Methylation Analysis Of Bovine Spermatogenesis And Stem Cells Differentiate Into Sperm Cells In Vitro

Study on the DNA methylation during bovine spermatogenesis and search for genes related to spermatogenesis.Then combined with culture and induction of stem cells in vitro to establish a complete system for inducing stem cells into functional germ cells.Thereby promoting animal genetic breeding,variety improvement and genetically modified varieties.Spermatogenesis consists of three stages including mitosis,meiosis and sperm formation,in which DNA methylation plays an important role.The normal expression of DNA methylation regulates the correct occurrence of sperm,while its abnormal expression causes abnormal sperm function.In this study,four different types of stem cells during bovine spermatogenesis were obtained by Laser Capture Microdissection(LCM).The study was done to analyze the DNA methylation changes in spermatogenesis and filtered out the functional gene.Besides,the bovine testicular mesenchymal stem cells(B-TMSCs)were successfully isolated and cultured in vitro,and RA was used to induce the TMSCs differentiate into stages of meiosis.The aim was to provide a theoretical basis for inducing differentiation of B-TMSCs into germ cells in vitro.LCM was used to obtain spermatogonial stem cells(SSCs),primary sperm cells(PSCs),round sperm cells(RSCs)and long sperm cells(LSCs).Single-cell whole Genome Bisulfite Sequencing(sc-WGBS)was used to analyze DNA methylation changes in the spermatogenesis of bovine.The results of the study indicated that the level of DNA methylation was dynamically changed during spermatogenesis.The tendency of SSCs to PSCs were hypermethylation.PSCs to RSCs were hypermethylation and hypomethylation cross appearance.RSCs to LSCs once again exhibited hypermethylation status.It is revealed that DNA methylation has a regulatory effect on spermatogenesis.Differentially methylated regions(DMR)gene showed there were 16134 differential genes in PSCs and SSCs comparation group.Besides,this group had the most significant difference in DNA levels.While there were 91 DMR genes in the RSCs and PSCs comparation group and 97 DMR genes in the LSCs and RSCs comparation group.In the PSCs and SSCs comparation group,there were various ubiquitin-protein ligase(E3)related genes(TRI11,SH3R1,ZNRF1,MARH8)and Histone-lysine-methyltransferase related genes(KMT2C,EHMT1,SMYD3,NSD3).The results demonstrated that E3 play an important role in spermatogenesis.And DNA methylation remains closely linked with histone modifications in spermatogenesis.KEGG analysis of DMR genes showed that the Rap1 signaling pathway in PSCs and SSCs comparation group had the most significant difference in DNA levels.This signaling pathway plays an important role in regulating germ cell-supporting cell adhesion and maybe an important signaling pathway in spermatogenesis.B-TMSCs were successfully isolated and cultured in vitro.RT-PCR analysis demonstrated that the B-TMSCs expressed CD105 and CD166,besides the flow cell analysis showed that the B-TMSCs expressed CD44,CD90,CD166 and positive rates were all above 98%.Colony forming efficiency indicated that the cell had the self-renew potential in vitro.The T-BMSCs were successfully inductedinto adipocytes and osteoblasts in vitro,which showed that the T-BMSCs have multi-directional differentiation potential.Besides,the T-BMSCs were induced into stages of meiosis by RA in vitro.Thus,B-TMSCs can be selected as the research object to do continue study combined with the differential genes obtained in this research.Study on the differentiation into germ cells in vitro will lay the theoretical foundation for the establishment of a complete induction of stem cells differentiation to form germ cells.