knowledge of how male and female germ cells diverge after sex determination comes from studies of mice. This is demonstrated in a recent publication from Bowles and colleagues in Developmental Cell, which documents the functional importance of fibroblast growth factor 9 (FGF9) signalling in murine fetal germ cells.3 The study reveals events immediately following sex determination which influence how germline cells in the testis respond to FGF9 in a manner that reinforces the process of masculinisation. The picture that is coming into focus is of a signalling network which mediates the correct timing of progressive changes, thereby ensuring that germ cells and the somatic cells which create their niche develop together. Retinoic acid (RA) produced by the mesonephros adjacent to the fetal gonad has recently been identified as the key stimulant for fetal germ cell entry into meiosis: its degradation by the Cyp26b1 enzyme within the testis ensures that most male germ cells continue mitosis and do not enter cell cycle arrest until postnatal processes trigger the first wave of spermatogenesis.4, 5 The additional and antagonistic involvement of FGF9 in driving male germline development was discerned by Bowles and colleagues from several experimental observations of germ cells in Cyp26b1-null testes. These XY germ cells are exposed to high levels of RA but do not fully adopt the phenotype of their female counterparts. Transcripts encoding meiotic markers that are usually upregulated in XX germ cells are only partially increased in XY Cyp26b1-null germ cells, and quite remarkably, the levels of the pluripotency markers Oct4 and Sox2 persist as usual for male germline cells. This provided a clue to the existence of another factor which sustained male pathway development, and FGF9 was an obvious candidate considering that previous studies had shown how its genetic deletion from mice caused male to female sex reversal.6 A remarkable observation reported in this manuscript was the identification in isolated embryonic day (E) 11.5 germ cells of a transcript encoding a fibroblast growth factor receptor isoform that binds FGF9 with high affinity. This FGFR2-IIIc transcript was characterized through reverse transcription-PCR. The authors have deduced, by extrapolation from previous publications, that this highlights a switch in receptor isoform synthesis between E10.5 germ cells7 and E11.5, the time frame identified for germline gender commitment in the testis.8 This isoform switch, which is expected to render the cells highly responsive to FGF9, is mediated by alternative mRNA splicing which alters the structure of the receptors' extracellular domain. Isoform switching in FGF receptors is a well-documented phenomenon that is known to trigger changes over a similar, short time span in other developmental processes, is linked with oncogenic changes and can be induced by exposure to other FGF ligands (e.g., Ref. 9). Identification of the local factor(s) which mediate this change in transcript processing within the developing testis will enhance our understanding of the processes of germline masculinisation. The results of this study have reinforced the knowledge that male and female germline developments are mutually exclusive. The authors have shown that FGF9 actions are antagonistic to, and independent of, RA signalling. FGF9 is initially present in both male and female gonads but it is greatly elevated in the testis by E11.5. The addition of FGF9 to urogenital ridge cultures did not alter Cyp26b1 transcript levels, nor did the addition of RA or absence of Cyp26b1 diminish FGF9 mRNA levels. However, the exposure of E11.5 XX urogenital ridges to FGF9 does diminish synthesis of the direct RA target, Stra8, and this appears to be by diminishing the responsiveness of XX germline cells to RA. As the field of in vitro germline genesis progresses in parallel, information about the pathway by which FGF9 drives male fate may offer the potential for efficiencies and gender selectivity in gamete derivation from embryonic and induced pluripotent stem cells. Similarly, the importance of learning how pluripotency is sustained is of clear importance to testicular cancer and may even be applicable to harnessing spermatogonial stem cells as a potential source of therapeutic materials. The study by Bowles, Koopman and colleagues8 represents another piece in the puzzle of sex determination which has recently progressed from being focussed on somatic cell events to one in which germline events are now being delineated. The use of mice to understand the fundamental events which shape our genetic material is essential, due to the ethical concerns and practical restrictions that limit work on relevant human samples. What do we know about FGF9 in human gonad biology? The study presented here suggests clear starting points for learning how diseases arising from intrinsic germline defects may emerge due to disregulation of FGF signalling.

A Male-Specific Role for p38 Mitogen-Activated Protein Kinase in Germ Cell Sex Differentiation in Mice1

Germ cell sex differentiation in the mouse embryo is denoted by meiosis entry in females and mitotic arrest in males. Because p38 mitogen-activated protein kinase (MAPK) signaling initiates mitotic arrest in other differentiating cell types, we investigated its potential role in XY germ cell differentiation in mice. We report that p38 MAPK is phosphorylated and therefore activated only in XY germ cells around the time of sex differentiation. Quantitative RT-PCR analysis showed that 14 known targets of p38 MAPK signaling are expressed in the embryonic gonads at this time and that five of these targets (Mapkapk5, Max, Myc, Hbp1, and Cebpa) have expression profiles similar to that of activated p38 MAPK. Inhibition of p38 MAPK signaling in XY germ cells ex vivo reduced expression of the pluripotency marker POU5F1 and increased the expression of Stra8 and SYCP3, premeiosis and meiosis markers, respectively, to levels approaching those observed in XX germ cells. These data suggest that p38 MAPK signaling antagonizes entry into meiosis in XY germ cells, instead directing them toward mitotic quiescence and a spermatogenic fate.

Transplanted XY germ cells produce spermatozoa in testes of XXY mice*

XXY mouse has been characterized as an experimental model for men with Klinefelter's syndrome (XXY male phenotype). To test whether donor XY germ cells could proliferate and differentiate in the XXY testicular environment, donor testicular cells from adult (2-3 months old) and immature (10 days old) XY green fluorescence protein (GFP) transgenic mice were transplanted into the seminiferous tubules of adult (4-7 months old) and young (6 weeks old) XXY recipient mice respectively. Twelve weeks after transplantation, GFP positive spermatogonia were found in 21.74% (five out of 23) of adult XXY recipients who received adult donor cells. The GFP positive segments of seminiferous tubules were observed in 44.44% (four out of nine) young XXY recipients who received donor cells from 10 days old GFP mice. We found using immunohistochemistry and cell morphology that donor-derived GFP positive germ cells were spermatogonia, spermatocytes, round spermatids and spermatozoa in some of the seminiferous tubules of young XXY recipient mice. The results demonstrated that the donor XY germ cells were able to qualitatively complete spermatogenesis in some of the seminiferous tubules of XXY mice.

Retinoblastoma 1 Protein Modulates XY Germ Cell Entry into G1/G0 Arrest During Fetal Development in Mice1

During mouse germ cell development, the first sign of sex differentiation occurs when XY germ cells enter G(1)/G(0) arrest from 12.5 days postcoitum (dpc). Retinoblastoma 1 (RB1), a potent cell cycle regulator, was investigated in XY germ cell arrest by studying germ cell proliferation in Rb1(-/-) mutant mouse embryos. Because mice homozygous for the Rb1 deletion die in utero around 14.5 dpc, we used ex vivo culture techniques to allow analysis of developing gonads to 16.5 dpc. In Rb1(-/-) gonads, we observed normal somatic cell development, assessed by immunofluorescence for markers HSD3B1 and anti-Müllerian hormone. However, at 14.5 dpc, when wild-type XY germ cells had arrested, we could detect actively proliferating germ cells using the proliferation markers MKI67, pHH3, and bromodeoxyuridine incorporation. The increased proliferation was reflected with a trend of increased germ cell number and expression of germ cell markers Ddx4 and Pou5f1 in the Rb1(-/-) testes. By 16.5 dpc, this phenotype was resolved such that the entire germ cell population had entered G(1)/G(0) arrest, although the total germ cell number remained elevated. At each stage analyzed, we saw no increase in expression of RB1 family members Rbl1 and Rbl2 in the Rb1(-/-) testes, but we saw a significant increase of cyclin-dependent kinase (CDK) inhibitor Cdkn1b and Cdkn2b expression. We conclude that Rb1 is required for correct germ cell entry into G(1)/G(0) arrest in the wild-type gonad at 14.5 dpc, but in its absence, upregulation of other cell cycle suppressors, including Cdkn1b and Cdkn2b, can induce delayed germ cell arrest.

Cell cycle analysis of fetal germ cells during sex differentiation in mice

Background information. Primordial germ cells in developing male and female gonads are responsive to somatic cell cues that direct their sex-specific differentiation into functional gametes. The first divergence of the male and female pathways is a change in cell cycle state observed from 12.5 dpc (days post coitum) in mice. At this time XY and XX germ cells cease mitotic division and enter G1/G0 arrest and meiosis prophase I respectively. Aberrant cell cycle regulation at this time can lead to disrupted ovarian development, germ cell apoptosis, reduced fertility and/or the formation of germ cell tumours.Results. In order to unravel the mechanisms utilized by germ cells to achieve and maintain the correct cell cycle states, we analysed the expression of a large number of cell cycle genes in purified germ cells across the crucial time of sex differentiation. Our results revealed common signalling for both XX and XY germ cell survival involving calcium signalling. A robust mechanism for apoptosis and checkpoint control was observed in XY germ cells, characterized by p53 and Atm (ataxia telangiectasia mutated) expression. Additionally, a member of the retinoblastoma family and p21 were identified, linking these factors to XY germ cell G1/G0 arrest. Lastly, in XX germ cells we observed a down-regulation of genes involved in both G1- and G2-phases of the cell cycle consistent with their entry into meiosis.Conclusion. The present study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors warranting further investigation in order to understand cases of aberrant cell cycle control in these specialized cells.

BAX-mediated cell death affects early germ cell loss and incidence of testicular teratomas in Dnd1Ter/Ter mice

A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1Ter/Ter) results in a significant early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129Sv/J background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost or what factors directly control the strain-specific phenotype variation. To determine the mechanism underlying early PGC loss we crossed Dnd1Ter/Ter embryos to a Bax-null background and found that germ cells were partially rescued. Surprisingly, on a mixed genetic background, rescued male germ cells also generated fully developed teratomas at a high rate. Double-mutant females on a mixed background did not develop teratomas, but were fertile and produced viable off-spring. However, when Dnd1Ter/Ter XX germ cells developed in a testicular environment they gave rise to the same neoplastic clusters as mutant XY germ cells in a testis. We conclude that BAX-mediated apoptosis plays a role in early germ cell loss and protects from testicular teratoma formation on a mixed genetic background.

Meiosis Onset Is Postponed to Postnatal Stages during Ovotestis Development in Female Moles

In mammals, germ cells are important both during development and for the function of female gonads, whereas male gonads may develop in the absence of germ cells. The gonads of female moles (genus Talpa) develop according to a testis-like pattern which results in the formation of ovotestes. In this paper, we studied the expression pattern of several pre-meiotic and meiotic germ cell markers, in order to establish the precise time of meiosis onset in the mole species T. occidentalis, and to investigate the location and possible role of germ cells in ovotestis organogenesis. Our results evidenced that: (1) the asymmetrical distribution of primordial germ cells, which concentrate in the cortex of the XX gonad, is brought about by germ cell depletion from the medulla between the s5a and s5b stages, (2) XX germ cells enter meiosis postnatally, which is quite exceptional among eutherian mammals, and (3) XX but not XY germ cells of moles express DMRT1 during premeiotic stages of development, an expression pattern not described previously in vertebrates.

Dazl deficiency leads to embryonic arrest of germ cell development in XY C57BL/6 mice

Genes of the DAZ family play critical roles in germ cell development in mammals and other animals. In mice, Dazl mRNA is first observed at embryonic day 11.5 (E11.5), but previous studies using Dazl-deficient mice of mixed genetic background have largely emphasized postnatal spermatogenic defects. Using an inbred C57BL/6 background, we show that Dazl is required for embryonic development and survival of XY germ cells. By E14.5, expression of germ cell markers (Mvh, Oct4, Dppa3/Stella, GCNA and MVH protein) was reduced in XY Dazl−/− gonads. By E15.5, most remaining germ cells in XY Dazl−/− embryos exhibited apoptotic morphology, and XY Dazl−/− gonads contained increased numbers of TUNEL-positive cells. The rare XY Dazl−/− germ cells that persisted until birth maintained a nuclear morphology that resembled that of wildtype germ cells at E12.5–E13.5, a critical developmental period when XY germ cells lose pluripotency and commit to a spermatogonial fate. We propose that Dazl is required as early as E12.5–E13.5, shortly after its expression is first detected, and that inbred Dazl−/− mice of C57BL/6 background provide a reproducible standard for exploring Dazl's roles in embryonic germ cell development.