Sox8 and Sox9 act redundantly for ovarian-to-testicular fate reprogramming in the absence of R-spondin1 in mouse sex reversals.
ABSTRACT: In mammals, testicular differentiation is initiated by transcription factors SRY and SOX9 in XY gonads, and ovarian differentiation involves R-spondin1 (RSPO1) mediated activation of WNT/?-catenin signaling in XX gonads. Accordingly, the absence of RSPO1/Rspo1 in XX humans and mice leads to testicular differentiation and female-to-male sex reversal in a manner that does not requireSry or Sox9 in mice. Here we show that an alternate testis-differentiating factor exists and that this factor is Sox8. Specifically, genetic ablation of Sox8 and Sox9 prevents ovarian-to-testicular reprogramming observed in XX Rspo1 loss-of-function mice. Consequently, Rspo1 Sox8 Sox9 triple mutant gonads developed as atrophied ovaries. Thus, SOX8 alone can compensate for the loss of SOX9 for Sertoli cell differentiation during female-to-male sex reversal.
Project description: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.
Project description:The transcription factors SRY and SOX9 and RSPO1/WNT4/?-Catenin signaling act as antagonistic pathways to drive testis and ovary development respectively, from a common gonadal primordium in mouse embryos. In this work, we took advantage of a double knockout mouse model to study gonadal development when Sox9 and Wnt4 are both mutated. We show that the XX gonad mutant for Wnt4 or for both Wnt4 and Sox9 develop as ovotestes, demonstrating that ectopic SOX9 function is not required for the partial female-to-male sex reversal caused by a Wnt4 mutation. Sox9 deletion in XY gonads leads to ovarian development accompanied by ectopic WNT/?-catenin signaling. In XY Sox9 mutant gonads, SRY-positive supporting precursors adopt a female-like identity and develop as pre-granulosa-like cells. This phenotype cannot be fully prevented by the deletion of Wnt4 or Rspo1, indicating that SOX9 is required for the early determination of the male supporting cell identity independently of repressing RSPO1/WNT4/?-Catenin signaling. However, in XY Sox9 Wnt4 double mutant gonads, pre-granulosa cells are not maintained, as they prematurely differentiate as mature granulosa cells and then trans-differentiate into Sertoli-like cells. Together, our results reveal the dynamics of the specific and independent actions of SOX9 and WNT4 during gonadal differentiation: SOX9 is essential in the testis for early specification of male-supporting cells whereas WNT4 functions in the ovary to maintain female-supporting cell identity and inhibit male-specific vascular and steroidogenic cell differentiation.
Project description:During mammalian sex determination, expression of the Y-linked gene Sry shifts the bipotential gonad toward a testicular fate by upregulating a feed-forward loop between FGF9 and SOX9 to establish SOX9 expression in somatic cells. We previously proposed that these signals are mutually antagonistic with counteracting signals in XX gonads and that a shift in the balance of these factors leads to either male or female development. Evidence in mice and humans suggests that the male pathway is opposed by the expression of two signals, WNT4 and R-SPONDIN-1 (RSPO1), that promote the ovarian fate and block testis development. Both of these ligands can activate the canonical Wnt signaling pathway. Duplication of the distal portion of chromosome 1p, which includes both WNT4 and RSPO1, overrides the male program and causes male-to-female sex reversal in XY patients. To determine whether activation of beta-catenin is sufficient to block the testis pathway, we have ectopically expressed a stabilized form of beta-catenin in the somatic cells of XY gonads. Our results show that activation of beta-catenin in otherwise normal XY mice effectively disrupts the male program and results in male-to-female sex-reversal. The identification of beta-catenin as a key pro-ovarian and anti-testis signaling molecule will further our understanding of the mechanisms controlling sex determination and the molecular mechanisms that lead to sex-reversal.
Project description:Sex-reversal cases in humans and genetic models in mice have revealed that the fate of the bipotential gonad hinges upon the balance between pro-testis SOX9 and pro-ovary beta-catenin pathways. Our central query was: if SOX9 and beta-catenin define the gonad's identity, then what do the gonads become when both factors are absent? To answer this question, we developed mouse models that lack either Sox9, beta-catenin, or both in the somatic cells of the fetal gonads and examined the morphological outcomes and transcriptome profiles. In the absence of Sox9 and beta-catenin, both XX and XY gonads progressively lean toward the testis fate, indicating that expression of certain pro-testis genes requires the repression of the beta-catenin pathway, rather than a direct activation by SOX9. We also observed that XY double knockout gonads were more masculinized than their XX counterpart. To identify the genes responsible for the initial events of masculinization and to determine how the genetic context (XX vs. XY) affects this process, we compared the transcriptomes of Sox9/beta-catenin mutant gonads and found that early molecular changes underlying the XY-specific masculinization involve the expression of Sry and 21 SRY direct target genes, such as Sox8 and Cyp26b1. These results imply that when both Sox9 and beta-catenin are absent, Sry is capable of activating other pro-testis genes and drive testis differentiation. Our findings not only provide insight into the mechanism of sex determination, but also identify candidate genes that are potentially involved in disorders of sex development.
Project description:We have previously established an in vivo requirement for GATA4 and FOG2 transcription factors in sexual differentiation. Fog2 null mouse fetuses or fetuses homozygous for a targeted mutation in Gata4 (Gata4(ki)), which cripples the GATA4-FOG2 interaction, exhibit a profound and early block in testis differentiation in both sexes. Others have shown that XX mice with the Ods transgenic insertion or the Wt1-Sox9 YAC transgene overexpress the testis differentiation gene, Sox9. Thus, these XX animals undergo dominant sex reversal by developing into phenotypically normal, but sterile, males. Now we have determined that Fog2 haploinsufficiency prevents (suppresses) this dominant sex reversal and Fog2+/-Wt1-Sox9 or Ods XX animals develop normally--as fertile females. The suppression of sex reversal in Fog2 heterozygous females results from approximately 50% downregulation of the expression from the transgene-associated allele of Sox9. The GATA4/FOG2-dependent sex reversal observed in the transgenic XX gonads has to rely on gene targets other than the Y chromosome-linked Sry gene. Importantly, Fog2 null or Gata4(ki/ki) embryos (either XX or XY) fail to express detectable levels of Sox9 despite carrying the Ods mutation or Wt1-Sox9 transgene. Fog2 haploinsufficiency leads to a decreased amount of SOX9-positive cells in XY gonads. We conclude that FOG2 is a limiting factor in the formation of a functional GATA4/FOG2 transcription complex that is required for Sox9 expression during gonadogenesis.
Project description:BACKGROUND: Up to now, two loci have been involved in XX sex-reversal in mammals following loss-of-function mutations, PIS (Polled Intersex Syndrome) in goats and R-spondin1 (RSPO1) in humans. Here, we analyze the possible interaction between these two factors during goat gonad development. Furthermore, since functional redundancy between different R-spondins may influence gonad development, we also studied the expression patterns of RSPO2, 3 and 4. RESULTS: Similarly to the mouse, RSPO1 shows a sex-dimorphic expression pattern during goat gonad development with higher levels in the ovaries. Interestingly, the PIS mutation does not seem to influence its level of expression. Moreover, using an RSPO1 specific antibody, the RSPO1 protein was localized in the cortical area of early differentiating ovaries (36 and 40 dpc). This cortical area contains the majority of germ cell that are surrounded by FOXL2 negative somatic cells. At latter stages (50 and 60 dpc) RSPO1 protein remains specifically localized on the germ cell membranes. Interestingly, a time-specific relocation of RSPO1 on the germ cell membrane was noticed, moving from a uniform distribution at 40 dpc to a punctuated staining before and during meiosis (50 and 60 dpc respectively). Interestingly, also RSPO2 and RSPO4 show a sex-dimorphic expression pattern with higher levels in the ovaries. Although RSPO4 was found to be faintly and belatedly expressed, the expression of RSPO2 increases at the crucial 36 dpc stage, as does that of FOXL2. Importantly, RSPO2 expression appears dramatically decreased in XX PIS-/- gonads at all three tested stages (36, 40 and 50 dpc). CONCLUSION: During goat ovarian development, the pattern of expression of RSPO1 is in agreement with its possible anti-testis function but is not influenced by the PIS mutation. Moreover, our data suggest that RSPO1 may be associated with germ cell development and meiosis. Interestingly, another RSPO gene, RSPO2 shows a sex-dimorphic pattern of expression that is dramatically influenced by the PIS mutation.
Project description:The new concept of mammalian sex maintenance establishes that particular key genes must remain active in the differentiated gonads to avoid genetic sex reprogramming, as described in adult ovaries after Foxl2 ablation. Dmrt1 plays a similar role in postnatal testes, but the mechanism of adult testis maintenance remains mostly unknown. Sox9 and Sox8 are required for postnatal male fertility, but their role in the adult testis has not been investigated. Here we show that after ablation of Sox9 in Sertoli cells of adult, fertile Sox8(-/-) mice, testis-to-ovary genetic reprogramming occurs and Sertoli cells transdifferentiate into granulosa-like cells. The process of testis regression culminates in complete degeneration of the seminiferous tubules, which become acellular, empty spaces among the extant Leydig cells. DMRT1 protein only remains in non-mutant cells, showing that SOX9/8 maintain Dmrt1 expression in the adult testis. Also, Sox9/8 warrant testis integrity by controlling the expression of structural proteins and protecting Sertoli cells from early apoptosis. Concluding, this study shows that, in addition to its crucial role in testis development, Sox9, together with Sox8 and coordinately with Dmrt1, also controls adult testis maintenance.
Project description: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/?-catenin signaling pathway in the somatic cells by the secreted protein RSPO1. Using mouse models, we now show that Rspo1 also activates the WNT/?-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 ?-catenin signaling acts within the germ cells to promote oogonial differentiation and entry into meiosis. Our results demonstrate that RSPO1/?-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.
Project description:Sertoli cells provide nutritional and physical support to germ cells during spermatogenesis. Sox8 encodes a high mobility group transcription factor closely related to Sox9 and Sox10. Sertoli cells produceSOX8 protein, and its elimination results in an age-dependent deregulation of spermatogenesis resulting in male infertility. This suggests that Sox8 is a critical regulator of Sertoli cell function for the maintenance of male fertility beyond the first wave of spermatogenesis. To better understand the roles of Sox8 in testicular development and maintenance of male fertility, we have performed a detailed analysis to explore its downstream genes. We have used mRNA expression profiling to identify affected genes in Sertoli cells in the mutant testes of 2-month-old mice. Expression profiling of the heterozygous and homozygous Sox8 mutant testes indicates alterations in several important spermatogenesis and blood-testis barrier genes, providing insight into the molecular basis of the defects in Sox8(-/-) testes beyond the first wave of spermatogenesis.
Project description:We aimed to identify the genetic cause in a cohort of 11 unrelated cases and two sisters with 46,XX SRY-negative (ovo)testicular disorders of sex development (DSD).Whole-exome sequencing (n = 9), targeted resequencing (n = 4), and haplotyping were performed. Immunohistochemistry of sex-specific markers was performed on patients' gonads. The consequences of mutation were investigated using luciferase assays, localization studies, and RNA-seq.We identified a novel heterozygous NR5A1 mutation, c.274C>T p.(Arg92Trp), in three unrelated patients. The Arg92 residue is highly conserved and located in the Ftz-F1 region, probably involved in DNA-binding specificity and stability. There were no consistent changes in transcriptional activation or subcellular localization. Transcriptomics in patient-derived lymphocytes showed upregulation of MAMLD1, a direct NR5A1 target previously associated with 46,XY DSD. In gonads of affected individuals, ovarian FOXL2 and testicular SRY-independent SOX9 expression observed.We propose NR5A1, previously associated with 46,XY DSD and 46,XX primary ovarian insufficiency, as a novel gene for 46,XX (ovo)testicular DSD. We hypothesize that p.(Arg92Trp) results in decreased inhibition of the male developmental pathway through downregulation of female antitestis genes, thereby tipping the balance toward testicular differentiation in 46,XX individuals. In conclusion, our study supports a role for NR5A1 in testis differentiation in the XX gonad.Genet Med 19 4, 367-376.