Preliminary Study On The Culture Of Buffalo Spermatogonial Stem Cell Like-Cells And Excavation Of Key Genes Related To Regulation

As an important livestock in the livestock industry of south China,buffaloes not only have high feed conversion rate,high nutritional value of dairy products,but also have strong disease resistance and rough feeding tolerance,which has high economic value.However,the breeding cycle of buffalo is long and the number of high-quality buffalo is small,so the cultivation of new buffalo varieties has become an urgent problem to be solved.Spermatogonial stem cells(SSCs)are ideal seed cells for genetic modification and germline conservation in mammals.Due to their broad application prospects in the fields of medicine and animal husbandry,they are receiving more and more attention.Long-term culture of buffalo SSCs has not been realized in vitro,which may be because we do not have enough understanding of the microenvironment of buffalo SSCs,and we know little about the related mechanism of self-renewal and differentiation of buffalo SSCs.In this study,the effects of serum-containculture system and serum-free culture system on the culture of SSC-like cells of juvenile buffalo were compared.The seminiferous tubules and spermatogonial stem cell-like cells(SSC-like cells)were used as research objects,and differential transcriptomics analysis was carried out between adult buffalo and juvenile buffalo by transcriptomics,in order to find out the key pathways and candidate genes regulating the state of buffalo SSC-like cells.The results of this study can provide some references for the study of buffalo SSCs self-renewal and proliferation related mechanisms and long-term in vitro culture system.The contents and results of this study are as follows:(1)We compared the effects of different systems on the culture of buffalo SSC-like cells in vitro.The results showed that compared with the culture system with unknown composition,the culture system with clear composition maintained the phenotype of juvenile buffalo SSC-like cells in vitro for a longer time(6 d vs 10 d)and could maintain the molecular characteristics of juvenile buffalo SSC-like cells better.(2)Transcriptomics techniques was used to analyze the seminiferous tubules of adult and juvenile buffaloes: A total of 17,299 genes were identified,including 13,174 genes with significant differences in expression,between the tubules of the testicles of juvenile buffaloes(4 months old)and adult buffaloes(2-3 years old).Compared with the adult buffalo seminiferous tubules,the biological process involved in the up-regulation genes in juvenile buffalo were mainly related to the expression of proteins on the surface of cell membrane,localization and recognition,which were closely related to the formation,recognition or homing of surface antigens of undifferentiated spermatogonial cells;while the biological processes involved in down-regulated genes were closely related to sperm differentiation.At the same time,we also enriched eight signaling pathways that may be closely related to self-renewal,proliferation,migration and other regulatory processes of buffalo spermatogonial stem cells,as well as four key genes: MYC,FZD10,ID4 and CCL27.(3)Using single cell transcriptome technology to analyze SSC-like cells between adult(2-3 years old)and juvenile(4 months old)buffalo,it was found that a total of 21,803 genes were identified,among which 1,419 genes with significant difference in expression level.Compared with adult buffalo SSC-like cells,the up-regulated genes of SSC-like cells in juvenile buffalo mainly involved in lipid metabolism,cell surface receptor signaling pathway,migration process and other biological processes related to maintenance of self-renewal status of stem cells and migration of stem cells;while the major biological processes involved in down-regulated genes are meiotic cycle,meiotic cell cycle and male gamete formation,which were related to gamete formation and differentiation.We have enriched 151 signaling pathways that may be closely related to the self-renewal,proliferation and migration of buffalo spermatogonial stem cells and candidate genes corresponding to the pathways.In conclusion,this study provides abundant reference data for further study on the mechanism of self-renewal of buffalo SSCs in vitro and long-term cultureby differential transcriptomics analysis of buffalo seminiferous tubules and SSC like-cells and in vitro.The data also laid a foundation for research on the in vitro culture of buffalo SSC like-cells.