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.
| Identifer | oai:union.ndltd.org:ADTP/279270 |
| Creators | Diana Gabriella Farkas Ross |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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