da Silva, Sara Luisa Morais; (1998) Sox9 : An Sry related gene involved in sex determination. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The process of sex determination involves a developmental decision which gives rise to the choice of male or female specific gonadal differentiation. These events ultimately lead to sexual differentiation, resulting in testis or ovary formation and subsequent male or female development of the embryo. The choice of either forming a testis or an ovary by the bipotential indifferent gonad, depends on the presence or absence of the Y-linked testis determining gene, SRY (Sex determining region on the Y chromosome). SRY is a member of a large family of embryonically expressed genes known as the SOX gene family (SRY - box related genes). One member of this family, SOX9, maps to chromosome 17 in humans and was recently shown to be the gene responsible for Campomelic dysplasia (CD). CD is a rare but frequently fatal syndrome, in which patients exhibit bowing of the long bones and other skeletal malformations. Of the reported CD cases that are chromosomally male, more than two thirds are phenotypically female. To begin to understand the role of Sox9 in sex determination its expression was analysed in detail during gonadal development in the mouse and in two situations where testis differentiation occurs in the absence of Sry. In the mouse, Sox9 was found to be expressed at a low level in the early genital ridge in both XX and XY embryos at a time prior to Sry expression in males. Its expression increases to a high level in testis, specifically in Sertoli cells where it is maintained throughout fetal development and in the adult testis. However, in the developing ovary the gene is downregulated coincident with the onset of Sry expression in XY embryos. In the chick, where there is no evidence for an Sry gene, Sox9 expression is also testis specific from the earliest sexually dimorphic stages. Also, in one type of experimental sex reversal in the mouse, Sox9 expression begins coincident with the morphological appearance of Sertoli-like cells in grafts of fetal ovaries transplanted to adult kidney capsules. Another pertinent question was to find genes that could be regulated by SOX9. The gene coding for the Anti-Mullerian hormone (Amh) is a good candidate. High levels of Sox9 expression just precede the onset of Amh transcription. There are two important binding sites located in the 5' region of Amh: an element that shares homology with demonstrated in vitro binding sites for the testis determining factor SRY and the Lymphoid enhancer factor 1 (LEF-1), and an element that closely resembles the AGGTCA "half site" defined for nuclear hormone receptors. The last one has been shown to be a binding site for SF1 (Steroidogenic Factor 1). Co-transfection and electrophoretic mobility shift (EMSA) assays were performed; the results indicating a co-operative effect between SOX9 and SF1 to activate the Amh promoter. A targeted mutation was introduced into the Sox9 gene via homologous recombination in ES cells. Mouse chimaeras were generated using two different methods; conventional injection into normal host blastocysts and generation of chimaeras with tetraploid host embryos. Both techniques showed that the majority of mouse chimaeras resulting from Sox9 +/- embryonic stem (ES) cells die around day 8.5 of gestation, presenting severe posterior malformations. These results are in agreement with the CD phenotype in which the patients show a variety of malformations, of which the posterior ones are the most severe, and a high mortality in uteru. All these results imply a very strong correlation in the gonad between high levels of Sox9 expression and Sertoli cell differentiation. Given the timing of expression and the XY sex reversal phenotype of CD patients, Sox9 may be a direct target for SRY in mammals, activate AMH production and be a critical Sertoli cell differentiation factor, perhaps in all vertebrates.

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