Identification of genes in the testis determination pathway has increased our understanding of the complexity of this decision pathway and provided molecular diagnosis and prevention for inherited disorders affecting sexual development. Sry is accepted as the gene encoding the testis-determining factor in mammals, yet genes directly affected by Sry are unknown. One such gene may be responsible for Sry-negative XX sex reversal, wherein an autosomal gene induces testis development in individuals lacking Sry and the Y chromosome. It is unlikely that these genes will be identified in humans because small family sizes and genetic heterogeneity are severe impediments to linkage analysis. Animal models lack these disadvantages and provide the means to study molecular events in embryonic tissue. Sex determination is expected to be similar among eutherian mammals, and steps common to all mammals should be applicable to humans. Sry-negative XX sex reversal in the canine model is inherited as an autosomal recessive trait. Affected individuals have a female karyotype (78,XX) and either testes (XX males) or ovotestes (XX true hermaphrodites). The canine syndrome is strikingly similar to familial Sry-negative XXSR in humans, in which XX males and XX true hermaphrodites occur as full siblings. The genetic etiology is unknown in both species. Characterization of the testis induction mechanism in the absence of Siy would greatly increase our understanding of testis induction at the molecular level. Our long term goal is to dissect the testis determination pathway, by characterizing genes having a testis induction role and explaining the molecular mechanism of their actions.
The specific aims of this proposal are: (1) Identify the autosomal gene responsible for canine Sry-negative XXSR, through linkage analysis and screening a BAC library. (2) Compare the gene expression pattern in gonads of affected embryos and controls during the normal period of testis determination (d28-32). (3) Compare the gene expression pattern in gonads of affected embryos and controls after the normal period of testis determination (d32-40).
For Aims 2 and 3 we will use quantitative reverse transcriptase polymerase chain reaction and whole mount in situ hybridization to compare embryonic gonadal expression of Sox9, MIS/AMh, Sf1, Daxl, Wt-1 and Sry. At the end of this grant period, we will have identified the gene causing this disorder and the step in the testis pathway that is altered by the mutation in timing, quantity, or location of gene expression.
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