Studies On Molecular Mechanism Of Porcine Immature Sertoli Cell Growth Regulated By MiR-762 And Genetic Improvement For Sperm Quality Traits

We have identified 129 miRNAs that are differentially expressed in immature and mature testes of Large White boars via a microarray approach in our pervious study.We focus on miR-762,and study its effect on pig spermatogenesis.Swine testicular(ST)cell line,isolated from swine fetal testes of 80 to 90 days,its great potential functions in boar reproductive research have not been fully explored,partly due to the unclear nature of ST cell line.Therefore,we characterized ST cells in this study.We also did association analysis between SNPs and sperm quality traits in three pig populations to explore the improved method of boar sperm quality.The main results in our study are as follows:1.We firstly identified ST cells as Sertoli cells by detecting the unique character of Sertoli cells in ST cells,two little satellite karyosomes attached to one nucleolus,via Feulgen staining and observation with light microscope and electron microscope.The purity of Sertoli cells was 100% via testing Sertoli cell marker proteins ABP and FASL in ST cells.We further confirmed ST cells were immature Sertoli cells by determining the mRNA expression of immature Sertoli cell marker gene KRT18 and mature Sertoli cell marker gene FSHR in ST cells,60-day and 180-day pig testes.The purity of immature Sertoli cells was 100% via testing immature Sertoli cell marker protein AMH in ST cells.2.Our study found that miR-762 directly bound the 3‘ untranslated region(3‘UTR)of ring finger protein 4(RNF4)and down-regulated RNF4 expression,indicating RNF4 is a target gene of miR-762.A single nucleotide polymorphism(SNP)in RNF4 3‘UTR which was significantly associated with porcine sperm quality traits led to a change in the miR-762 binding-site number.Moreover,miR-762 promoted the proliferation of and inhibited apoptosis in porcine immature Sertoli cells,and drove immature Sertoli cells into DNA synthesis phase(S phase)from pre-DNA-synthetic phase(G1 phase).RNF4 and miR-762 exerted a similar effect on immature Sertoli cell proliferation,cell cycle and cell apoptosis.miR-762 reduced AR protein and AR transcriptional regulatory activity in ST cells,while RNF4 had the opposite effect,and RNF4 co-localized with AR in ST cell nuclei,indicating that miR-762 affect immature Sertoli cells by partially weakening the AR transcriptional regulatory activity caused by decreased RNF4.We also detected that miR-762 accelerated DNA damage repair by testing ?-H2 AX which was a marker of unrepaired DNA breaks.All these findings suggested that mi R-762 might play a role in pig spermatogenesis by regulating immature Sertoli cell growth.3.Seven molecular markers in DAZL,H2 AFZ,RNF4,SPAG1,MMP9 and NR4A1 genes were genotyped and association analyzed with sperm quality traits in Duroc,Large White and Landrace pig populations.The results showed that 6 molecular markers in DAZL,H2 AFZ,RNF4,SPAG1 and NR4A1 genes are significantly associated with sperm quality traits(P<0.05).