Sox9 Driven Sex Determination in Mice with Limited Y Chromosome Gene Contribution

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

The window of sex determination is a short but critical time during which sex fate of the embryo is decided depending on the chromosomal predisposition inherited from the parents. This genetic information guides the direction of gonadal sex development towards acquisition of anatomical male or female sexual characteristics. In most mammals, male sex determination is initiated by expression of the Y chromosome encoded Sry. The duration of SRY signaling is transient - just long enough to ensure upregulation of autosomally encoded Sox9. This way, SRY acts as a molecular switch that initiates differentiation of the bipotential gonad into a testis. However, once SRY signaling ceases, Sox9 remains at the center of male-specific molecular signaling profile that ensures masculinization of the genital ridges. The focus of my dissertation is on the effects of Sry-to-Sox9 replacement on the development of genital ridges, as well as spermatogenesis and fertility in adult males. Work presented here explores the hypothesis that transgenic overexpression of Sox9 can replace the function of endogenous Sry at the time of sex determination and can sufficiently support spermatogenesis and fertility in adult males. The sex-determining function of transgenic Sox9 was tested using two genetic models: (1) mice that carried X chromosome and Y chromosome lacking the endogenous Sry (XYTdym1); (2) mice that carried a single X chromosome and lacked all endogenous Y chromosome genes (XO). The progression of spermatogenesis in XYTdym1 males transgenic for Sox9 (XYTdym1Sox9) was assessed through various sperm and testicular analyses such as sperm counts, motility, and morphology evaluation, as well testis weight, shape, histology, vasculature assessment and mRNA expression levels. The fertility of XYTdym1Sox9 males was tested through fecundity trials and in vitro (IVF). These investigations revealed that despite some testicular abnormalities transgenic overexpression of Sox9 transgene in the context of YTdym1 can replace the function endogenous Sry and allows for sufficient levels of spermatogenesis and fertility. In the mouse only two Y chromosome genes were shown to be necessary for successful assisted reproduction: Sry and spermatogonial proliferation factor Eif2s3y. When Sry and Eif2s3y were transgenically added to mice carrying a single X chromosome (XO), these mice (XEif2s3yOSry) developed as phenotypic males and produced haploid gametes. The second part of my dissertation, focuses on replacement of Sry and Eif2s3y with their non Y chromosome encoded homologues, Sox9 and Eif2s3x. We found that males with Y chromosome genes limited to two (XEif2s3yOSry), one (XOSry,Eif2s3x and XEif2s3yOSox9) or none (XOSox9,Eif2s3x) can father offspring with assisted reproduction. However, we also observed that these males had various abnormalities of the seminiferous epithelium and testicular interstitium. Moreover, sometimes the gonads of the transgenic males resembled ovotestes and had elevated expression of ovarian markers. These findings led to a hypothesis that pathologies observed in males with limited Y chromosome genes arise at the time of sex determination due to altered signaling in the genital ridges. To test this hypothesis, I compared gonadal development in males without Y chromosome and in which sex determination is initiated either by Sry or Sox9, and spermatogenesis is driven by either Eif2s3y or Eif2s3x. My findings support the initial hypothesis and suggest that all XO males with limited Y chromosome genes have altered gonadal development at the critical time of sex determination. The failure to completely antagonize female specific signaling and establish characteristic male-like gonadal morphology is especially prominent in genotypes that have sex determination driven by transgenic overexpression of Sox9.