Expression And Function Of PRSS23 In Bovine Follicular Granulosa Cells

Cattle are very important for animal husbandry,and are widely used in meat and milk industry.Cattle are of temperance and not seasonal affective.Singleton and low ovulation rate are important factors that restrict the expansion of cattle.So the present research is important for improving the ability of cattle reproduction.Serine protease 23(PRSS23)is associated with bovine follicular development,but the effect of PRSS23 on bovine follicular development is not clear.The Hereford cattle ovarian samples used in this study were collected from Shanxi Wenshui slaughterhouse.We proceed bioinformatics analysis for PRSS23 of bovine follicular granulosa cells,In order to distinguish the dominant follicle(DF)and the subordinate follicle(SF),estrogen(E2)and progesterone(P4)concentration in follicular fluid was determined using ELISA method.We explored the expression and localization of PRSS23 in DF and SF on molecular and protein level.Finally,bovine follicular granulosa cells were cultured in vitro and used for exploring the roles of PRSS23 in bovine follicular development.E2 concentration and the apoptosis of granulosa cells were detected after PRSS23 gene was silenced.This study will provide theoretical basis for further research the PRSS23 regulatory mechanism in bovine follicular development.The experimental research results and conclusions are as follows:1.The total length of PRSS23 mRNA CDS of bovine follicular granulosa cells was 1128bp,encoding 375 amino acids.Bioinformatics analysis showed that the protein has a signal peptide with 24 amino acids,no transmembrane zone,9 potential O-glycosylation sites,1 potential N-glycosylation site and 40 potential phosphorylation sites.It belongs to the serine protease superfamily of trypsin,and has a functional area of 140?245AA.The sequence similarity analysis showed that sheep and goats were the most closely related ruminants,and had the lowest similarity with primates,chimpanzees and gibbons,and also had the lowest affinity with mice and rats.2.The concentration of E2 and P4 of follicular fluid were determined using a competitive ELISA method.Results showed that in the 11 groups of largest follicles and the second largest follicles,the NO.2,NO.5 and NO.10 group of follicles are in accordance with the characters of DF and SF.E2 concentration in DF was significantly higher than that in SF,and the P4 concentration in SF was significantly higher than that in DF.3.qRT-PCR results showed that the expression of PRSS23 mRNA in bovine SF was significantly higher than that in DF(P<0.0/1).Immunohistochemical results showed that PRSS23 was expressed in different sizes of bovine follicular granulosa cells.Western Blotting results showed that the expression of PRSS23 in bovine SF was significantly higher than that in DF(P<0.01).4.Gene silencing of PRSS23 showed that PRSS23 restrained the proliferation of granulosa cells and the secretion of E2,which may promote the atresia of bovine follicles.