Effects Of Sry Silencing On Mouse Embryos Sex Determination Genes And Gonad Development

SRY (sex-determining region on the Y chromosome, Sry in mouse) is the Sex determination gene of Y chromosome. It is a"master gene"for initiatied male sex differentiation in the bipotential indifferent gonads of mammals. Since Sry was discovered numberous genes have been suggested in the literature comprising the chain of gene expression for male differentiation. There are many evidences to show that SRY is at the top of the cascade of genetic activity leading to male development in mammals. SRY gene is difficlut to study in vivo that hampered the gene research process.Untill now, the direct target genes of SRY in vivo haven't been identified. In order to identify the SRY gene molecular action and it regulation mechanism, we inhibited the Sry gene expression by RNAi and then examined the expression changes of other sex-related genes. Six genes (WT1, Sox9, SF1, GATA4, AMH and DAX1), which are suggested to be closely related to Sry regulation were studied, and we also examined the effects of mouse embryos'gonads development where Sry silencing. Our works provide a new approach to research development genes in post implantation-staged embryos and make a great effort to reveal the Sry regular action. The main research content and conclusions as follows:1. Re-Construction of Sry small interference RNA expressing vector and evalued it expressed in mouse embryos. An EGFP gene was inserted into the siRNA expression vector pSilencer4.1/Sry565 and control vector pSilencer 4.1-CMV neo, and then the vectors pSilencer4.1/Sry565-EGFP (pSry565-EGFP) and pSilencer 4.1-CMV EGFP vector (pControl-EGFP) were constrcted. The siRNA expression vector pSry565-EGFP and control vector pControl-EGFP were injected into gestated mouse through tail vein at 9.5dpc (days post coitum). Embryos were collected at 11.5dpc. A Nikon-inverted fluorescence microscope was used to determine EGFP expression. The results showed that using tail vine injection could sucsesfully transfer plasmids into mouse embryos and the plasmids were widely expressed in the embryos and have no tissue or organ bias.2. Effects of siRNA expression vector on inhibiting Sry expression. At 11.5dpc the embryos were collected and all embryos have sex determined. Male embryos were used to examine Sry mRNA by RT-PCR and SRY protein was examined by Western-blot. The result indicated that pSry565-EGFP vector could significantly inhibite Sry gene expression. An inhibition rate of 80% was obtained as determined by Western-blot analysis and RT-PCR.3. Effects of Sry silencing on the expression of WT1, Sox9, SF1, GATA4, DAX1 and AMH. The sex-related genes, including WT1, Sox9, SF1, GATA4, DAX1 and AMH, were examined at 11.5dpc by RT-PCR. Our results showed that the mRNA level of WT1 was significantly increased in Sry silencing embryos gonad compared to those in the control embryos. However, the expression level of Sox9, which was thought to be the most likely target gene of Sry in the Sry cascade, was not changed significantly. Also, no obvious change was found for GATA4, SF1 and DAX1 in Sry silencing embryos gonad, AMH gene was not detected in Sry silencing embryos gonad and not in control embryos. The results indicated that the expression of WT1 was significantly increased in the Sry silencing embryos. In contrast, no dramatic change was found for other sex-related genes by silencing of Sry.4. Effects of Sry silencing on mouse embryos gonads. Male embryos gonads tissue were collected at12.5dpc, 13.5dpc, 15.5dpc respectively, and histological section were made to observe the mouse embryos gonads development. After the F1 generation mouse were bone 5 weeks and 3 month their testis were examined. The results confirmed that the mouse embryos gonads development was hampered, and the mouse testis dysplasia. The results confirmed Sry silencing could significant interfere gonad development.