Effect Of PLZF On The Characteristics Of Dairy Goat Male Germline Stem Cells

Male germline stem cells(mGSCs)locate on the basal membrane of the seminiferous tubules of the testes,which can form sperms and maintain the stability of their populations by self-renewal.And mGSCs are the only male adult stem cells that can pass the genetic information to the next generation through natural reproductive process in vivo.Prominelocytic leukemia zinc finger(PLZF),acts as a multi-functional transcription regulator,was involved in many biological processes,such as embryonic development,differentiation,proliferation and apoptosis.In recent years,PLZF,as a highly conserved multi-functional transcription factor involved in many important physiological development process,has aroused people's attention.PLZF is a widely recognized marker gene of mGSCs,which is expressed in undifferentiated spermatogonia.PLZF plays an important role in the regulation of mGSCs' pluripotency maintenance,the balance between differentiation and self-renewal,and the fertility of male individuals.As an important transcription factor,PLZF is expressed in a strict tissue-specific and stage-specific fashion during spermatogenesis,involved in maintaining the dynamic balance of mGSCs between self-renewal and differentiation.Although progresses have been made in understanding the molecular mechanisms underlying PLZF regulation of self-renewal versus differentiation in mGSCs,these achievements were made in model animal mice.Especially,the role of PLZF in livestock mGSCs is less reported.Exploring the effect of PLZF on the dairy goat mGSCs is of great significance to further understand the mechanism of spermatogenesis.In this study,we constructed a PLZF overexpression lentiviral vector and a PLZF interference lentiviral vector,and used them to infect the dairy goat mGSCs,then screened several cell lines overexpressed and interferenced PLZF.The effects of PLZF on pluripotency,self-renewal,differentiation,proliferation and apoptosis characteristics of dairy goat mGSCs were investigated by immunofluorescence,qPCR,Western blot,EdU staining,cell growth curve,CCK8,and flow cytometry.1.The construction of PLZF overexpression and interference lentiviral vectors,and screening of stable transduced cell lines.We successfully constructed the PLZF overexpression lentiviral vector pPLZF-CDH and PLZF interference lentiviral vector pshPLZF.The lentiviral vectors were then packaged and transduced to the dairy goat mGSC.Cell lines overexpressed or interfered PLZF were obtained after screening.2.PLZF promotes the pluripotency and self-renewal of dairy goat mGSCs and inhibits their differentiationBy immunofluorescence staining,qPCR and Western blot,we found that the expression level of GFR?1,Vasa,CD49 f,CD90,Oct4,Etv5 were significantly up-regulated while the expression of c-Kit was significantly declined.Further,we also found that Tet1 transcription was obviously down-regulated by qPCR,while Dnmt3 a was distinctly up-regulated,which suggested that PLZF promotes the methylation of DNA.3.PLZF inhibited the proliferation of dairy goat mGSCs and promoted its apoptosis.We found that PLZF overexpressed mGSCs showed decreased cell growth rate,cell cycle arrested at G0/G1 phase and increased cell apoptosis through cell growth curve,CCK8,flow cytometry detection of cell cycle,immunofluorescence staining,qPCR and western blot.Cell transplantation assay showed that testes transplanted with PLZF overexpressed cells were smaller,and the quantity and quanlity of cell clusters in the seminiferous tubules were not as good as the control groups.While testes transplanted with PLZF interfered dairy goat mGSCs were bigger than the control groups,and the quantity and quanlity of cell clusters in the seminiferous tubules were better than the control groups.