Ryohei Sekido

Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer

The mammalian Y chromosome acts as a dominant male determinant as a result of the action of a single gene, Sry, whose role in sex determination is to initiate testis rather than ovary development from early bipotential gonads. It does so by triggering the differentiation of Sertoli cells from supporting cell precursors, which would otherwise give follicle cells. The related autosomal gene Sox9 is also known from loss-of-function mutations in mice and humans to be essential for Sertoli cell differentiation; moreover, its abnormal expression in an XX gonad can lead to male development in the absence of Sry. These genetic data, together with the finding that Sox9 is upregulated in Sertoli cell precursors just after SRY expression begins, has led to the proposal that Sox9 could be directly regulated by SRY. However, the mechanism by which SRY action might affect Sox9 expression was not understood. Here we show that SRY binds to multiple elements within a Sox9 gonad-specific enhancer in mice, and that it does so along with steroidogenic factor 1 (SF1, encoded by the gene Nr5a1 (Sf1)), an orphan nuclear receptor. Mutation, co-transfection and sex-reversal studies all point to a feedforward, self-reinforcing pathway in which SF1 and SRY cooperatively upregulate Sox9 and then, together with SF1, SOX9 also binds to the enhancer to help maintain its own expression after that of SRY has ceased. Our results open up the field, permitting further characterization of the molecular mechanisms regulating sex determination and how they have evolved, as well as how they fail in cases of sex reversal.

Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation

In mammals, the transcription factor SRY, encoded by the Y chromosome, is normally responsible for triggering the indifferent gonads to develop as testes rather than ovaries. However, testis differentiation can occur in its absence. Here we demonstrate in the mouse that a single factor, the forkhead transcriptional regulator FOXL2, is required to prevent transdifferentiation of an adult ovary to a testis. Inducible deletion of Foxl2 in adult ovarian follicles leads to immediate upregulation of testis-specific genes including the critical SRY target gene Sox9. Concordantly, reprogramming of granulosa and theca cell lineages into Sertoli-like and Leydig-like cell lineages occurs with testosterone levels comparable to those of normal XY male littermates. Our results show that maintenance of the ovarian phenotype is an active process throughout life. They might also have important medical implications for the understanding and treatment of some disorders of sexual development in children and premature menopause in women.

Fgf9 and Wnt4 Act as Antagonistic Signals to Regulate Mammalian Sex Determination

The genes encoding members of the wingless-related MMTV integration site (WNT) and fibroblast growth factor (FGF) families coordinate growth, morphogenesis, and differentiation in many fields of cells during development. In the mouse, Fgf9 and Wnt4 are expressed in gonads of both sexes prior to sex determination. Loss of Fgf9 leads to XY sex reversal, whereas loss of Wnt4 results in partial testis development in XX gonads. However, the relationship between these signals and the male sex-determining gene, Sry, was unknown. We show through gain- and loss-of-function experiments that fibroblast growth factor 9 (FGF9) and WNT4 act as opposing signals to regulate sex determination. In the mouse XY gonad, Sry normally initiates a feed-forward loop between Sox9 and Fgf9, which up-regulates Fgf9 and represses Wnt4 to establish the testis pathway. Surprisingly, loss of Wnt4 in XX gonads is sufficient to up-regulate Fgf9 and Sox9 in the absence of Sry. These data suggest that the fate of the gonad is controlled by antagonism between Fgf9 and Wnt4. The role of the male sex-determining switch— Sry in the case of mammals—is to tip the balance between these underlying patterning signals. In principle, sex determination in other vertebrates may operate through any switch that introduces an imbalance between these two signaling pathways.

Sex determination and SRY: down to a wink and a nudge?

Sex-determining region Y (Sry) is the crucial gene that initiates male sex determination in most mammals. Although several components of the pathway regulating sexual differentiation have been elucidated, the mechanism of Sry action within this was unclear. However, recent discoveries in cellular, genetic and molecular aspects of gonad development are shedding light on the precise role of SRY in the regulation of Sox9, a crucial downstream target gene. SRY is thought to act synergistically with SF1, a nuclear receptor, through an enhancer of Sox9 to promote Sertoli cell differentiation in mice. In this review, we focus on the regulation of these genes and their interaction with other genes involved in promoting testis or ovary development. We also explore the common features between sex determination in mammals and in other vertebrates that lack Sry.

Identification of SOX3 as an XX male sex reversal gene in mice and humans

Sex in mammals is genetically determined and is defined at the cellular level by sex chromosome complement (XY males and XX females). The Y chromosome-linked gene sex-determining region Y (SRY) is believed to be the master initiator of male sex determination in almost all eutherian and metatherian mammals, functioning to upregulate expression of its direct target gene Sry-related HMG box-containing gene 9 (SOX9). Data suggest that SRY evolved from SOX3, although there is no direct functional evidence to support this hypothesis. Indeed, loss-of-function mutations in SOX3 do not affect sex determination in mice or humans. To further investigate Sox3 function in vivo, we generated transgenic mice overexpressing Sox3. Here, we report that in one of these transgenic lines, Sox3 was ectopically expressed in the bipotential gonad and that this led to frequent complete XX male sex reversal. Further analysis indicated that Sox3 induced testis differentiation in this particular line of mice by upregulating expression of Sox9 via a similar mechanism to Sry. Importantly, we also identified genomic rearrangements within the SOX3 regulatory region in three patients with XX male sex reversal. Together, these data suggest that SOX3 and SRY are functionally interchangeable in sex determination and support the notion that SRY evolved from SOX3 via a regulatory mutation that led to its de novo expression in the early gonad.

Fibroblast growth factor receptor 2 regulates proliferation and Sertoli differentiation during male sex determination

Targeted mutagenesis of Fgf9 in mice causes male-to-female sex reversal. Among the four FGF receptors, FGFR2 showed two highly specific patterns based on antibody staining, suggesting that it might be the receptor-mediating FGF9 signaling in the gonad. FGFR2 was detected at the plasma membrane in proliferating coelomic epithelial cells and in the nucleus in Sertoli progenitor cells. This expression pattern suggested that Fgfr2 might play more than one role in testis development. To test the hypothesis that Fgfr2 is required for male sex determination, we crossed mice carrying a floxed allele of Fgfr2 with two different Cre lines to induce a temporal or cell-specific deletion of this receptor. Results show that deletion of Fgfr2 in embryonic gonads phenocopies deletion of Fgf9 and leads to male-to-female sex reversal. Using these two Cre lines, we provide the first genetic evidence that Fgfr2 plays distinct roles in proliferation and Sertoli cell differentiation during testis development.

SRY: A transcriptional activator of mammalian testis determination

Sry (sex-determining region Y) is the sex-determining gene on the mammalian Y chromosome, which encodes a transcription factor containing a DNA-binding domain characteristic of some high mobility group proteins (HMG box). It is the founder member of the Sox (Sry-related HMG box) gene family and is therefore classified in the Sox A group. In mice, the transient expression of Sry between 10.5 and 12.5 dpc triggers the differentiation of Sertoli cells from the supporting cell precursor lineage, which would otherwise give rise to granulosa cells in ovaries. However, little was known about the target genes of SRY and molecular mechanisms how SRY leads to testis development. Recent work has provided evidence that SRY binds directly to a testis-specific enhancer of Sox9 (TES) and activates Sox9 expression in co-operation with steroidogenic factor 1 (SF1). Furthermore, this SRY action is limited to a certain time period during embryogenesis.

Failure of SOX9 Regulation in 46XY Disorders of Sex Development with SRY, SOX9 and SF1 Mutations

Background In human embryogenesis, loss of SRY (sex determining region on Y), SOX9 (SRY-related HMG box 9) or SF1 (steroidogenic factor 1) function causes disorders of sex development (DSD). A defining event of vertebrate sex determination is male-specific upregulation and maintenance of SOX9 expression in gonadal pre-Sertoli cells, which is preceded by transient SRY expression in mammals. In mice, Sox9 regulation is under the transcriptional control of SRY, SF1 and SOX9 via a conserved testis-specific enhancer of Sox9 (TES). Regulation of SOX9 in human sex determination is however poorly understood. Methodology/Principal Findings We show that a human embryonal carcinoma cell line (NT2/D1) can model events in presumptive Sertoli cells that initiate human sex determination. SRY associates with transcriptionally active chromatin in NT2/D1 cells and over-expression increases endogenous SOX9 expression. SRY and SF1 co-operate to activate the human SOX9 homologous TES (hTES), a process dependent on phosphorylated SF1. SOX9 also activates hTES, augmented by SF1, suggesting a mechanism for maintenance of SOX9 expression by auto-regulation. Analysis of mutant SRY, SF1 and SOX9 proteins encoded by thirteen separate 46,XY DSD gonadal dysgenesis individuals reveals a reduced ability to activate hTES. Conclusions/Significance We demonstrate how three human sex-determining factors are likely to function during gonadal development around SOX9 as a hub gene, with different genetic causes of 46,XY DSD due a common failure to upregulate SOX9 transcription.

Excess DAX1 leads to XY ovotesticular disorder of sex development (DSD) in mice by inhibiting steroidogenic factor-1 (SF1) activation of the testis enhancer of SRY-box-9 (Sox9).

Human DAX1 duplications cause dosage-sensitive sex reversal (DSS) whereby chromosomally XY individuals can develop as females due to gonadal dysgenesis. However, the mechanism of DSS-adrenal hypoplasia congenita on X, gene 1 (DAX1) action in the fetal testis is unknown. We show that in fetal testes from XY Dax1-overexpressing transgenic mice, the expression of the key testis-promoting gene sex-determining region on Y (SRY)-box-9 (Sox9) is reduced. Moreover, in XY Sox9 heterozygotes, in which testis development is usually normal, Dax1 overexpression results in ovotestes, suggesting a DAX1-SOX9 antagonism. The ovarian portion of the XY ovotestes was characterized by expression of the granulosa cell marker, Forkhead box-L2, with complete loss of the Sertoli cell markers, SOX9 and anti-Müllerian hormone, and the Leydig cell marker CYP17A1. However, the expression of SRY and steroidogenic factor-1 (SF1), two key transcriptional regulators of Sox9, was retained in the ovarian portion of the XY ovotestes. Using reporter mice, Dax1 overexpression reduced activation of TES, the testis enhancer of Sox9, indicating that DAX1 might repress Sox9 expression via TES. In cultured cells, increasing levels of DAX1 antagonized SF1-, SF1/SRY-, and SF1/SOX9-mediated activation of TES, due to reduced binding of SF1 to TES, providing a likely mechanism for DSS.

A MicroRNA (mmu-miR-124) Prevents Sox9 Expression in Developing Mouse Ovarian Cells

ABSTRACT In mammals, sex differentiation depends on gonad development, which is controlled by two groups of sex-determining genes that promote one gonadal sex and antagonize the opposite one. SOX9 plays a key role during testis development in all studied vertebrates, whereas it is kept inactive in the XX gonad at the critical time of sex determination, otherwise, ovary-to-testis gonadal sex reversal occurs. However, molecular mechanisms underlying repression of Sox9 at the beginning of ovarian development, as well as other important aspects of gonad organogenesis, remain largely unknown. Because there is indirect evidence that micro-RNAs (miRNA) are necessary for testicular function, the possible involvement of miRNAs in mammalian sex determination deserved further research. Using microarray technology, we have identified 22 miRNAs showing sex-specific expression in the developing gonads during the critical period of sex determination. Bioinformatics analyses led to the identification of miR-124 as the candidate gene for ovarian development. We knocked down or overexpressed miR-124 in primary gonadal cell cultures and observed that miR-124 is sufficient to induce the repression of both SOX9 translation and transcription in ovarian cells. Our results provide the first evidence of the involvement of a miRNA in the regulation of the gene controlling gonad development and sex determination. The miRNA microarray data reported here will help promote further research in this field, to unravel the role of other miRNAs in the genetic control of mammalian sex determination.