Study On Regulation Effects Of Epiregulin,Epigen And SCF On Goat Spermatogonial Stem Cell In Virto Culture

Spermatogonial stem cells(SSCS)is an adult stem cell(ASCs)located on the basement membrane of seminiferous tubules in male animals.As a kind of germ cells with self-renewal ability and differentiation potential.Spermatogonial stem cells are an important part of the initiation process of spermatogenesis.The current in vitro culture and transplantation of SSC is of significance for the study of the recovery of male reproductive function,the production of transgenic animals and the research of the mechanism of male germ cell differentiation.Although at present,in vitro culture system of SSC mice is mature.Many cytokine involved the regulation of SSCs self-renewal have been identified.However,many cytokines can affect SSC but stillun discovered.Therefore,to further study proliferation and differentiation of the potential cytokines which is possible to participate in SSCs regulation.To explore the functions of the potential cytokines.Discovering new regulatory factors of spermatogonial stem cells.To provide reference for long-term in vitro culture system of other mammalian SSCs.Epiregulin is one of the members of the epidermal growth factor(EGF)family,which canregulate normal cell proliferation and inhibits proliferation of tumor epithelial cells through the epidermal growth factor receptor(EGFR).The Epigen is present in the testis,heart,liver,and acts as a ligand for the ErbB receptor to promote mitosis in the cell.Stem cell factor(SCF)as a multifunctional cytokine that can bind to its ligand c-kit regulates the survival,proliferation and differentiation of many cells.SCF is widely present in progenitor cells,hematopoietic stem cells and germ cells.Previous studies have shown that SCF has very important physiological functions in the regulation of reproductive stem cell growth.To investigate the effects of Epiregulin,Epigen and SCF on the key genes of goat SSCs proliferation and cell pool maintenance and key proteins of their proliferation pathways during in vitro culture.Provides relevant theoretical references for the cultivation of other mammalians SSCs.The main results of this study are as follows:1.Goat seminiferous tubules were collect by digestion of hyaluronidase and clostridiopeptidase A.The single cell suspension was enrich in SSC by differential adhesion method.The results showed that the SSC marker gene of enriched spermatogonial stem cells was significantly higher than adherent Sertoli cells.The enriched SSCs were identify by fluorescence staining of SSCs surface markers CD9 and GFRA1.The results showed that we got a relatively high purity SSCs.2.The results showed that the concentration of 20 ng/mL Epiregulin,100 ng/mL Epigen,and 20 ng/mL SCF can significantly promoted the proliferation of goat SSCs(P < 0.05).The effect of concentration of 20 ng/mL Epiregulin and 100 ng/mL Epigen was significant higher than other groups(P< 0.05),but there was no significant difference between the two groups(P> 0.05).The addition of Epiregulin,Epigen,and SCF can increased the cell viability of SSCs.The cell viability of the concentration of 20 ng/mL Epiregulin is 1.4 ± 0.08 significantly higher than other groups.Studies have shown that the concentration of 20 ng/mL Epiregulin and 100 ng/mL Epigen can effectively promote the proliferation of SSCs.3.Compared with the control group,the expression of LHX 1,C-RET,ETV5 and GFRA1,the key genes for SSCs proliferation,was significantly higher than control group.However,the concentration of 20 ng/mL Epireguli and SCF could upregulate the differentiation-related genes STRA8 and C-KIT of SSCs?STRA8 and C-KIT expression of the concerntration of 20 ng/mL SCF was significantly higher than 20 ng/mL Epireguli(P<0.05).However,the expression of OCT4 and PLZF have not significantly different between all groups(P> 0.05).WB test show that while adding 100 ng / mL Epigen,20 ng / mL Epireguli and 20 ng/mL SCF can increase the expression of AKT and p-AKT.The addition of Epiregulin,Epigen and SCF to SSCs basic medium can promote the proliferation of SSCs.Adding 100 ng/mL Epigen and 20 ng/mL Epiregulin can significantly promote the proliferation of SSCs and significantly up-regulate the key genes of SSCs proliferation and AKT expression.