Embryonic Testis Research Articles

Basic Helix-Loop-Helix Transcription Factor TCF21 Is a Downstream Target of the Male Sex Determining Gene SRY

The cascade of molecular events involved in mammalian sex determination has been shown to involve the SRY gene, but specific downstream events have eluded researchers for decades. The current study identifies one of the first direct downstream targets of the male sex determining factor SRY as the basic-helix-loop-helix (bHLH) transcription factor TCF21. SRY was found to bind to the Tcf21 promoter and activate gene expression. Mutagenesis of SRY/SOX9 response elements in the Tcf21 promoter eliminated the actions of SRY. SRY was found to directly associate with the Tcf21 promoter SRY/SOX9 response elements in vivo during fetal rat testis development. TCF21 was found to promote an in vitro sex reversal of embryonic ovarian cells to induce precursor Sertoli cell differentiation. TCF21 and SRY had similar effects on the in vitro sex reversal gonadal cell transcriptomes. Therefore, SRY acts directly on the Tcf21 promoter to in part initiate a cascade of events associated with Sertoli cell differentiation and embryonic testis development.

SRY Directly Regulates the Neurotrophin 3 Promoter During Male Sex Determination and Testis Development in Rats1

Neurotrophin 3 (Ntf3) is expressed in Sertoli cells and acts as a chemo-attractant for cell migration from the mesonephros into the developing testis, a process critical to the early morphological events of testis cord formation. The male sex-determining gene Sry initiates the process of testicular development. Sox9 is a key regulator of male sex determination and is directly regulated by SRY. Information on other downstream target genes of SRY is limited. The current study demonstrates an interaction of SRY with the Ntf3 promoter both in vitro and in vivo. The Ntf3 promoter in both rat and mouse contains at least one putative SRY binding site in the -0.6 kb promoter region. In a luciferase reporter assay system, both SRY and SOX9 stimulated the Ntf3 promoter in vitro through an interaction with this SRY-binding motif. In an immunoprecipitation-based pull-down assay, recombinant SRY protein bound the Ntf3 promoter fragment containing an intact SRY binding site, whereas the same protein did not interact with the fragment containing a mutated SRY motif. Specific antibodies against SRY were used in a chromatin immunoprecipitation (ChIP) assay of embryonic testis and were found to precipitate the Ntf3 promoter region. The SRY ChIP assay confirmed the direct interaction between SRY and the Ntf3 promoter in vivo during male sex determination. Observations suggest that SRY physically interacts with the Ntf3 promoter during male sex determination to coordinate cell migration in the testis to form testis cords.

Conditional ablation of Gata4 and Fog2 genes in mice reveals their distinct roles in mammalian sexual differentiation

Assembly of functioning testis and ovary requires a GATA4–FOG2 transcriptional complex. To define the separate roles for GATA4 and FOG2 proteins in sexual development of the testis we have ablated the corresponding genes in somatic gonadal cells. We have established that GATA4 is required for testis differentiation, for the expression of Dmrt1 gene, and for testis cord morphogenesis. While Sf1Cre-mediated excision of Gata4 permitted normal expression of most genes associated with embryonic testis development, gonadal loss of Fog2 resulted in an early partial block in male pathway and sex reversal. We have also determined that testis sexual differentiation is sensitive to the timing of GATA4 loss during embryogenesis. Our results now demonstrate that these two genes also have non-overlapping essential functions in testis development.

Two distinct origins for Leydig cell progenitors in the fetal testis

During the differentiation of the mammalian embryonic testis, two compartments are defined: the testis cords and the interstitium. The testis cords give rise to the adult seminiferous tubules, whereas steroidogenic Leydig cells and other less well characterized cell types differentiate in the interstitium (the space between testis cords). Although the process of testis cord formation is essential for male development, it is not entirely understood. It has been viewed as a Sertoli-cell driven process, but growing evidence suggests that interstitial cells play an essential role during testis formation. However, little is known about the origin of the interstitium or the molecular and cellular diversity within this early stromal compartment. To better understand the process of mammalian gonad differentiation, we have undertaken an analysis of developing interstitial/stromal cells in the early mouse testis and ovary. We have discovered molecular heterogeneity in the interstitium and have characterized new markers of distinct cell types in the gonad: MAFB, C-MAF, and VCAM1. Our results show that at least two distinct progenitor lineages give rise to the interstitial/stromal compartment of the gonad: the coelomic epithelium and specialized cells along the gonad–mesonephros border. We demonstrate that both these populations give rise to interstitial precursors that can differentiate into fetal Leydig cells. Our analysis also reveals that perivascular cells migrate into the gonad from the mesonephric border along with endothelial cells and that these vessel-associated cells likely represent an interstitial precursor lineage. This study highlights the cellular diversity of the interstitial cell population and suggests that complex cell–cell interactions among cells in the interstitium are involved in testis morphogenesis.

Xenoestrogen-Induced Sex Reversal of Chicken Embryonic Testes Involves Specific CpG Dinucleotide Demethylation at a DNA Hypomethylation Dip in CYP19A1/Aromatase Promoter.

Xenoestrogen-Induced Sex Reversal of Chicken Embryonic Testes Involves Specific CpG Dinucleotide Demethylation at a DNA Hypomethylation Dip in CYP19A1/Aromatase Promoter.

Focal Müllerian duct retention in male mice with constitutively activated β-catenin expression in the Müllerian duct mesenchyme

Müllerian-inhibiting substance (MIS), which is produced by fetal Sertoli cells shortly after commitment of the bipotential gonads to testicular differentiation, causes Müllerian duct (MD) regression. In the fetal female gonads, MIS is not expressed and the MDs will differentiate into the internal female reproductive tract. We have investigated whether dysregulated β-catenin activity affects MD regression by expressing a constitutively activated nuclear form of β-catenin in the MD mesenchyme. We show that constitutively activated (CA) β-catenin causes focal retention of MD tissue in the epididymides and vasa deferentia. In adult mutant mice, the retained MD tissues express α-smooth muscle actin and desmin, which are markers for uterine differentiation. MD retention inhibited the folding complexity of the developing epididymides and usually led to obstructive azoospermia by spermatoceles. The MDs of urogenital ridges from mutant female embryos showed less regression with added MIS in organ culture compared with control MDs when analyzed by whole mount in situ hybridization for Wnt7a as a marker for the MD epithelium. CA β-catenin did not appear to affect expression of either MIS in the embryonic testes or its type II receptor (AMHR2) in the MD mesenchyme nor did it inhibit pSmad1/5/8 nuclear accumulation, suggesting that dysregulated β-catenin must inhibit MD regression independently of MIS signaling. These studies suggest that dysregulated Wnt/β-catenin signaling in the MD mesenchyme might also be a contributing factor in persistent Müllerian duct syndrome, a form of male pseudohermaphroditism, and development of spermatoceles.

Activin A, a product of fetal Leydig cells, is a unique paracrine regulator of Sertoli cell proliferation and fetal testis cord expansion

Formation of tubular structures relies upon complex interactions between adjacent epithelium and mesenchyme. In the embryonic testes, dramatic compartmentalization leads to the formation of testis cords (epithelium) and the surrounding interstitium (mesenchyme). Sertoli cells, the epithelial cell type within testis cords, produce signaling molecules to orchestrate testis cord formation. The interstitial fetal Leydig cells, however, are thought only to masculinize the embryo and are not known to be involved in testis cord morphogenesis. Contrary to this notion, we have identified activin A, a member of the TGF-beta protein superfamily, as a product of the murine fetal Leydig cells that acts directly upon Sertoli cells to promote their proliferation during late embryogenesis. Genetic disruption of activin betaA, the gene encoding activin A, specifically in fetal Leydig cells resulted in a failure of fetal testis cord elongation and expansion due to decreased Sertoli cell proliferation. Conditional inactivation of Smad4, the central component of TGF-beta signaling, in Sertoli cells led to testis cord dysgenesis and proliferative defects similar to those of Leydig cell-specific activin betaA knockout testes. These results indicate that activin A is the major TGF-beta protein that acts directly on Sertoli cells. Testicular dysgenesis in activin betaA and Smad4 conditional knockout embryos persists into adulthood, leading to low sperm production and abnormal testicular histology. Our findings challenge the paradigm that fetal testis development is solely under the control of Sertoli cells, by uncovering an active and essential role of fetal Leydig cells during testis cord morphogenesis.

Changes in the tubular compartment of the testis of Gallus domesticus during development

1. The aim of the present study was to analyze histological and stereological changes in the tubular compartment in Gallus domesticus testes, as well as the variations in the number and size of their cells, from the start of morphological differentiation of the gonads (8-d chick-embryo) until the adult reproductive stage (28 weeks old). 2. In embryonic chick testes, the total volume occupied by the interstitial tissue is greater than that occupied by the tubular compartment, but in the post-hatched chick the total volume of tubular compartment exceeds that of the interstitial tissue. 3. From day 1 until 28 weeks of age, the seminiferous tubules increased in total volume, diameter, and epithelial height, which was directly related to the increase in the number of Sertoli and germ cells and the size of Sertoli cells. 4. In the testes of one-day- and 6-week-old chicks, Sertoli cells were the most abundant cell type in the seminiferous tubules due to hyperplasia, but in 28-week-old birds the germ cells were the most abundant cell type. Hypertrophy rather than hyperplasia of Sertoli cells appears to be responsible for the increase in the total volume of seminiferous tubules. 5. There are marked age-dependent changes in the tubular compartment of chick testes that help to understand the histological and stereological events occurring during normal development.

Fetal Leydig Cells: Progenitor Cell Maintenance and Differentiation

In most eutherian mammals, sexually dimorphic masculinization is established by androgen-producing fetal Leydig cells in the embryonic testis. Fetal Leydig cells, which lack expression of the testis-determining gene SRY, arise after the appearance of SRY-expressing Sertoli cells. Therefore, the appearance and differentiation of fetal Leydig cells are probably regulated by factors derived from Sertoli cells. Results from mouse genetic models have revealed that maintenance and differentiation of fetal Leydig cell population depends upon a balance between differentiation-promoting and differentiation-suppressing mechanisms. Although paracrine signaling via Sertoli cell-derived Hedgehog ligands is necessary and sufficient for fetal Leydig cell formation, cell-cell interaction via Notch signaling and intracellular transcription factors such as POD1 are implicated as suppressors of fetal Leydig cell differentiation. This review provides a model that summarizes the recent findings in fetal Leydig cell development.

321. HEDGEHOG SIGNALLING COMPONENTS IN DEVELOPING RAT TESTIS

Hedgehog (Hh) signalling regulates normal development of many tissues and is upregulated in some cancers. Mice which lack the testicular desert hedgehog (Dhh) ligand exhibit disrupted embryonic gonad formation and male infertility in adulthood. However, the roles and sites of Hedgehog (Hh) signalling activity in the developing rat testis are unknown. Transcripts encoding Hh pathway components in embryonic and juvenile rat testes were localised by in situ hybridization with DIG-labelled cRNA probes. On embryonic day (E) 17.5, Sertoli cells contained transcripts encoding Dhh and both gonocytes and Sertoli cells contained the Ptc2 and Smo receptor transcripts. The cytoplasmic regulators (fused [Fu], suppressor of fused [SuFu]) and the three Gli transcriptional mediators were also detected in gonocytes. On E21.5 and postnatal day 4, all transcripts were present in Sertoli cells, but not gonocytes. To test the function of Hh signalling at the onset of rodent spermatogenesis, the newborn (day 1) mouse testis was cultured in hanging drop cultures. Addition of the selective Hh signalling inhibitor, cyclopamine, yielded a significantly lower level of the Hh target, Gli1 mRNA, as measured by real time PCR compared to vehicle controls. This confirms Hh pathway activity and inhibition in this system. Meiotic markers, SYCP3 and Stra8, were consistently upregulated (n = 6) following cyclopamine addition, however the fold-change varied between experiments. Overall, the cellular expression data indicate that Hh signals are active in both embryonic and juvenile rodent testes. The finding that Hh genes analysed in this study are expressed in both Sertoli and germ cells shows that both cell types are potential Hh targets, depending on the developmental stage of testis. The upregulation of candidates for Dhh target genes in the juvenile mouse testis in vitro suggests that Hedgehog signaling downregulates the expression of meiotic genes in gonocytes via paracrine mechanisms.

Estrogenic in vitro assay on mouse embryonic Leydig cells

We and others have reported that mouse embryonic testes contain a subpopulation of somatic cells expressing estrogen receptor alpha (ERalpha). In order to provide evidence for a possible direct estrogen effect on mammalian testes from the early stage of their differentiation, here we devised a method for the in vitro culture of the ERalpha-expressing cells from 12.5 days post coitum mouse testes and their transfection with plasmids containing the classical estrogen responsive element (ERE) or the alternative estrogen AP-1 responsive element upstream of the luciferase reporter gene (ERE-Luc and AP-1-Luc). StAR immunopositivity of the most part of the ERalpha+ cells grown in culture and subjected to the estrogenic assay, allowed their identification as embryonic Leydig cells. Maximum induction of the ERE-Luc activity was achieved with 10 nM 17-beta estradiol (E2), from 1.7 to 3-fold in such cells and from 2.3 to 5.7-fold in MCF-7 cells used for comparison; the anti-estrogen ICI 182.780 abolished such effects. AP-1-Luc was less sensitive to E2 in both cell types (10 nM E2, 1.2 to 2.7-fold increase in embryonic Leydig cells; about 3-fold in MCF-7 cells) and the effect was not ICI-dependent. Eventually, we stimulated the transfected cells with various xenoestrogens such as lindane, bisphenol A or mono-(2-ethylhexyl) pthalate and with the phytoestrogen zeralenone obtaining evidence for ERE-Luc, but not AP-1-Luc stimulation in embryonic Leydig cells. These results represent evidence of functional ERalpha-dependent genomic pathways in embryonic Leydig cells and describe an in vitro assay suitable for evaluating the activity of putative estrogenic compounds on such cells.

Cyp26b1 Expression in Murine Sertoli Cells Is Required to Maintain Male Germ Cells in an Undifferentiated State during Embryogenesis

In mammals, germ cells within the developing gonad follow a sexually dimorphic pathway. Germ cells in the murine ovary enter meiotic prophase during embryogenesis, whereas germ cells in the embryonic testis arrest in G0 of mitotic cell cycle and do not enter meiosis until after birth. In mice, retinoic acid (RA) signaling has been implicated in controlling entry into meiosis in germ cells, as meiosis in male embryonic germ cells is blocked by the activity of a RA-catabolizing enzyme, CYP26B1. However, the mechanisms regulating mitotic arrest in male germ cells are not well understood. Cyp26b1 expression in the testes begins in somatic cells at embryonic day (E) 11.5, prior to mitotic arrest, and persists throughout fetal development. Here, we show that Sertoli cell-specific loss of CYP26B1 activity between E15.5 and E16.5, several days after germ cell sex determination, causes male germ cells to exit from G0, re-enter the mitotic cell cycle and initiate meiotic prophase. These results suggest that male germ cells retain the developmental potential to differentiate in meiosis until at least at E15.5. CYP26B1 in Sertoli cells acts as a masculinizing factor to arrest male germ cells in the G0 phase of the cell cycle and prevents them from entering meiosis, and thus is essential for the maintenance of the undifferentiated state of male germ cells during embryonic development.

Foxl2functions in sex determination and histogenesis throughout mouse ovary development

BackgroundPartial loss of function of the transcription factor FOXL2 leads to premature ovarian failure in women. In animal models, Foxl2 is required for maintenance, and possibly induction, of female sex determination independently of other critical genes, e.g., Rspo1. Here we report expression profiling of mouse ovaries that lack Foxl2 alone or in combination with Wnt4 or Kit/c-Kit.ResultsFollowing Foxl2 loss, early testis genes (including Inhbb, Dhh, and Sox9) and several novel ovarian genes were consistently dysregulated during embryonic development. In the absence of Foxl2, expression changes affecting a large fraction of pathways were opposite those observed in Wnt4-null ovaries, reinforcing the notion that these genes have complementary actions in ovary development. Loss of one copy of Foxl2 revealed strong gene dosage sensitivity, with molecular anomalies that were milder but resembled ovaries lacking both Foxl2 alleles. Furthermore, a Foxl2 transgene disrupted embryonic testis differentiation and increased the levels of key female markers.ConclusionThe results, including a comprehensive principal component analysis, 1) support the proposal of dose-dependent Foxl2 function and anti-testis action throughout ovary differentiation; and 2) identify candidate genes for roles in sex determination independent of FOXL2 (e.g., the transcription factors IRX3 and ZBTB7C) and in the generation of the ovarian reserve downstream of FOXL2 (e.g., the cadherin-domain protein CLSTN2 and the sphingomyelin synthase SGMS2). The gene inventory is a first step toward the identification of the full range of pathways with partly autonomous roles in ovary development, and thus provides a framework to analyze the genetic bases of female fertility.

Expression and evolutionary conservation of the tescalcin gene during development

The tescalcin gene ( Tesc) encodes an EF-hand calcium-binding protein that interacts with the sodium/hydrogen exchanger, NHE1. Previous studies indicated that Tesc was expressed in mouse embryonic testis, but not in ovary, during the critical period of testis and ovary determination. In this paper we compared the expression of Tesc in embryonic tissues of chicken and mouse. Tesc expression was sexually dimorphic in the embryonic gonads of both mouse and chicken. Tescalcin (TESC) was detected in both Sertoli cells and germ cells. In the embryonic brain of both mouse and chicken, Tesc was highly expressed in the nasal placode and in fibers extending from the olfactory epithelium to the primordial olfactory bulb. Tesc was expressed in the embryonic heart of both chicken and mouse. In mouse Tesc expression was also detected in embryonic adrenal. These studies indicate very specific expression of Tesc in various tissues in chicken and mouse during embryologic development, and conservation of Tesc expression in both species.

Testis cord differentiation after the sex determination stage is independent of Sox9 but fails in the combined absence of Sox9 and Sox8

Sox9 and Sox8 are transcription factors expressed in embryonic and postnatal Sertoli cells of the mouse testis. Sox9 inactivation prior to the sex determination stage leads to complete XY sex reversal. In contrast, there is normal embryonic testis development in Sox8 mutants which are initially fertile, but later develop progressive seminiferous tubule failure and infertility. To determine whether Sox9 is required for testis development after the initial steps of sex determination, we crossed Sox9 flox mice with an AMH-Cre transgenic line thereby completely deleting Sox9 in Sertoli cells by E14.0. Conditional Sox9 null mutants show normal embryonic testis development and are initially fertile, but, like Sox8 −/− mutants, become sterile from dysfunctional spermatogenesis at about 5 months. To see whether Sox8 may compensate for the absence of Sox9 during embryonic testis differentiation, we generated a Sox9 conditional knockout on a Sox8 mutant background. In the double mutants, differentiation of testis cords into seminiferous testis tubules ceases after P6 in the absence of one Sox8 allele, and after P0 in the absence of both Sox8 alleles, leading to complete primary infertility. Sox9,Sox8 double nullizygous testes show upregulation of early ovary-specific markers and downregulation of Sertoli intercellular junctions at E15.5. Their very low Amh levels still cause complete regression of the Müllerian duct but with reduced penetrance. This study shows that testis cord differentiation is independent of Sox9, and that concerted Sox9 and Sox8 function in post E14.0 Sertoli cells is essential for the maintenance of testicular function.

Identification of Hedgehog Signaling Outcomes in Mouse Testis Development Using a Hanging Drop-Culture System1

The Hedgehog (Hh) signaling pathway affects fetal testis growth. Recently, we described the dynamic cellular production of Hh signaling pathway components in juvenile and adult rodent testes. The Hh signaling is understood to regulate cord formation in the fetal testis, but minimal knowledge exists regarding how Hh signaling impacts the postnatal testis. To investigate this, we employed hanging drop cultures, which are used routinely in embryoid body formation. This approach has the advantage of using small media volume, and we examined its suitability for short-term culture of both murine embryonic gonads and adult testis tubules. The effects of cyclopamine, a specific Hh signaling inhibitor, were examined following culture of Embryonic Day 11.5 urogenital ridges (as control) and adult seminiferous tubule fragments for 24-48 h using histological, cell proliferation, and gene expression analyses. Cultured embryonic testes displayed generally normal cord structure, anti-Müllerian hormone (Amh) expression, and cell proliferation; known Hh target gene expression (Gli1, osteopontin, official symbol Spp1, and Amh) was altered in response to cyclopamine. Cultured adult tubules exhibited some loss of seminiferous epithelium organization over 48 h. Spermatogonia continued to proliferate, however, and no significant loss of viability was noted overall. Addition of cyclopamine significantly affected levels of Gli1, Igfbp6, Ccnd2 (cyclin D2), Ccnb1 (cyclin B1), Spp1, Kit, and Amh mRNAs; these genes have been shown previously to be expressed in Sertoli and germ cells. These novel results identify Hh target genes in the testis and demonstrate this signaling pathway likely affects cell survival and differentiation in the context of normal adult testis.

Testis Development, Fertility, and Survival in Ethanolamine Kinase 2-Deficient Mice

Ethanolamine kinase 2 (Eki2) was previously isolated from a differential expression screen designed to identify candidate genes involved in testis development and differentiation. In mouse, Eki2 is specifically up-regulated in Sertoli cells of the developing testis at the time of sex determination. Based on this expression profile, Eki2 was considered a good candidate testis-determining gene. To investigate a possible role of Eki2 in testis development, we have generated a mouse with targeted disruption of the Eki2 gene by using an EGFP replacement strategy. No abnormalities were detected in the Eki2-deficient mice with regard to embryonic and adult testis morphology, differentiation, function, or fertility. Furthermore, no significant differences were observed in litter sizes, pup mortality rates, or distribution of the sexes among the offspring. Ethanolamine kinases are involved in the biosynthesis of phosphatidylethanolamine, a major membrane phospholipid. Expression analysis indicates that the absence of an apparent phenotype in the Eki2-deficient mice may be due to compensation by Eki2-family members or the activation of an alternative pathway to generate phosphatidylethanolamine. Expression of EGFP in this mouse model enabled the isolation of gonad cell populations, providing a useful resource from which to obtain relatively pure early steroidogenic cells for further studies.

Insulin-Like Growth Factor 1 Is Expressed in Mouse Developing Testis and Regulates Somatic Cell Proliferation

Testicular development occurs prenatally in mammals. The developmental underlying mechanism is only partially understood. The aim of the present investigation was to study the expression of the gene coding for insulin-like growth factor 1 (Igf-1) and Igf-1 type 1 receptor (Igf-1r) and their respective proteins in mouse Sertoli and Leydig cells at gestation day 12 (E12)-E18. Moreover, we sought to determine the effect of IGF-1 on the proliferation of both cell types and to establish the signal transduction mechanism involved in the IGF-1 pathway. Transcripts for the Igf-1 and Igf-1r genes were found in Sertoli and Leydig cells from E12-E18. Highest IGF-1 and IGF-Ir protein expression levels were found in both cell types at E18. Exogenous IGF-1 administration increased Sertoli and Leydig cell proliferation at E14-E18 in vitro. Inhibition of the pathway mitogen-activated extracellular signal-regulated protein kinase (MEK) 1/2 with UO126 diminished the proliferation of the Sertoli and Leydig cells in vitro. We propose that IGF-1 and IGF-1r regulate Sertoli and Leydig cell proliferation through the MEK/extracellular-signal-regulated kinase (ERK) 1/2 signal transduction pathway, leading to development and growth of the mouse embryonic testis.

Sdmg1 is a conserved transmembrane protein associated with germ cell sex determination and germline-soma interactions in mice.

In mammals, the supporting cell lineage in an embryonic gonad communicates the sex-determining decision to various sexually dimorphic cell types in the developing embryo, including the germ cells. However, the molecular nature of the sex-determining signals that pass from the supporting cells to the germ cells is not well understood. We have identified a conserved transmembrane protein, Sdmg1, owing to its male-specific expression in mouse embryonic gonads. Sdmg1 is expressed in the Sertoli cells of embryonic testes from 12.5 dpc, and in granulosa cells of growing follicles in adult ovaries. In Sertoli cells, Sdmg1 is localised to endosomes, and knock-down of Sdmg1 in Sertoli cell lines causes mis-localisation of the secretory SNARE Stx2 and defects in membrane trafficking. Upregulation of Sdmg1 appears to be part of a larger programme of changes to membrane trafficking pathways in embryonic Sertoli cells, and perturbing secretion in male embryonic gonads in organ culture causes male-to-female germ cell sex reversal. These data suggest that changes that occur in the cell biology of embryonic Sertoli cells may facilitate the communication of male sex-determining decisions to the germ cells during embryonic development.

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.