Influences Of CD49f And MiR-302on Biological Properties Of Dairy Goat Male Germline Stem Cells Cultured In Vitro

Dairy goat own great potentialities in production of milk and meat, in particular, thebreast bioreactor of goat has huge valuable when it was used to produce biomedicine products.One of the current hotspots is to carry out genetic breeding and transgenic cloned using malegermline stem cells (mGSCs) as donor cells. Male GSCs are the material basis ofspermatogenesis and own the capability of dedifferentiating into pluripotent stem cells underappropriate conditions. CD49f is an efficient marker for both mouse and human mGSCs,connected tightly with pluripotency. MiR-302can regulate cell pluripotency and reprogramhuman somatic cells into pluripotent stem cells. However, the mechanisms of mGSCsdedifferentiating into pluripotent stem cells are still need to be elucidated. Here, we exploredthe applicability of CD49f as an efficient marker for enriching and identifying dairy goatmGSCs; Optimization the culture conditions of dairy goat mGSCs based on our previousreports; Exploring the biological property difference by in vitro culture of CD49f positive andnegative cells; Transduced immortalized genes into dairy goat mGSCs to explore whetherdairy goat mGSCs could be immortalized and the characteristics of the immortalized cells;Study the influences of miR-302on dairy goat mGSCs by transducing miR-302intoimmortalized dairy goat mGSCs; what’s more, the mechnisms of CD49f and miR-302actedon dairy goat mGSCs were explored based on previous reports. We discovered that:(1) Homology analysis show that the sequences of goat CD49f mRNA and amino acidwhich obtained by electronic cloning own more than80%similarity among human, mouse,cattle, rat and goat; Immunofluroenscene staining results show that CD49f positive cellslocalized on the basement of seminiferous tubules and collocated with CD90, PLZF, ETV5,H3K9me3and PGP9.5which had been reported as the markers of mGSCs. We selectedCD49f positive cells selected through MACS using CD49f antibody express the markersCD49f, CD90, VASA and PLZF; cultured the cells on MEF feeder can found SSC coloniesand their biological properties are similar to mGSCs of other species as previously reported.(2) SSC medium containing GDNF, EGF and bFGF is more suitable for culturing dairygoat mGSCs compared to the dedifferentiation medium and N2B27medium; Polylysinetreated culture plates promote the colonies formation simply and efficiently, but repress cellproliferation. Addition of5μmol/L P53inhibitor can improve the proliferation ability. (3) Cultured CD49f negative and positive cells on laminin and polylysine in vitro withSSC medium show that CD49f positive cells form SSC colonies efficiently, express moreCD49f, PLZF, OCT4, PCNA, Pou3f1and SOX2, but less P21. Induced by RA can promoteCD49f positive cells to form sperm-like haploid cells, meiosis associated genes expressedwhen determined through immunofluroscense staining. Analyze the causes found CD49fpositive cells expressed more miR-302b, OCT4, SOX2, PLZF and CDK2than CD49fnegative cells other than P21. Immunofluroscense staining results show that CD49f positivecells expressed OCT4, Ki67in nucular, however, CD49f negative cells expressed in plasm.(4) Transduced lentivirus containing SV40large T antigen and Bmi1gene into dairy goatmGSCs immortalized dairy goat mGSCs. Cell proliferation assay show that immortalizeddairy goat mGSCs own greater cell proliferation ability, RT-PCR and immunofluenscencestaining than those normal mGSCs, which shows that CD90, CD49f, BLIMP1, C-Myc,NANOG, PLZF, OCT4, STRA8, TERT, Gfra1, CD117, VASA, SOX2, CDK2, CyclinD1,PCNA and PLZF, differentiation potential examination and transplantation results allidentified that immortalized dairy goat mGSCs own all the characteristics of mGSCs. Celladherent ability improved while apoptosis repressed after the lentivirus containing miR-302was transduced into immortalized dairy goat mGSCs. QPCR identified that miR-302repressed P21, promoted CD49f expression. Primarily, we ensured miR-302acted throughP21repression and CD49f improvement.(5) Combined with the conclusions of previous reports, we identified that theproliferation of CD49f positive cells were regulated by PI3K pathway. Bioinformaticsanalysis and experiments dissected molecular mechnisms of the effects of CD49f andmiR-302on dairy goat mGSCs, also the mechanisms of the interaction of CD49f andmiR-302. The results show that CD49f upregulate C-Myc, E-Cadherin and OCT4to regulatepluripotency and cell proliferation; MiR-302regulate the expression of E-Cadherin, OCT4and P21to change the ability of cell adherent and proliferation; the interactions of miR-302and CD49f acted through the role of media molecular just as E-Cadherin and OCT4.Conclusively, our work ensures CD49f as an efficient marker for identification andenrichment of dairy goat mGSCs, and we have optimized culture system of dairy goat mGSCswithout feeder in vitro. After immortalized dairy goat mGSCs line was established, we foundmiR-302can promote cell adherence and repress cell apoptosis. Combined with theconclusions of previous reports, we primarily dissected the mechanisms of the effects ofCD49f and miR-302on dairy goat mGSCs. Our work will be of great use in the future studyof the biological properties and molecular of dairy goat mGSCs.