Mammalian Sex Determination

In mammals, the sex of an individual is determined at conception. When a Y-bearing sperm fertilizes a X-bearing egg, the sex of the embryo is male while a X-bearing sperm will produce a female embryo. In the presence of the Y chromosome, specifically the sex-determining region Y (SRY) gene on this chromosome, the fetus develops testes, which in turn synthesize male sex hormones that are critical for further development of male phenotypic characteristics. In the absence of a Y chromosome, the fetus develops ovaries, synthesizes female sex hormones, important for the development of female phenotypes. Hence, SRY is critical in switching on the male sex determination and differentiation. SRY induces the differentiation of the supporting cells in the indifferent gonad to Sertoli cells, which subsequently establish tight and adherens junctions among themselves to form the testis cord encapsulating the germ cells. The testis cord is the first visible vasculature of the embryonic testis, which is followed by a cascade of events leading to the development of a fully functional testis and secondary male sex characteristics. SRY encodes a nuclear protein harboring a unique DNA/protein-binding domain, called high mobility group (HMG) box. It is a founding member of a family of transcription factors, termed SRY-box (SOX) proteins that contain a conserved SRY-type HMG box. SRY and SOX genes encode transcription factors that play important roles in stem cell pluripotency and cell fate determination and differentiation during embryogenesis. SRY interacts with the transcription factor SF1 and activates a related gene, SOX9, which assumes the SRY functions, establishes its own gene regulatory program and continues the testis differentiation.

SRY Forms Various Nuclear Complexes with Co-Factors

Our research focuses on elucidating the molecular mechanisms by which SRY mediates its testis determination and differentiation functions. Besides interacting with SF1, we demonstrated that SRY also interacts with a variety of partner proteins to achieve its transcriptional and nuclear functions (Figure 1). SRY interacts with beta-catenin and inhibits the WNT signaling critical in activation of ovarian differentiation genes, and with SF1/SP1 and activates testis differentiation genes. SRY also binds the KRAB-O protein, which is a member of the KRAB-ZF superfamily of proteins that are capable of recruiting a powerful gene silencing complex(es) consisting KAP1 and heterochromatin 1 (HP1). Hence, through the molecular bridge KRAB-O, SRY could repress its targets by bringing a silencing complex to their vicinity. SRY binds the poly(ADP- ribose) polymerase 1 (PARP1), which polymerizes ADP-ribose onto chromatin proteins and modulates the accessibility of transcription factors and other nuclear proteins to the DNA. By recruiting such a powerful chromatin modulator, SRY could remodel the chromatin structure and determines the epigenetic landscape, thereby establishing the fate, of the supporting cells into Sertoli lineage.

Figure 1. SRY interacts with a variety of nuclear partners to mediate different nuclear functions. A) SRY binds to β-catenin and inhibits Wnt signaling regulation of ovarian genes. SRY also interacts with KRAB-O bridging molecule and recruits the KAP1-HP1 gene repressor complex to the vinicity and represses its target genes. B) SRY binds SF1 or SP1 and activates sex-determinig genes, such as SOX9. C) SRY binds PARP1, which polymerizes ADP-ribose onto chromatin proteins and regulates the accessibility of transcription factors to its target genes. D) SRY and SOX9 bind to the same target genes and regulate their expression.

Hierarchy of Gene Regulation in Male Sex Determination

Despite the elucidation of the regulatory hierarchy SRY and SOX9 in mediating male sex determination, numerous questions remain. First, is Sox9 the only target gene for SRY? If not, what are the other target genes for the SRY-containing transcription complexes? Second, what are the functions of the SRY targets? Third, because SOX9 is responsible for propagating testis differentiation beyond SRY actions and their DNA binding high-mobility group boxes are functionally interchangeable, do SRY and SOX9 bind and regulate similar target genes in the same developmental pathway? To address these questions, we have conducted a comprehensive global chromatin immunoprecipitation (ChIP) and promoter tiling study to identify the targets for SRY and SOX9 at the time of sex determination in the laboratory mouse.

In the laboratory mouse, SRY expression in the embryonic gonads starts from E10.5, peaks at E11.5 and diminishes at E12.5. SOX9 activation starts a few hours after that of SRY and continues beyond SRY expression. Accordingly, we performed global ChIP studies with specific antibodies against the mouse SRY and SOX9 at E11.5 and E12.5 respectively in biological replicates. Our results show that SRY binds and regulates a variety of known sex determining genes. Importantly SRY and SOX9 bind to the same regions at the promoters of many common targets in Sertoli cells, involved in testis differentiation. SRY, but not SOX9, binds to numerous known ovarian differentiation genes and represses their activation through WNT/b-catenin and other signaling pathways. Sertoli cell-Sertoli cell junction signaling, important for testis cord formation, is the top canonical pathway among the SRY and SOX9 targets. Hence, SRY determines Sertoli cell fate by repressing ovarian and activating testicular differentiation genes, promotes early Sertoli cells to form testis cord, and then passes on its functions to SOX9, which regulates a set of common targets and also activates its own gene regulatory program, beyond SRY actions, in sex determination (Figure 2).

Figure 2. SRY expression is activated at E10.5 embryonic gonad in the mouse. It interacts with a variety of nuclear factors and forms transcription and chromatin modulating complexes with different functions. They switch on male sex determination by turning off ovarian differentiating genes and turning on testicular differentiating genes, including Sox9. SOX9 continues the SRY functions by regulating a large portion of SRY targets and activating its own gene regulatory program, beyond SRY actions. SRY and SOX9 mediate many early testis differentiation processes, including determining the fate of the Sertoli cells, promoting Sertoli cell proliferation, inhibiting ovarian differentiation, inducing the Sertoli cell-Sertoli cell junction signaling, and eventually forming testis cord encapsulating the germ cells at E12.5 stage, when SRY expression diminishes.