The Regulation Of FSTL3 Gene On Year-round Estrus In Sheep

The purposes of this research is to analyze the relationship between FSTL3 and year-round estrous in sheep. The results could provide theoretical basis for regulating the seasonal reproduction in sheep. FSTL3 gene which may partly regulate year-round estrous in TGF-beta pathway was screened through RNA-Seq technology performed previously. In this study, the Small Tail Han sheep and Tan sheep have been used as a model to analyze the expression of FSTL3 gene in ovary at different reproductive status. The RNAi was used to explore the effect of silencing FSTL3 gene expression in granulosa cells of Small Tail Han sheep. The real-time PCR, Western-Blot and immunoflurescence technique was used to explore the effect of inhibiting action ofFSTL3 genetic expression. Furthermore, the E2 and P4 concentrations were analyzed by RIA in cell culture medium. The results are listed as follows:1. Real-time PCR was taken to measure relative gene expression of FSTL3 in ovary of Small Tail Han sheep and Tan sheep. It was found that the expression of FSTL3 in both sheep showed regular fluctuation with the estrous cycle:FSTL3 gene expression reached peak during dioestrum and declined to the lowest in estrus. In autumn, there is no difference in the expression of FSTL3 between Small Tail Han sheep and Tan sheep. By the spring, FSTL3 expression of Tan sheep remained stable while Small Tail Han sheep kept fluctuation. To analyze the relation between FSTL3 and year-round estrous in Small Tail Han sheep, a further study on granulosa cells would be necessary.2. Immunofluorescent staining of FSHR in cultured granulosa cell showed that over 95% of cells tested are positive for FSHR.3. The RNAi was used to explore the effect of silencing FSTL3 gene expression on Small Tail Han sheep granulosa cells. Real-time PCR showed that the inhibition efficiency of 3 pairs of siRNAs are 62.33%、49.27%、85.84% respectively.4. The protein expression of FSTL3 in granulosa cell was detected by western blot and immunofluorescent staining. We found that using the RNAi can cause the inhibition of the expression of FSTL3 gene.5. The E2 and P4 concentrations in cell culture medium were analyzed by RIA.When follicular granulosa cells were treated with RNAi, it could promote secretion of E2 (P<0.05) while P4 was decreased in follicular granulosa cells culture medium(P< 0.01). It was suggested FSTL3 could partly regulate year-round estrous in sheep.