Expression And Function Of PRSS35 In Bovine Follicle Granulosa Cells

The cattle is a uniparous animal,and only a follicle develops and finally ovulates in one estrus cycle,while a mass of follicles that are involved in the development of the ovaries are closed and apoptotic during growing.Low ovulation rate has been a key factor in the large-scale reproduction of cattle,sheep and other uniparous animals.In the early stage of our research group,cattle were used as the experimental animal model.Using high-throughput depth sequencing technology to screen 10 genes related to the follicle development of cattle estrus cycle,we found that serine protease 35(PRSS35)was associated with the development of bovine follicles.Used as a research object of the bovine ovaries from the cattle slaughterhouse in wenshui county,Shanxi Province,the CDS sequence of bovine ovarian PRSS35 gene was firstly amplified and carried out bioinformatics analysis.Then the dominant and subordinate of cattle follicles was selected and the PRSS35 protein was also positioned in bovine follicle granulosa cells.After,from two levels of gene and protein,PRSS35 was researched the influence on the different stages of bovine follicle granulosa cells.Finally,using gene siltation,siRNA transfected granulosa cells to study the effect of PRSS35 gene on bovine follicle granulosa cells.This study will make a theoretical and practical basis for exploring the role of PRSS35 in the development of bovine follicles.The results and conclusions of this study are as follows:1.The total length of the cattle PRSS35 gene CDS sequence is 1239 bp which coded 412 amino acids.The PRSS35 protein's isoelectric point is 9.82,the protein belongs to basic protein and the molecular weight is 46.5KDa.It is an unstable hydrophilic secretory protein with 20 amino acids of signal peptide,eleven O-glycosylation sites,two N-glycosylation sites and three phosphorylation sites,and has a typical TrypSpc domain named trypsin kind serine protease domain structure.Evolutionary analysis showed that cattle,along with the buffalo of the Bovinae,and the sheep and goats of the Bovidae,were more intimate relationship than the rat and mice of the rodentia.2.The concentration of E2 and P4 was determined by ELISA from 12 cattles ovarian.The largest follicle E2 concentration of three cattles significantly higher than the second largest follicle(P<0.05)and that of the P4 concentration significantly lower than the second largest follicle(P<0.05).So,the three cattles of the largest follicle and the second largest follicle were determined as dominant follicle and subordinate follicles,respectively.3.The immunohistochemical results showed that PRSS35 protein was both expressed in the granulosa cell layer of cattle dominant and subordinate follicles.By Real-time PCR,the expression of PRSS35 mRNA in cattle dominant follicle granulosa cells was significantly higher than the subordinate follicles(P<0.001).Western Blot data showed that the expression of PRSS35 protein in cattle dominant follicle granulosa cells was significantly higher than the subordinate follicles(P<0.01).4.The gene silencing data showed that when the PRSS35 gene was silenced,the number of apoptosis of follicle granulosa cells increased,the amount of E2 of follicular fluid also reduced.It is indicates that PRSS35 gene promotes the proliferation of bovine follicle granulosa cells and plays a positive role in the growth and development of bovine follicles.