Molecular Mechanism Of Expression And Regulation Of GPx5 Gene In Sheep Epididymis

The epididymis is one of the vital organs of the male reproductive system, responsible for sperm maturation, transport and storage. The realization of the function of epididymis depends on its vigorous physical activity, and the generation of free radicals is inevitable. Excessive accumulation of free radicals is bound to cause oxidative stress of epididymis, the sperm plasma membrane and DNA oxidative damage occurs, greatly affect the ability of the sperm motility and fertilization, which results in a decreased ability to male livestock breeding. This study discusses GPx5 gene expression characteristics of space and time on the sheep epididymis and androgen effects on sheep GPx5 gene expression of epididymis andconstruct epididymal epithelial cells in vitro, at the cellular level to study GPx5 gene expression patterns. Using RNAi technology in preliminary identification GPx5 gene transcription level of antioxidant mechanism. GPx5 gene plays a key role as sheep male fertility index. This study provide a basis for molecular auxiliary marker and GPx5 gene research in the future to lay the theoretical foundation. This study Lay the foundation for research that GPx5 gene and antioxidant mechanism of epididymis.1. Expression and locational analysis of GPx5 in the epididymidis of sheep with different ages. The study was conducted to explore the expression of Glutathione peroxidase 5 (GPx5) of sheep epididymis at different parts and different ages. The mRNA and protein expression levels of GPx5 and the localization of GPx5 in different parts of sheep epididymis at different developmental periods were determined by fluorescence quantitative PCR, immunoblotting and immunofluorescence methods, respectively. The quantitative real-time PCR results showed that the mRNA expression level of GPx5 was significantly higher than the other portion at epididymal caput of 8-month-old sheep (P< 0.05). The mRNA expression level of GPx5 in 8-month-old sheep epididymis caput was significantly higher than the mRNA expression level of GPx5 in the 2-month-old sheep and 5-year-old sheep epididymis caput (P< 0.05). RT-PCR experiments showed that the band present at 120bp. The result indicated that GPx5 gene were expressed at sperm of sheep. Western blot results showed that GPx5 protein was not expressed in epididymis of 2-month-old. All parts of the epididymis in 8-month-old sheep were expressed and the epididymal cauda of 5-year-old sheep were not expressed. Western blot analysis showed that the protein expression level of GPx5 was significantly higher than the other portion at epididymal caput of 8-month-old sheep (P< 0.05). The protein expression of GPx5 in epididymal caput of 8-month-old sheep was significantly higher than the protein expression of GPx5 in epididymal caput of 5-year-old sheep (P< 0.05). Immunofluorescence results displayed that GPx5 protein positive signals in the epididymis of the 2-month-old was not detected. All parts of the epididymis in 8-month-old sheep had positive fluorescent signal, and the fluorescence intensity of epididymal caput and corpus were significantly higher than the cauda of epididymis, and that was found the positive fluorescent signal in epididymal tube’s liquid. The epididymal caput and corpus in 5-year-old sheep had the positive fluorescent signal. Moreover, the fluorescence widely distributed in the nucleus and cytoplasm of epithelium cell in epididymal duct. Epididymal sperm had the positive fluorescent signal too. All ages sheep had the mRNA expression of GPx5, but it was significantly different in different portions. GPx5 protein distributed at the epithelium cell in epididymal duct. However, it was negative in epididymidis of sexually immature 2-month-old and epididymal cauda of 5-year-old sheep. These results indicated that the protein expression of GPx5 was closely related with sexual development stages of sheep.2. Effect of androgen on the GPx5 gene expression of sheep epididymis. The study was conducted to explore the effect of androgen on the specific expression gene GPx5 expression of sheep epididymis and method were using enzyme-linked immuno sorbent assay (ELISA);real-time fluorescent quantitative PCR and western blotting (WB). ELISA results showed that the content of DHT; T and AR in the serum and epididimis were significantly increased except for serum T after testosterone propionate(TP) treatment (P< 0.05). Real-time PCR results showed that the mRNA expression level of GPx5 and AR of TP group was significantly higher than the control group (P< 0.05). WB analysis results showed that the expression level of protein GPx5 in sheep pididymis at TP group was significantly higher than the control group (P< 0.05). These results indicated that sheep epididymis GPx5 gene expression was regulated by the androgen.3. Mechanical shear combined with enzyme digestion method, for the first time, primitive cells in vitro successful isolation and culture of epididymis epithelium. The tissue-specific expression of cytokeratin-18 (CK18) and GPx5 protein in epididymal epithelial cells was identified by immunofluorescent staining. Used RT-PCR method to detect GPx5-mRNA. Under microscopic observation the cells, they are strongly attached to each other and present pebbles shape. CK18 and GPx5 immunofluorescence detected positive signal at the cytoplasm of epididymal epithelial cells. RT-PCR experiments showed that the band present at 120bp. The result indicated that GPx5 gene were expressed at cytoplasm of epididymal epithelial cells.4. Preparation and identification of recombinant adenoviruses carrying short hairpin RNA targeting GPx5 of sheep. Using RNA interference and the recombinant adenovirus technology, The objective of this study was to silence the expression of GPx5 using the RNA interference by recombinant adenovirus。 The AdMax^TM interference system was used in this experiment. We designed and synthesized 3 pairs of complementary single-strand DNA oligonucleotides (GPx5 shRNA1、GPx5 shRNA2、GPx5 shRNA3) which targeting 3 different sites of GPx5 mRNA and then oligonucleotides were cloned into shuttle vector pHBAd-U6-Scramble-CMV-GFP. The recombinant adenovirus particles (AD-GPx5 shRNA1、AD-GPx5 shRNA2、AD-GPx5 shRNA3) were produced and further amplified by transfecting HEK-293 cells. The titer of adenovirus reached 1 X 1010PFU/ml、1.26×1010PFU/ml,1.58X 1010PFU/ml,respectively, determined by TCID 50 assays. Real-time Quantitative PCR indicated that AD-GPx5 shRNA2 and AD-GPx5-shRNA3 had better interference efficiency by infecting sheep epididymal epithelial primary cells after 24 and 36 h, in which mRNA expression levels of GPx5 gene were reduced 42%,40%,44% and 65%,,75%,73%,respectively, SOD active and GSH content of AD-GPx5 shRNA2 group was significantly higher than the AD-GFP group (P< 0.05). So AD-GPx5-shRNA2 and AD-GPx5-shRNA3 has been proved that it has significant interference effect on expression of GPx5. GPx5 gene recombinant adenovirus can effective jamming GPx5 mRNA expression and interference GPx5 gene cause other factors of oxidation increased.