I-43: Identification of SOX3 as an XX MaleSex Reversal Gene in Mice and Jumans

Background: Mammals utilise an XX/XY system of sex determination in which the Y-linked gene SRY (Sexdetermining region Y) exerts a dominant masculinising influence on sexual development. Sex chromosome homology and comparative sequence studies suggest that SRY evolved from the related SOX3 gene on the X chromosome, although there is no direct functional evidence to support this hypothesis. The overall objective of this study was to determine whether SOX3 expression in the bipotential gonad can trigger male development by functioning as a surrogate for SRY. To address this important question, we utilised a unique transgenic mouse model in which Sox3 expression is ectopically activated in the gonad. In addition, to determine whether SOX3 gain-offunction could cause XX male sex reversal in humans, we screened a cohort of SRY-negative 46 XX male sex reversal patients for chromosomal rearrangements at the SOX3 locus. Materials and Methods: We generated a Sox3 transgenic (Tg) mouse line termed Sr (Sex reversed) using a 36 kb mouse Sox3 genomic fragment containing an IRES-EGFP reporter cassette inserted into the 3’ UTR of the Sox3 gene. This transgene was ectopically expressed in the developing XX gonads due to a position effect. To determine whether SOX3 rearrangements are associated with XX male sex reversal, we screened 16 SRY-negative 46, XX male patients for copy number variation using Affymetrix 6.0 whole genome SNP (7 patients) and Illumina 1M (9 patients) microarrays. Results: Analysis of more than 500 adult Sr transgenic mice revealed that the Sox3 transgene induced complete XX male sex reversal with a penetrance of approximately 80%. As the genetic switch that triggers male development occurs during embryonic development, we performed a careful analysis of early gonad development in XX hemizygous transgenic (Tg/+) embryos. Key early events of the testis differentiation pathway including Sox9 upregulation, Sertoli cell differentiation, testis cord formation, and generation of a male-specific vasculature all occurred in XX Tg/+ gonads. These data suggest that SOX3 may function as a molecular switch, activating the testis differentiation pathway via a mechanism that is functionally analogous to SRY. To investigate this further, we performed cotransfection assays using the recently identified Sox9 testis-specific enhancer element (TESCO) and showed that SOX3, like SRY, had modest transactivation activity and functioned synergistically with steroidogenic factor-1 (SF1) in this context. In addition, genetic rescue experiments showed that Sox3 failed to induce XX testis development in gonads that lacked Sox9. Consistent with the Sr mouse model, we also identified unique genomic rearrangements of the SOX3 regulatory region in three patients with XX male sex reversal. Conclusion: Together, these data indicate that Sox3 gain-of-function in the developing gonad induces testis development by functioning as a surrogate for Sry. Our findings also provide important functional evidence to support the longstanding hypothesis that Sox3 is the evolutionary precursor of Sry and suggest that rearrangements of SOX3 are a relatively frequent cause of XX male sex reversal in humans.