Mouse Testis Development Research Articles

Germ cells influence cord formation and Leydig cell gene expression during mouse testis development.

It is widely accepted that, during the development of testes in the mammalian embryo, male germ cells are influenced by signals from the surrounding somatic cells, but not vice versa, so that germ cells are dispensable for the formation of testes. We now demonstrate that development of the mouse fetal testis is compromised in the absence of germ cells. Using two- and three-dimensional imaging techniques, we reveal that W(e)/W(e) mutant testes devoid of germ cells have misshapen and poorly organized cords. We also found that mutant gonads have fewer Sertoli cells than normal and that the Leydig cells express key markers at higher than normal levels. These observations point to the existence of germ cell-derived signals that directly or indirectly affect the Sertoli and Leydig cell populations, and provide a new paradigm for the organogenesis of the mammalian testes.

Effects of silver nanoparticles on neonatal testis development in mice.

BackgroundMetal nanoparticles (MNPs) play an important role in consumer products. An increasing use of MNPs has raised concerns about potential risks for human health. Therefore, in vivo tests of MNPs are urgently required. Using mice as a model animal, the aim of the present study was designed to investigate the effect of biologically synthesized silver nanoparticles (AgNPs) on spermatogenesis in neonatal mice.MethodsAgNPs were synthesized using Bacillus funiculus. The prepared nanoparticles were characterized using various analytical techniques such as UV–visible spectroscopy, X-ray diffraction, Fourier transform-infrared spectroscopy, and transmission electron microscopy. The prepared AgNPs were used to investigate testis development in neonatal mice. Institute of Cancer Research neonatal male mice were used in all experiments and were treated with different doses (0, 1, and 5 mg/kg) of AgNPs five times (interval of 3 days from postnatal day [PND] 8–21) by abdominal subcutaneous injection.ResultsThe results showed that the sperm abnormalities such as quality and quantity were significantly increased by the synthesized AgNPs. The diameter of the convoluted tubules shrank significantly in mice treated with AgNPs on PND28 and PND42. The results of reverse transcription-quantitative polymerase chain reaction indicated that the E1f1ay, Gsta4, and Fdx1 genes were up-regulated, and the Amh, Cx43, and Claudin-11 genes were down-regulated in response to AgNPs exposure on PND28; however, these genes recovered at PND60. AgNPs had no effect on the recombination levels of chromosomes in germ cells.ConclusionThese results demonstrated the adverse effects of AgNPs on the male reproductive tract, particularly spermatogenesis and the quality of sperm. This study suggests that the development of nanomaterials should be safer and non-toxic to the living organisms and the potential reprotoxicity of AgNPs should be investigated more carefully.

Expression of CCM2 and CCM3 during mouse gonadogenesis.

Three cerebral cavernous malformation (CCM) proteins, CCM1, CCM2, and CCM3, regulate cell-cell adhesion, cell shape and polarity, and most likely cell adhesion to extracellular matrix. Recently, CCM2 and CCM3 are known to be expressed in control and varicocele-induced rat testes, but little is known about these proteins during gonadogenesis. This led us to study the CCM proteins during the mouse gonadogenesis. Neonatal (PND 0), postnatal, and adult mice testes and ovaries were obtained from mice. CCM2 and CCM3 expression were analyzed during mouse testicular and ovarian development by immunohistochemistry and quantitative real-time PCR. The results showed that in both sexes, Ccm2 and Ccm3 mRNA and protein were first detectable after gonadogenesis when the gonads were well differentiated and remained present until the adult stage. In the testis, CCM2 and CCM3 expression were restricted to the nuclei of Sertoli cells, suggesting a conserved role in testicular differentiation. In the ovary, the CCM2 and CCM3 proteins were localized in the cytoplasm of oocytes, suggesting an unexpected role during oogenesis. Quantitative real-time PCR (qRT-PCR) results showed that expression of Ccm2 and Ccm3 genes could play a role in the regulation of mouse gonadogenesis translational activation upon testicular and ovarian development. The localization of CCM2 and CCM3 proteins show their different functions for CCM2 and CCM3 which may have important roles in testicular and ovarian differentiation. In conclusion, CCM2 and CCM3 may be involved in establishing the differential expression pattern in developing mouse testis and ovary.

Expression profiling of long noncoding RNAs in neonatal and adult mouse testis.

In recent years, advancements in genome-wide analyses of the mammalian transcriptome have revealed that long noncoding RNAs (lncRNAs) is pervasively transcribed in the genome and an increasing number of studies have demonstrated lncRNAs as a new class of regulatory molecules are involved in mammalian development (Carninci et al. (2005); Fatica and Bozzoni (2014)), but very few studies have been conducted on the potential roles of lncRNAs in mammalian testis development. To get insights into the expression patterns of lncRNA during mouse testis development, we investigated the lncRNAs expression profiles of neonatal and adult mouse testes using microarray platform and related results have been published (Sun et al., PLoS One 8 (2013) e75750.). Here, we describe in detail the experimental system, methods and validation for the generation of the microarray data associated with our recent publication (Sun et al., PLoS One 8 (2013) e75750.). Data have been deposited to the Gene Expression Omnibus (GEO) database repository with the dataset identifier GSE43442.

Expression of Dickkopf-like1 protein (Dkkl1) in mouse testis

Objective To investigate the expression and the distribution of Dickkopf-like1 (Dkkl1) protein during the development of mouse testis. Methods Testes cDNA samples from BALB/c mice in different postnatal days were hybridized with mouse whole genome affymetrix chip to screen the spermatogenesis-related genes. The characteristics of the selected genes were analyzed by various bioinformatics tools. The mRNA expression of Dkkl1 at different stages of testis development and different tissues in mouse were analyzed by RT-PCR. The protein localization of Dkkl1 in mouse testis was assesed by immunohistochemistry. Results By analyzing the gene chip signals of mouse testis aged 4 d, 9 d, 18 d, 35 d, 54 d and 6 months, Dkkl1 was identified with a differential expression in the developmental stages of testis. RT-PCR analysis showed that the expression of Dkkl1 mRNA was firstly detected on 15 d testis tissue and gradually upregulated during the testis developing to the adult stage. The Dkkl1 protein was predominantly located in spermatocytes and round spermatids in mouse testis. Conclusion The expression of Dkkl1 is gradually upregulated during the development of mouse testis and corresponds to the mouse spermatogenesis. It may play a critical role in male mammalian spermatogenesis.

Stage-specific signaling pathways during murine testis development and spermatogenesis: A pathway-based analysis to quantify developmental dynamics.

Shifting the field of developmental toxicology toward evaluation of pathway perturbation requires a quantitative definition of normal developmental dynamics. This project examined a publicly available dataset to quantify pathway dynamics during testicular development and spermatogenesis and anchor toxicant-perturbed pathways within the context of normal development. Genes significantly changed throughout testis development in mice were clustered by their direction of change using K-means clustering. Gene Ontology terms enriched among each cluster were identified using MAPPfinder. Temporal pathway dynamics of enriched terms were quantified based on average expression intensity for all genes associated with a given term. This analysis captured processes that drive development, including the peak in steroidogenesis known to occur around gestational day 16.5 and the increase in meiosis and spermatogenesis-related pathways during the first wave of spermatogenesis. Our analysis quantifies dynamics of pathways vulnerable to toxicants and provides a framework for quantifying perturbation of these pathways.

Expression of histone H3 lysine 4 methylation and its demethylases in the developing mouse testis.

Histone H3 lysine 4 methylation (H3K4me) is an epigenetic modification associated with gene activation and is dynamically regulated by histone methylases and demethylases. To date, the expression patterns of H3K4me and its demethylases in the developing testis remain unclear. The present study was designed to detect the expression of H3K4me1/2/3 and its demethylases LSD1, RBP2 and SMCX in 21-, 40- and 60-day-old mouse testes by using immunohistochemistry, quantitative real-time polymerase chain reaction (PCR) and Western blot. The immunohistochemical results demonstrated that the expression patterns of the same protein were similar in testes at different ages and that the positive staining cell types were mainly Leydig cells, type A and B spermatogonia, leptotene spermatocytes and spermatids for H3K4me1/2/3, Leydig cells, type A spermatogonia, zygotene and pachytene spermatocytes, spermatids, and Sertoli cells for LSD1 and type A and B spermatogonia for RBP2. Immunostaining for SMCX was not detected in testes. Quantitative real-time PCR and Western blot showed that the amounts of LSD1, RPB2 and SMCX mRNA and protein were age-dependent, were significantly reduced with increasing age and exhibited a negative correlation with the protein levels of H3K4me1/2/3. Thus, H3K4me, which is modified by its demethylases, probably plays a role in male spermatogenesis and testis development.

Cellular microenvironment dictates androgen production by murine fetal Leydig cells in primary culture.

Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3-5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture.

Identification and validation of suitable reference genes for RT-qPCR analysis in mouse testis development.

RT-qPCR is a commonly used method for evaluating gene expression; however, its accuracy and reliability are dependent upon the choice of appropriate reference gene(s), and there is limited information available on suitable reference gene(s) that can be used in mouse testis at different stages. In this study, using the RT-qPCR method, we investigated the expression variations of six reference genes representing different functional classes (Actb, Gapdh, Ppia, Tbp, Rps29, Hprt1) in mice testis during embryonic and postnatal development. The expression stabilities of putative reference genes were evaluated using five algorithms: geNorm, NormFinder, Bestkeeper, the comparative delta C(t) method and integrated tool RefFinder. Analysis of the results showed that Ppia, Gapdh and Actb were identified as the most stable genes and the geometric mean of Ppia, Gapdh and Actb constitutes an appropriate normalization factor for gene expression studies. The mRNA expression of AT1 as a test gene of interest varied depending upon which of the reference gene(s) was used as an internal control(s). This study suggested that Ppia, Gapdh and Actb are suitable reference genes among the six genes used for RT-qPCR normalization and provide crucial information for transcriptional analyses in future studies of gene expression in the developing mouse testis.

Epigenetic Regulation of Sox30 Is Associated with Testis Development in Mice

DNA methylation is involved in tissue-specific and developmentally regulated gene expression. Here, we screened a novel methylation gene Sox30, whose methylation might contribute to its regulation and testis development in mice. Sox30 is a member of Sox transcription factors, and is considered to be involved in spermatogonial differentiation and spermatogenesis. However, the precise function and regulatory expression pattern remain unclear. In the present study, we found that Sox30 is highly expressed in adult testes but not in ovaries. Sox30 expression begins in early development, and in the testes, it is specifically increased coincidentally with development until adulthood. Moreover, Sox30 is expressed not only in testis germ cells, but also in sertoli cells. Sox30 is hypo-methylated in testis, epididymis and lung of adult mice, in which Sox30 is expressed. By contrast, Sox30 is hypermethylated in ovary, heart, brain, liver, kidney, spleen, pancreas, muscle, intestine, pituitary gland, blood and hippocampus of adult mice, in which the Sox30 is absent. Importantly, decreased methylation at CpG islands of Sox30 is observed in mouse developmental testes after birth, which is associated with enhanced Sox30 expression. However, the hypermethylated status of Sox30 is maintained in ovaries that does not express Sox30 during this period. Further, following demethylation treatment using 5-aza-dC, Sox30 expression is restored in GC2, TM3 and TM4 cell lines. This observation convincingly confirms that methylation really contributes to Sox30 silencing. In summary, we show that Sox30 expression is under the control of DNA methylation status, and this expression pattern is associated with testis development in mice.

The expression pattern of PARP-1 and PARP-2 in the developing and adult mouse testis

Although the importance of the PARP family members in the adult testis has already been acknowledged, their expression in the developing testis has not been addressed. We performed immunohistochemistry by using PARP-1 and PARP-2 antibodies on the developing mouse testis at embryonic day (E) 15.5, E17.5, postnatal day (PN) 0, PN3, PN9, PN20 and adult. Our results showed that at embryonic and early postnatal days, the expression of PARP-1 was in the nuclei of gonocytes and spermatogonia. PARP-1 was positive in interstitial cells with nuclear localization at all studied ages. At embryonic and early postnatal days, the expression of PARP-2 was in the cytoplasm of gonocytes and spermatogonia. During the progress of spermatogenesis, PARP-2 was localized in the cytoplasm of pre-leptotene spermatocytes on PN9, in the cytoplasm of pachytene spermatocytes on PN15 and in the cytoplasm of round spermatids on PN20. In the adult, PARP-2 staining can still be observed in the cytoplasm of spermatogonia, but to a much lesser degree than in the round and elongating spermatids. For all the studied ages, PARP-2 was positive in Sertoli cells and interstitial cells with cytoplasmic localization. Our results indicate that PARP proteins are present in germ and somatic cells during testis development in mice.

A site-specific, single-copy transgenesis strategy to identify 5' regulatory sequences of the mouse testis-determining gene Sry.

The Y-chromosomal gene SRY acts as the primary trigger for male sex determination in mammalian embryos. Correct regulation of SRY is critical: aberrant timing or level of Sry expression is known to disrupt testis development in mice and we hypothesize that mutations that affect regulation of human SRY may account for some of the many cases of XY gonadal dysgenesis that currently remain unexplained. However, the cis-sequences involved in regulation of Sry have not been identified, precluding a test of this hypothesis. Here, we used a transgenic mouse approach aimed at identifying mouse Sry 5′ flanking regulatory sequences within 8 kb of the Sry transcription start site (TSS). To avoid problems associated with conventional pronuclear injection of transgenes, we used a published strategy designed to yield single-copy transgene integration at a defined, transcriptionally open, autosomal locus, Col1a1. None of the Sry transgenes tested was expressed at levels compatible with activation of Sox9 or XX sex reversal. Our findings indicate either that the Col1a1 locus does not provide an appropriate context for the correct expression of Sry transgenes, or that the cis-sequences required for Sry expression in the developing gonads lie beyond 8 kb 5′ of the TSS.

Expression analysis of PAWP during mouse embryonic stem cell-based spermatogenesis in vitro

Postacrosomal sheath WW domain-binding protein (PAWP) is a novel sperm protein identified as a candidate sperm-borne, oocyte-activating factor (SOAF). However, regulation of PAWP gene expression is poorly understood. Therefore, we examined the PAWP gene expression across different stages of mouse embryonic stem cell (ESC)-based spermatogenesis in vitro and compared this expression at different stages of mouse testis development in vivo. Expression of PAWP was also examined in mouse embryonic fibroblasts (MEF), Sertoli cell, and the NIH3T3 cancerous cell line. We used a transgenic mouse ESC line C57BL/6J expressing Stra8-EGFP that was plated in murine ESC medium. To induce differentiation, cells were cultured on gelatin-coated medium with Retinoic Acid (RA) treatment. We applied reverse transcription-PCR and real-time PCR to analyze the differential expression of PAWP mRNA during different stages of mouse ESC differentiation in vitro parallel with mouse testis development in vivo and in cell lines. We found that expression of PAWP is increased during testis development in vivo with greatest expression at postmeiotic phase. It is also highly expressed in mouse ESC-derived germ-like cells after 30d of RA induction in vitro. PAWP is remarkably expressed in mouse ESC and NIH3T3 cell line. These results indicate that PAWP plays a role in spermatogenesis and germ cell development. Moreover, we suggest PAWP as one of the markers that could be looked in ESC studies as a confirmed testis-specific gene. We also suggest an additional possible role for PAWP in proliferation of cancerous cell in general.

Analysis ofSPATA19gene expression during male germ cells development, lessons from in vivo and in vitro study

Spermatogenesis-associated-19 (SPATA19) is a novel spermatogenesis related gene that has important biological functions in reproduction. However, the expression aspects of SPATA19 gene are not yet well understood. We analysed SPATA19 gene expression during mouse testis development in vivo (5, 15 and 25 days old mouse testis), in parallel with mouse embryonic stem cells (ESCs) differentiation into male germ-like cells in vitro (5, 11, 19 and 27 days after differentiation), as well as Sertoli cell, mouse embryonic fibroblasts (MEF), and NIH3T3 cancerous cell line. We cultured transgenic mouse ESCs line C57BL/6J expressing Stra8-EGFP in ESC's medium and induced their differentiation by retinoic acid (RA) treatment, and meiotic cells were then sorted by FACS. RT-PCR and real-time PCR were used for analysis of SPATA19 mRNA expression. The expression of SPATA19 mRNA is increased during mouse testis development in vivo and it is highest in 25-days-old mouse testis. SPATA19 is also expressed most highly in mouse ESC-derived germ-like cells after 27 days of RA induction and NIH3T3 cell line but no expression was found in mouse ESC and Sertoli cell line. These findings demonstrate that the expression of SPATA19 increases during male germ cells development. We also suggest an additional possible role for SPATA19 in male germ cell differentiation and tumourigenic process.

Wt1 deficiency causes undifferentiated spermatogonia accumulation and meiotic progression disruption in neonatal mice

Spermatogenesis is a complex process involving the regulation of multiple cell types. As the only somatic cell type in the seminiferous tubules, Sertoli cells are essential for spermatogenesis throughout the spermatogenic cycle. The Wilms tumor gene, Wt1, is specifically expressed in the Sertoli cells of the mouse testes. In this study, we demonstrated that Wt1 is required for germ cell differentiation in the developing mouse testes. At 10 days post partum, Wt1-deficient testes exhibited clear meiotic arrest and undifferentiated spermatogonia accumulation in the seminiferous tubules. In addition, the expression of claudin11, a marker and indispensable component of Sertoli cell integrity, was impaired in Wt1(-/flox); Cre-ER(TM) testes. This observation was confirmed in in vitro testis cultures. However, the basal membrane of the seminiferous tubules in Wt1-deficient testes was not affected. Based on these findings, we propose that Sertoli cells' status is affected in Wt1-deficient mice, resulting in spermatogenesis failure.

Cell-type-specific regulation of genes involved in testicular lipid metabolism: fatty acid-binding proteins, diacylglycerol acyltransferases, and perilipin 2

Male germ cell differentiation entails the synthesis and remodeling of membrane polar lipids and the formation of triacylglycerols (TAGs). This requires fatty acid-binding proteins (FABPs) for intracellular fatty acid traffic, a diacylglycerol acyltransferase (DGAT) to catalyze the final step of TAG biosynthesis, and a TAG storage mode. We examined the expression of genes encoding five members of the FABP family and two DGAT proteins, as well as the lipid droplet protein perilipin 2 (PLIN2), during mouse testis development and in specific cells from seminiferous epithelium. Fabp5 expression was distinctive of Sertoli cells and consequently was higher in prepubertal than in adult testis. The expression of Fabp3 increased in testis during postnatal development, associated with the functional differentiation of interstitial cells, but was low in germ cells. Fabp9, together with Fabp12, was prominently expressed in the latter. Their transcripts increased from spermatocytes to spermatids and, interestingly, were highest in spermatid-derived residual bodies (RB). Both Sertoli and germ cells, which produce neutral lipids and store them in lipid droplets, expressed Plin2. Yet, while Dgat1 was detected in Sertoli cells, Dgat2 accumulated in germ cells with a similar pattern of expression as Fabp9. These results correlated with polyunsaturated fatty acid-rich TAG levels also increasing with mouse germ cell differentiation highest in RB, connecting DGAT2 with the biosynthesis of such TAGs. The age- and germ cell type-associated increases in Fabp9, Dgat2, and Plin2 levels are thus functionally related in the last stages of germ cell differentiation.

Multinucleated cells are involved in normal development and apoptosis in mouse testes

Multinucleated cells are present in impaired spermatogenesis and in the senescent testis. Following accumulating evidence from our previous studies on the identification of multinucleated cells during normal testicle development, the current study further investigated the possible mechanism and role of these multinucleated cells. Healthy male Kunming mice were used in the present study. The association between multinucleated cells and cell apoptosis were analyzed using TUNEL analysis and immunohistochemistry. The results showed that multinucleated cells are widespread in the testicular tissue of seminiferous tubules on postnatal days 23, 27, 30, 33, 36, 40, 47, 50 and 54 suggesting that these cells are involved in the process of normal development of mouse testis. Histochemical analysis revealed a lack of proliferating cell nuclear antigen, cyclin D1 protein expression in multinucleated cells, suggesting that these cells are not involved in the G1 and S phases of the cell cycle and cell proliferation. Increased expression of Bax and caspase 3 was detected, revealing that multinucleated cells may be associated with cell apoptosis during testicular development. To the best of our knowledge, this study demonstrated for the first time that multinucleated cells are present during normal testicular development and may be associated with spermatogonial stem cell apoptosis. Therefore, multinucleated cells may be important in the spermatogenesis process.

Effects of TGFbeta2 on Wild-Type and Tgfbr3 Knockout Mouse Fetal Testis1

TGFBR3 (betaglycan), a TGFbeta superfamily coreceptor, is essential for normal seminiferous cord and Leydig cell development in the fetal mouse testis and has been associated with testicular dysgenesis syndrome in men. However, the mechanisms underlying TGFBR3-regulated testis development are unclear. We tested the hypothesis that loss of Tgfbr3 compromises the functions of TGFbeta2 in the differentiating fetal testis. Analysis of expression of transcripts encoding the TGFbeta superfamily members showed a predominance of TGFbeta mRNAs during the critical window of development when testis structure is established (11.5-14.5 days postcoitum [dpc]). When cultured under basal conditions for 2 days, explants of 13.5 dpc wild-type fetal testis/mesonephros complexes exhibited structure and gene expression profiles resembling those observed in vivo between 13.5-15.5 dpc. Similarly, development of Tgfbr3 knockout testis explants recapitulated the dysgenesis and decreased somatic cell marker expression previously observed in vivo. TGFbeta2 treatment partially rescued cord development in 11.5-13.5 dpc Tgfbr3 knockout explants but did not significantly alter somatic or germ cell gene expression. In contrast, TGFbeta2 treatment of wild-type explants disrupted cord structure and significantly downregulated the somatic and steroidogenic cell markers Amh, Sf1, Star, Cyp11a, Hsd3b1, and Cyp17a1. We conclude that 1) the compromised cord development in Tgfbr3 null fetal testis is due to, at least in part, disrupted TGFbeta2 function; 2) the reduction in steroidogenesis observed in the Tgfbr3 null testis may be regulated by additional TGFBR3 ligands, rather than TGFbeta2; and 3) both cord maintenance and somatic cell development are highly sensitive to the levels of TGFbeta2.

Mouse Testis Development and Function Are Differently Regulated by Follicle-Stimulating Hormone Receptors Signaling During Fetal and Prepubertal Life

It is currently admitted that Follicle-Stimulating Hormone (FSH) is physiologically involved in the development and function of fetal/neonatal Sertoli cells in the rat but not the mouse. However, FSH is produced by both species from late fetal life onwards. We thus reinvestigated the role of FSH in mouse testis development at day 0 (birth) 6, 8 and 10 post-partum (dpp) by using mice that lack functional FSH receptors (FSH-R−/−). At birth, the number and proliferative index of Sertoli cells were significantly lower in FSH-R−/− mice than in wild type neonates. Claudin 11 mRNA expression also was significantly reduced in FSH-R−/− testes at 0 and 8 dpp, whereas the mRNA levels of other Sertoli cell markers (Transferrin and Desert hedgehog) were comparable in FSH-R−/− and wild type testes. Conversely, AMH mRNA and protein levels were higher at birth, comparable at 6 dpp and then significantly lower in FSH-R−/− testes at 8–10 dpp in FSH-R−/− mice than in controls. Although the plasma concentration of LH and the number of Leydig cells were similar in FSH-R−/− and control (wild type), testosterone concentration and P450c17 mRNA expression were significantly increased in FSH-R−/− testes at birth. Conversely, at 10 dpp when adult Leydig cells appear, expression of the steroidogenic genes P450scc, P450c17 and StAR was lower in FSH-R−/− testes than in controls. In conclusion, our results show that 1) like in the rat, signaling via FSH-R controls Sertoli cell development and function during late fetal life in the mouse as well; 2) paracrine factors produced by Sertoli cells are involved in the FSH-R-dependent regulation of the functions of fetal Leydig cells in late fetal life; and 3) the role of FSH-R signaling changes during the prepubertal period.

GADD45G Functions in Male Sex Determination by Promoting p38 Signaling and Sry Expression

Male sex determination in mammals is induced by Sry, a gene whose regulation is poorly understood. Here we show that mice mutant for the stress-response gene Gadd45g display complete male-to-female sex reversal. Gadd45g and Sry havea strikingly similar expression pattern in the genital ridge, and they are coexpressed in gonadal somatic cells. In Gadd45g mutants, Sry expression is delayed and reduced, and yet Sry seemed to remain poised for expression, because its promoter is demethylated on schedule and is occupied by active histone marks. Instead, p38 MAPK signaling is impaired in Gadd45g mutants. Moreover, the transcription factor GATA4, which is required for Sry expression, binds tothe Sry promoter invivo in a MAPK-dependent manner. The results suggest that a signaling cascade, involving GADD45G → p38 MAPK → GATA4 → SRY, regulates male sex determination.