Peptidyl arginine deiminase 2 (Padi2) is expressed in Sertoli cells in a specific manner and regulated by SOX9 during testicular development.

Peptidyl arginine deiminases (PADIs) are enzymes that change the charge of proteins through citrullination. We recently found Padi2 was expressed exclusively in fetal Sertoli cells. In this study, we analyzed the transcriptional regulation of Padi2 and the role of PADI2 in testicular development. We showed SOX9 positively regulated Padi2 transcription and FOXL2 antagonized it in TM3 cells, a model of Sertoli cells. The responsive region to SOX9 and FOXL2 was identified within the Padi2 sequence by reporter assay. In fetal testes from Sox9 knockout (AMH-Cre:Sox9flox/flox) mice, Padi2 expression was greatly reduced, indicating SOX9 regulates Padi2 in vivo. In vitro analysis using siRNA suggested PADI2 modified transcriptional regulation by SOX9. However, Padi2-/- XY mice were fertile and showed no apparent reproductive anomalies. Although, PADI2 is known as an epigenetic transcriptional regulator through H3 citrullination, no significant difference in H3 citrullination between wildtype and Padi2-/- XY gonads was observed. These results suggest Padi2 is a novel gene involved in testis development that is specifically expressed in Sertoli cells through the regulation by SOX9 and FOXL2 and PADI2 supports regulation of target genes by SOX9. Analysis of the Padi2-/- XY phenotype suggested a redundant factor compensated for PADI2 function in testicular development.

In mammalian foetal testes, SOX9 regulates expression of its target genes by binding to genomic regions with conserved signatures.

In mammalian embryonic gonads, SOX9 is required for the determination of Sertoli cells that orchestrate testis morphogenesis. To identify genetic networks directly regulated by SOX9, we combined analysis of SOX9-bound chromatin regions from murine and bovine foetal testes with sequencing of RNA samples from mouse testes lacking Sox9. We found that SOX9 controls a conserved genetic programme that involves most of the sex-determining genes. In foetal testes, SOX9 modulates both transcription and directly or indirectly sex-specific differential splicing of its target genes through binding to genomic regions with sequence motifs that are conserved among mammals and that we called 'Sertoli Cell Signature' (SCS). The SCS is characterized by a precise organization of binding motifs for the Sertoli cell reprogramming factors SOX9, GATA4 and DMRT1. As SOX9 biological role in mammalian gonads is to determine Sertoli cells, we correlated this genomic signature with the presence of SOX9 on chromatin in foetal testes, therefore equating this signature to a genomic bar code of the fate of foetal Sertoli cells. Starting from the hypothesis that nuclear factors that bind to genomic regions with SCS could functionally interact with SOX9, we identified TRIM28 as a new SOX9 partner in foetal testes.

SOX9 regulates expression of the male fertility gene Ets variant factor 5 (ETV5) during mammalian sex development.

In humans, dysregulation of the sex determining gene SRY-box 9 (SOX9) leads to disorders of sex development (DSD). In mice, knock-out of Sox9 prior to sex determination leads to XY sex reversal, while Sox9 inactivation after sex determination leads to spermatogenesis defects. SOX9 specifies the differentiation and function of Sertoli cells from somatic cell precursors, which then orchestrate the development and maintenance of other testicular cell types, largely through unknown mechanisms. Here, we describe a novel testicular target gene of SOX9, Ets variant factor 5 (ETV5), a transcription factor responsible for maintaining the spermatogonial stem cell niche. Etv5 was highly expressed in wild-type XY but not XX mouse fetal gonads, with ETV5 protein localized in the Sertoli cells, interstitial cells and germ cells of the testis. In XY Sox9 knock-out gonads, Etv5 expression was strongly down-regulated. Similarly, knock-down of SOX9 in the human Sertoli-like cell line NT2/D1 caused a decrease in ETV5 gene expression. Transcriptomic analysis of NT2/D1 cells over-expressing SOX9 showed that ETV5 expression was increased in response to SOX9. Moreover, chromatin immunoprecipitation of these cells, as well as of embryonic mouse gonads, showed direct binding of SOX9 to ETV5 regulatory regions. We demonstrate that SOX9 was able to activate ETV5 expression via a conserved SOX site in the 5' regulatory region, mutation of which led to loss of activation. In conclusion, we present a novel target gene of SOX9 in the testis, and suggest that SOX9 regulation of ETV5 contributes to the control of male fertility.

Testicular differentiation occurs in absence of R-spondin1 and Sox9 in mouse sex reversals.

In mammals, male sex determination is governed by SRY-dependent activation of Sox9, whereas female development involves R-spondin1 (RSPO1), an activator of the WNT/beta-catenin signaling pathway. Genetic analyses in mice have demonstrated Sry and Sox9 to be both required and sufficient to induce testicular development. These genes are therefore considered as master regulators of the male pathway. Indeed, female-to-male sex reversal in XX Rspo1 mutant mice correlates with Sox9 expression, suggesting that this transcription factor induces testicular differentiation in pathological conditions. Unexpectedly, here we show that testicular differentiation can occur in XX mutants lacking both Rspo1 and Sox9 (referred to as XX Rspo1(KO)Sox9(cKO) ()), indicating that Sry and Sox9 are dispensable to induce female-to-male sex reversal. Molecular analyses show expression of both Sox8 and Sox10, suggesting that activation of Sox genes other than Sox9 can induce male differentiation in Rspo1(KO)Sox9(cKO) mice. Moreover, since testis development occurs in XY Rspo1(KO)Sox9(cKO) mice, our data show that Rspo1 is the main effector for male-to-female sex reversal in XY Sox9(cKO) mice. Thus, Rspo1 is an essential activator of ovarian development not only in normal situations, but also in sex reversal situations. Taken together these data demonstrate that both male and female sex differentiation is induced by distinct, active, genetic pathways. The dogma that considers female differentiation as a default pathway therefore needs to be definitively revised.

RSPO1/ß-catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary.

Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog gene and factors synthesized by the somatic Sertoli cells promote gonocyte differentiation. In the female, ovarian differentiation requires activation of the WNT/ß-catenin signaling pathway in the somatic cells by the secreted protein RSPO1. Using mouse models, we now show that Rspo1 also activates the WNT/ß-catenin signaling pathway in germ cells. In XX Rspo1(-/-) gonads, germ cell proliferation, expression of the early meiotic marker Stra8, and entry into meiosis are all impaired. In these gonads, impaired entry into meiosis and germ cell sex reversal occur prior to detectable Sertoli cell differentiation, suggesting that ß-catenin signaling acts within the germ cells to promote oogonial differentiation and entry into meiosis. Our results demonstrate that RSPO1/ß-catenin signaling is involved in meiosis in fetal germ cells and contributes to the cellular decision of germ cells to differentiate into oocyte or sperm.

XY Sox9 embryonic loss-of-function mouse mutants show complete sex reversal and produce partially fertile XY oocytes.

Gonadal differentiation is the first step of mammalian sex determination. The expression of the Y chromosomal testis determining factor Sry leads to up-regulation of the transcription factor Sox9 which promotes testis differentiation. Previous studies showed that Sox9 deficiency induces expression of ovarian markers in XY mutant fetal gonads before they die. To better understand the genome-wide transcriptional profile underlying this process we compared samples from XY Sf1:Cre(Tg/+); Sox9(flox/flox) mutant gonads in which Sox9 is ablated in Sertoli-precursor cells during early stages of gonad development to XX Sox9(flox/flox) ovaries and XY Sox9(flox/flox) testes at E13.5. We found a complex mRNA signature that indicates wide-spread transcriptional de-regulation and revealed for XY mutants at E13.5 an intermediate transcript profile between male and female gonads. However, XY Sf1:Cre(Tg/+); Sox9(flox/flox) mutant gonads develop as ovaries containing XY developing follicles at P0 but less frequently so than in XX control ovaries. Furthermore, we studied the extent to which developing XY mutant ovaries are able to mediate adult fertility and observed that XY oocytes from XY mutant ovaries are competent for fertilization; however, two thirds of them fail to develop beyond two-cell stage embryos. Taken together, we found that XY Sf1:Cre(Tg/+); Sox9(flox/flox) females are capable of producing viable offspring albeit at a reduced level.

Transient development of ovotestes in XX Sox9 transgenic mice.

The sex of an individual results from the paternal transmission of the SRY gene located on the Y chromosome. In turn, SRY initiates Sox9 expression, a transcription factor required for testicular differentiation. Ectopic activation of SOX9 in XX Wt1:Sox9 transgenic mice induces female-to-male sex reversal in adult mice. Here we show that complete sex reversal is preceded by a transient phase of ovotestis differentiation with XX Wt1:Sox9 transgenic gonads containing a testicular central region and one or both ovarian poles indicating that Wt1:Sox9 is not as efficient as Sry to induce male development. In XX Wt1:Sox9(Tg/+) gonads, transgenic Sox9 is expressed earlier than Sox9 in XY gonads and is able to induce the expression of EGFP, knocked into the 3' UTR of Sox9 indicating that SOX9 is involved in the initiation and maintenance of its own expression. However, the delayed onset of expression of endogenous Sox9-EGFP suggests that this activation requires other factors, whose expression depends on SOX9. In the testicular regions of the XX Wt1:Sox9 ovotestes, proliferation of the XX fetal germ cells is hampered and they differentiate as pro-spermatogonia. This indicates that XX germ cells are not competent to respond to proliferative signals released from a testicular environment. In the ovarian regions, despite the continuous mRNA expression of the WT1:Sox9 transgene, the SOX9 protein does not accumulate suggesting that regulation of this gene in ovarian cells involves post-transcriptional mechanisms. Finally, ovarian cells of the XX Wt1:Sox9 ovotestis undergo apoptosis during late embryogenesis leading to complete female-to-male sex reversal of the transgenic mice at birth.