H19 LncRNA Regulates Proliferation And Apoptosis Of Bovine Male Germline Stem Cell Via IGF-1 Signaling

Male germline stem cells(m GSCs)are unique adult germ cells with self-renewal potential and spermatogenesis function in the testis.However,further studies are needed to establish a long-term cultural system of m GSCs in vitro,especially for large animals such as bovine m GSCs.In this study,we first established a stable immortalized bovine male germline stem cell line by transducing Simian virus 40(SV40)large T antigen.The proliferation of these cells was improved significantly.These cells could express spermatogonial stem cell(SSC)-specific markers,such as PLZF,PGP9.5,VASA,LIN28,and CD49 F,both in the m RNA and protein levels.Additionally,these cells could be differentiated into three germ layer cells to enter meiosis,form colonies,and proliferate in the seminiferous tubules of busulfan-induced infertile mice.The immortalized bovine m GSCs maintain the criteria of m GSCs.In addition,we used the immortalized m GSCs line as the research material.We found the IGF-1 pathway played an important role in regulating proliferation and apoptosis of m GSCs.When the IGF-1R was blocked,the proliferous ability of m GSCs was declined and the apoptosis of m GSCs was increased.We also found that the long non-coding RNA H19 could regulate the IGF-1 signaling pathway,sequentially regulating the proliferation and apoptosis of m GSCs.1.The isolation,purification and identification of bovine mGSCsBovine mGSCs were purified by magnetic activated cell sorting(MACS)with THY1 antibody,and the GFRA-1 positive cells were significantly higher than control group.We identified these cells through morphology,PCR and immunofluorescence staining.These purified cells expressed SSC-specific marker like PLZF,GFRA-1,PGP9.5(also named as UCHL-1)?LIN28A.Germ cell marker:VASA,pluripotent marker: OCT-4,NANOG,SOX-2,KLF-4,self-renewal marker: ETV-5.These results suggest that our isolated cells were putative bovine m GSCs.Next,we have successfully established the immortalized bovine m GSCs by transducing SV40 large T antigen,the cells grew well after 30 passages in vitro,and the culture time was 6±1 months.The proliferation of these cells was improved significantly,and the demand of serum and cytokines or other nutrition was lower than wild type.2.Immortalization of the bovine Male Germline Stem CellsThe immortalized cell line did not lose the characters of SSC-specific markers identified by PCR,immunofluorescence staining and Western blot.The cultured cells expressed SSC-specific markers(GFRA1,PGP9.5(also known as UCHL-1),and LIN28),germ cell marker(VASA),self-renewal marker(ETV-5),and pluripotent marker(OCT4).The cells also expressed SSC-specific markers(PLZF and LIN28)and pluripotent markers(OCT4,NANOG,SOX2,and KLF4).These cells were positive for pancreatic and duodenal homeobox 1(PDX1),an endoderm lineage protein;a-smooth muscle(ASM),a mesoderm lineage protein and NESTIN,an ectoderm lineage protein,as compared with control.In addition,the expression levels of meiosis markers(STRA8,SCP3,DAZL,and ESCO2)in RA stimulation group were higher than in the control group.Immunofluorescence showed that the percentage of positive cells for STRA8(pre-meiosis marker),DAZL,and ESCO2(meiotic marker)in RA treatment group was higher than that of the control group.3.H19 lnc RNA alters Self-renewal of bovine male germline stem cell via IGF-1 signalingIn this study,we used different concentrations of IGF-1(20ng/m L,40 ng/m L,60ng/m L)to stimulate the immortalized bovine m GSCs,and we found IGF-1(40 ng/ml)could promote the proliferation of bovine m GSCs in a dose-dependent manner.Next,we blocked IGF-1R by IGF-1 specific receptor inhibitor(PPP),and then we found the proliferation of bovine m GSCs was reduced identificated by CCK-8,EDU,western blot,flow cytometry.Finally,we investigated the mechanism of IGF-1R signaling on the survival of bovine m GSCs.We found that the IGF-1 signaling pathway could be inhibited and the cell proliferation was inhibited when the non-encoding RNA H19 was interfered by si RNA.The study demonstrated H19 could play an important role in regulating the proliferation and apoptosis of m GSCs.