Canning, Claire Ann; (2003) SRY and DAX1 in mammalian sex determination. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mammalian sex determination in its simplest form can be considered as a cell fate decision, dependent on SRY/Sry, the testis determining gene. SRY, a member of the HMG box family of transcription factors, is expressed in the indifferent XY gonad at around 10.5 - 12.0 dpc in mice and induces differentiation of the Sertoli cells which then influence other cell types in the gonad and the rest of the embryo to develop as male. In the absence of SRY/Sry expression, follicle cells develop and the fate of the bipotential gonad is switched to that of an ovary, and the embryo develops as female. Sry is rapidly evolving gene, and the human and mouse genes share no homology outside of the HMG box DNA binding domain. Despite this sequence divergence, we describe that human SRY functions identical to its murine homologue, and causes male sex determination in XX transgenic mice. We have examined the expression and localisation of SOX9, a likely target of SRY, and show that in XX gonads transgenic for human SRY, SOX9 expression is identical to that observed in XY control gonads. The lack of sequence similarity outside of the HMG box domains questions how SRY functions in regulating target genes. Our results demonstrate that the CAG repeat domain present in the mouse SRY protein is not required for SRY function during sex determination and we propose that SRY may act simply as an architectural transcription factor. In comparison to testis differentiation, little is known about female development and ovary differentiation, however it appears that an X-linked gene, DAX1/Dax1, may act as an antagonist of the male pathway. To address whether Dax1 can function as an anti-testis factor, we chose the Estrogen Receptor ligand inducible system to misexpress Dax1 before the onset of Sry expression. In vitro assays were established to test the function of this system and to ensure that DAX1 function was also retained. The initial Estrogen Receptor DAX1 fusion protein (DAX1:ER) was retained in the cytoplasm of transfected cells in the absence of ligand, and responded to ligand administration by localising to the nucleus. A reporter assay demonstrated that full length DAX1 repressed the transcriptional activation of the Amh promoter. However, DAX1:ER was unable to repress this activation and was therefore non functional. An additional fusion protein (ERglyDAX1) retained the repressive function of DAX1, however, it did not remain in the cytoplasm of transfected cells in the absence of ligand. Reasons why these two fusion proteins failed to function in the desired manner are discussed.

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