Functional Study On BAD Gene Affecting Year-round Estrus Trait In Sheep

In China, most of sheep breeds are seasonal estrous, which limits the production efficiency of sheep industry. The year-round estrus could improve the ewe productivity. To change seasonal estrus at genetic level and cultivate new breeds, it is important to understand the molecular mechanism of year-round estrus of sheep. BAD gene was a key differentially expressed gene between the year-round estrous and seasonal estrous sheep ovaries from the result of RNA-seq performed by our team. To identify the result of RNA-seq and investigate the regulatory mechanism of BAD in year-round estrus, here we identified the BAD gene function. The follicles of Small Tail Han sheep were selected in this study, granulose cells were isolated by the aspiration method, and a proper granulose cells culture system was constructed. To identify the purity of granulose cells, cell immunofluorescence technique was used to detect the expression of FSHR protein on granulose cells. The most effective BAD-siRNA was selected and transfected onto granulose cells in vitro. The expression of BAD gene at mRNA level was detected by real-time quantitative PCR. The expression of BAD at protein level was detected by Western-blot and cell immunofluorescence technique. P4 and E2 levels secreted by granulose cells were detected by radioimmunoassay. The results were listed as follows. Real-time quantitative PCR revealed that BAD gene expression at proestrus was significantly higher(P<0.05) than that at estrus, diestrus and anestrus. BAD gene expression at different estrous cycle sheep ovaries was corresponded with the result in RNA-seq. FSHR positive rates in granulose cells were over 98%. It showed that the granulose cells we cultured had high purity. Real-time quantitative PCR indicated that BAD gene expression decreased 68.82% at mRNA level in 48 hours after BAD-siRNA transfection, and Western-blot and cell immunofluorescence technique revealed that BAD expression was also significantly decreased. The results showed that P4 secretion level was significantly increased(P<0.01), and E2 secretion level was decreased but not significantly(P>0.05). In conclusion, BAD gene might regulate P4 secretion level to control sheep year-round estrus.