A role for TRPC3 in mammalian testis development.

SOX9 is a key transcription factor for testis determination and development. Mutations in and around the SOX9 gene contribute to Differences/Disorders of Sex Development (DSD). However, a substantial proportion of DSD patients lack a definitive genetic diagnosis. SOX9 target genes are potentially DSD-causative genes, yet only a limited subset of these genes has been investigated during testis development. We hypothesize that SOX9 target genes play an integral role in testis development and could potentially be causative genes in DSD. In this study, we describe a novel testicular target gene of SOX9, Trpc3. Trpc3 exhibits high expression levels in the SOX9-expressing male Sertoli cells compared to female granulosa cells in mouse fetal gonads between embryonic day 11.5 (E11.5) and E13.5. In XY Sox9 knockout gonads, Trpc3 expression is markedly downregulated. Moreover, culture of E11.5 XY mouse gonads with TRPC3 inhibitor Pyr3 resulted in decreased germ cell numbers caused by reduced germ cell proliferation. Trpc3 is also expressed in endothelial cells and Pyr3-treated E11.5 XY mouse gonads showed a loss of the coelomic blood vessel due to increased apoptosis of endothelial cells. In the human testicular cell line NT2/D1, TRPC3 promotes cell proliferation and controls cell morphology, as observed by xCELLigence and HoloMonitor real-time analysis. In summary, our study suggests that SOX9 positively regulates Trpc3 in mouse testes and TRPC3 may mediate SOX9 function during Sertoli, germ and endothelial cell development.

A "Four Core Genotypes" rat model to distinguish mechanisms underlying sex-biased phenotypes and diseases.

Background: We have generated a rat model similar to the Four Core Genotypes mouse model, allowing comparison of XX and XY rats with the same type of gonad. The model detects novel sex chromosome effects (XX vs. XY) that contribute to sex differences in any rat phenotype. Methods: XY rats were produced with an autosomal transgene of Sry , the testis-determining factor gene, which were fathers of XX and XY progeny with testes. In other rats, CRISPR-Cas9 technology was used to remove Y chromosome factors that initiate testis differentiation, producing fertile XY gonadal females that have XX and XY progeny with ovaries. These groups can be compared to detect sex differences caused by sex chromosome complement (XX vs. XY) and/or by gonadal hormones (rats with testes vs. ovaries). Results: We have measured numerous phenotypes to characterize this model, including gonadal histology, breeding performance, anogenital distance, levels of reproductive hormones, body and organ weights, and central nervous system sexual dimorphisms. Serum testosterone levels were comparable in adult XX and XY gonadal males. Numerous phenotypes previously found to be sexually differentiated by the action of gonadal hormones were found to be similar in XX and XY rats with the same type of gonad, suggesting that XX and XY rats with the same type of gonad have comparable levels of gonadal hormones at various stages of development. Conclusion: The results establish a powerful new model to discriminate sex chromosome and gonadal hormone effects that cause sexual differences in rat physiology and disease.

Somatic FGFR2 is Required for Germ Cell Maintenance in the Mouse Ovary.

During sex determination in the mouse, fibroblast growth factor 9 signals through the fibroblast growth factor receptor 2c isoform (FGFR2c) to trigger Sertoli cell and testis development from 11.5 days post coitum (dpc). In the XX gonad, the FOXL2 and WNT4/RSPO1 pathways drive granulosa cell and ovarian development. The function of FGFR2 in the developing ovary, and whether FGFR2 is required in the testis after sex determination, is not clear. In fetal mouse gonads from 12.5 dpc, FGFR2 shows sexually dimorphic expression. In XX gonads, FGFR2c is coexpressed with FOXL2 in pregranulosa cells, whereas XY gonads show FGFR2b expression in germ cells. Deletion of Fgfr2c in XX mice led to a marked decrease/absence of germ cells by 13.5 dpc in the ovary. This indicates that FGFR2c in the somatic pregranulosa cells is required for the maintenance of germ cells. Surprisingly, on the Fgfr2c-/- background, the germ cell phenotype could be rescued by ablation of Foxl2, suggesting a novel mechanism whereby FGFR2 and FOXL2 act antagonistically during germ cell development. Consistent with low/absent FGFR2 expression in the Sertoli cells of 12.5 and 13.5 dpc XY gonads, XY AMH:Cre; Fgfr2flox/flox mice showed normal testis morphology and structures during fetal development and in adulthood. Thus, FGFR2 is not essential for maintaining Sertoli cell fate after sex determination. Combined, these data show that FGFR2 is not necessary for Sertoli cell function after sex determination but does play an important role in the ovary.

FGF9 variant in 46,XY DSD patient suggests a role for dimerization in sex determination.

46,XY gonadal dysgenesis (GD) is a Disorder/Difference of Sex Development (DSD) that can present with phenotypes ranging from ambiguous genitalia to complete male-to-female sex reversal. Around 50% of 46,XY DSD cases receive a molecular diagnosis. In mice, Fibroblast growth factor 9 (FGF9) is an important component of the male sex-determining pathway. Two FGF9 variants reported to date disrupt testis development in mice, but not in humans. Here, we describe a female patient with 46,XY GD harbouring the rare FGF9 variant (missense mutation), NM_002010.2:c.583G > A;p.(Asp195Asn) (D195N). By biochemical and cell-based approaches, the D195N variant disrupts FGF9 protein homodimerisation and FGF9-heparin-binding, and reduces both Sertoli cell proliferation and Wnt4 repression. XY Fgf9D195N/D195N foetal mice show a transient disruption of testicular cord development, while XY Fgf9D195N/- foetal mice show partial male-to-female gonadal sex reversal. In the general population, the D195N variant occurs at an allele frequency of 2.4 × 10-5 , suggesting an oligogenic basis for the patient's DSD. Exome analysis of the patient reveals several known and novel variants in genes expressed in human foetal Sertoli cells at the time of sex determination. Taken together, our results indicate that disruption of FGF9 homodimerization impairs testis determination in mice and, potentially, also in humans in combination with other variants.

Testis Determination Requires a Specific FGFR2 Isoform to Repress FOXL2.

Male sex determination in mammals relies on sex determining region Y-mediated upregulation of sex determining region-box 9 (SOX9) expression in XY gonads, whereas Wnt family member (WNT)/R-spondin 1 signaling and forkhead box L2 (FOXL2) drive female sex determination in XX gonads. Fibroblast growth factor (FGF) 9 signaling ensures sustained SOX9 expression through repression of one of the ovarian pathways (WNT signaling), whereas the significance of FGF-mediated repression of the FOXL2 pathway has not been studied. Previously, we demonstrated that FGFR2 is the receptor for FGF9 in the XY gonad. Whether a specific isoform (FGFR2b or FGFR2c) is required was puzzling. Here, we show that FGFR2c is required for male sex determination. Initially, in developing mouse embryos at 12.5 to 13.5 days postcoitum (dpc), XY Fgfr2c-/- gonads appear as ovotestes, with SOX9 and FOXL2 expression predominantly localized to the posterior and anterior gonadal poles, respectively. However, by 15.5 dpc, XY Fgfr2c-/- gonads show complete male-to-female sex reversal, evident by the lack of SOX9 and ectopic expression of FOXL2 throughout the gonads. Furthermore, ablation of the Foxl2 gene leads to partial or complete rescue of gonadal sex reversal in XY Fgfr2c-/- mice. Together with previous findings, our data suggest that testis determination involves FGFR2c-mediated repression of both the WNT4- and FOXL2-driven ovarian-determining pathways.