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SRY (Sex determining Region of Y chromosome) is the master gene initiating testis determination in mammals. To shed light on the molecular mechanism of SRY action during testicular differentiation, we examined the effects of TAT-HMG fusion protein on gonadal sex differentiation in culture. HMG is the DNA binding motif of SRY and "TAT" is a protein transduction domain. Each pair of CD1 mouse gonadal primordia at 11.5 days post coitum (dpc) was cultured with or without TAT-HMG dissolved in dimethyl sulfoxide (DMSO) up to 3 days. Immunocytochemical labeling and Real-time RT-PCR of Sry, Sox9 and Mis indicated that DMSO blocked testicular differentiation, Sertoli cell differentiation and testis cords formation, downstream of SRY. TUNEL showed a massive mesenchymal cell death, which might be responsible for disruption of testis cord formation. Treatment with TAT-HMG rescued Sertoli cell differentiation, probably by up regulation of Sry, but not testis cord formation or cell death.

Sex determination in mammals is regulated by the sex-determining region on the Y chromosome (SRY); the presence of SRY activates the male developmental pathway and suppresses the gene network necessary for female gonad development. Mutations in sex determination genes lead to various abnormal sexual phenotypes, including sex reversal syndrome in which the genetic and phenotypic sex do not match. Sex reversal syndrome has been reported in humans, mouse, and several domestic species. In horses, SRY-negative XY sex reversal syndrome has been well described and is caused by deletions on the Y chromosome. However, the molecular causes of the SRY-positive condition in horses and other mammals are not known. This research investigated five horses affected with SRY-positive XY sex reversal syndrome. Sequencing of the coding exon region of the SRY gene in the five cases showed 99-100% alignment with the sequences of normal males. Genotyping of two closely related individuals with 46 normal male controls on an equine SNP50 Beadchip identified two statistically significant SNPs in a ~16 Mb region on the long arm of horse chromosome 3 (ECA3q). The region was analyzed using Gene Ontology (GO) and Gene Relationships Across Implicated Loci (GRAIL) to select functionally relevant candidate genes for sequencing. Further analysis of the entire horse genome was done through array comparative genomic hybridization (aCGH), which investigated possible structural rearrangements, such as copy number variants (CNVs). Deletions of olfactory receptor genes were detected on multiple chromosomes and confirmed through quantitative real-time PCR (qPCR). A homozygous deletion on ECA29 in a region containing genes of the aldo-keto reductase gene family, known to play a role in interconverting sex hormones between active forms and inactive forms, was discovered in two sex reversed animals. The findings were confirmed through qPCR and fluorescence in situ hybridization (FISH), and experiments to define the specific breakpoints of the deletion through PCR have been initiated. This research represents the first systematic search in the horse genome for mutations and CNVs related to sex determination. The findings contribute to better understanding of the molecular mechanisms of sex determination in horses and other mammals, including humans.

Abstract Biotechnological strategies aimed at producing male-only offspring have the potential to improve the yield of the Australian beef industry. As a proof-of-concept project, I aimed to target the primary male sex-determining gene Sry to the X chromosome in mice, to produce a transgenic XY male that would transmit Sry – and hence maleness – to both XX and XY offspring. In this project I aimed to target a 14.5 kb DNA fragment containing Sry to an X-chromosome locus that escapes X-inactivation. After considering many potential loci, a targeting strategy and construct were designed for the SMCX locus, which is well conserved between mouse, human and bovine. A targeting vector with 5kb and 3kb arms of homology was also constructed without Sry, to target the locus. Attempts to introduce the 14.5 kb Sry fragment into the construct were unsuccessful, and a smaller construct, containing only the coding sequence of the Sry gene driven by a strong promoter, is currently being made. In order to translate this transgenic approach into cattle, other facets of bovine sex determination required investigation. First, it was important to identify the necessary regulatory regions upstream of bovine SRY needed for the gene to be functional, and secondly to investigate the timing of testis development in male bovine embryos. To enable sequence comparison, I sequenced upstream of the bovine and goat SRY gene and through bioinformatic analysis identified regulatory regions common to several mammals. I identified four regions of high homology upstream of bovine SRY conserved between human, goat, and pig, but not mouse. These regions are likely to be important for the regulation of the gene in these species, as they share unique transcription factor binding sites. From this research I concluded that 9 kb upstream of bovine SRY were likely to be useful in transgenic strategies to produce sex-reversed cattle. Although I attempted to use a 15 kb bovine genomic fragment containing SRY to sex reverse XX mice, this project was unsuccessful. I also investigated the expression pattern of genes known to have a role in sex determination, including SRY, in early bovine embryos. I identified the major time points important for male sex determination, including the first appearance of the gonadal ridge from the mesonephros at day 31, the onset of SRY expression and its peak at day 39, and the appearance of testis cords at day 42, along with the pattern of expression of many other genes downstream of SRY. This information will allow future researchers to check that transgenic SRY expression is occurring at the correct time and place for it to be able to cause XX sex reversal in cattle. I also identified some of the major time points important for female sex determination, including that ovigerous cords form between CRL 37-91 in female bovine embryos. In addition I show the cellular differentiation of the cortex and medulla at this time. I have also predicted the female germ cell entry into meiosis around CRL 40 in bovine embryos through the use of qRT-PCR for STRA8 and SYCP3. This is the first detailed account of gene expression profiles in early female bovine embryos, unfortunately the data is incomplete due to an uneven distribution of embryo ages due to the difficulty of obtaining embryos from timed matings. Hopefully in the future obtaining more female embryos of the missing stages can complete the female data. This project has provided additional basic knowledge about bovine sex-determination events to ensure the possibility of making single-sex livestock a real possibility in the future. The similarity between human and bovine developmental time frames also points to cattle being a good alternative model for human development, and emphasises the need for further research in species other than mouse, with the aim of ultimately understanding our own biology.

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