I-17: The Mechanism of Gonadal Sex Determination

Background In mammals, a single exon gene SRY on the Y-chromosome is activated in the XY gonadal primordium and initiates a cascade of molecular and morphological events leading to testicular differentiation. SRY-encoded protein (SRY) is a transcription factor harboring a HMG-box DNAbinding motif that upregulates SOX9, which encodes another transcription factor sharing the DNA binding motif with SRY. SOX9 upregulates other genes, such as FGF9 and PGD2, which in turn stabilize SOX9 expression and contribute to testis cord organization. By contrast, in the absence of SRY or SOX9, ovarian differentiation is initiated in the XX gonad by the activation of genes, such as WNT4 and FOXL2, whose protein products antagonize testicular differentiation in gonadal somatic cells. These molecular mechanisms of sex determination have been well established by examining the mice with a deletion or mutation of individual genes involved. However, naturally occurring sex reversal often involves asymmetric gonadal sex, e.g., development of a testis and an ovary within an individual, the mechanism of which remains largely unexplained. The goal of our study is to understand the mechanism of sex determination in the B6.YTIR mouse, which develops various combinations of gonadal structures. MaterialsAndMethods The B6.YTIR mouse was established by repeating backcrosses to place the Y chromosome of a local Mus. musculus domesticus variant caught in Tirano, Italy (TIR) onto the C57BL/6J (B6) genetic background, and propagated in our mouse colony (currently at the N60 backcross generation). B6.YTIR males were crossed with B6 females, and bilateral urogenital complexes were isolated from each fetus at 11.5 – 14.5 days postcoitum (dpc) while cranial biopsy was taken for genotyping by PCR amplification of the Zfy sequence. The accurate developmental stage at 11.5 – 12.5 dpc was determined by the number of tail somites (ts) from the base of the genital tubercle. The urogenital complex was fixed and processed for immunofluorescence localization of SRY, SOX9, and MVH (DDX4) in histological sections whereas the gonad separated from the mesonephros was subjected to RTPCR of Sry, Sox9, and Wnt4. Gonadal structures beyond 14.5 dpc were evaluated morphologically. Results In the B6.YTIR gonad, Sry was expressed in a comparable manner to the normal B6.XY gonad, but consequent Sox9 expression was delayed and decreased after the peak at 20 ts. Although SOX9-positive cells were seen in the entire gonad at 19-20 ts, they disappeared except for the central region, in which testis cords were formed (ovotestes) by 14.5 dpc, or disappeared from the entire gonad, in which ovarian structures developed (ovaries). Wnt4 expression pattern was similar to that in the normal B6.XX gonad. A fetal ovotestis usually developed into a small testis by regression of ovarian components by birth while a fetal ovary continued to develop into a mature ovary full of follicles. The frequency of ovotestis development was more frequent on the left side and influenced by the maternal age. Conclusion Inefficient upregulation of Sox9 allows for partial or complete sex reversal despite normal expression of Sry/SRY. However, the ultimate gonadal sex is influenced by non-genetic factor(s).