Preliminary Study On Factors Affecting Buffalo Spermatogonial Stem Cell Enrichment And Proliferation In Vitro

Buffalo is one of the important livestock in southern China.Due to its low reproductive efficiency and lack of excellent germplasm resources,it has seriously restricted the development of the buffalo industry.Spermatogonial Stem Cells(SSCs)are one of the most promising seed cells for animal germplasm resources improvement and new variety cultivation,and have become a hot research topic in the field of animal reproduction.At present,the in vitro culture system of mouse SSCs is relatively mature,but there are few related studies on buffalo.It is well known that the in vitro culture system of SSCs cannot meet the long-term culture needs of buffalo SSCs.This study aims to optimize the current in vitro culture system of buffalo SSCs by exploring relevant factors affecting buffalo spermatogonia enrichment and in vitro proliferation.The main research results obtained are as follows:1.To explore the factors affecting the preparation of buffalo sertoli feeder cells.The buffalo testicular cell suspension was adhered for 6 h,and the adherent cells were stained with sertoli cell-specific antibody WT1 and the purity was over 95%.Sertoli cells were treated with different concentrations of mitomycin C.The results showed that the cell proliferation of the 3.0 ?g/ml mitomycin C treatment group was significantly lower than that of the control group and the 1.5 ?g/ml mitomycin C treatment group,but with other treatment groups.There was no significant difference,so the mitomycin C treatment concentration of the sertoli feeder cells was preferably 3.0 ?g/ml.Different generations of sertoli cells(0-10 generations)were collected,and the sertoli cell-specific expression genes were analyzed by qRT-PCR.The results showed that the expression levels of GDNF in Sertoli cells of P6,P7 and P8 generations were significantly higher than those of others.The SCF and FGF2 of P7 and P8 generations were significantly higher than other generation cells.2.The expression of SSCs-related genes in buffalo of different ages was observed.The DDX4 antibody and PGP9.5 antibody were used to locate the SSCs in the buffalo testis tissue.It was found that the SSCs are mainly distributed near the basement membrane of seminiferous tubules.QRT-PCR was used to analyze the expression of SSCs related genes in pre-puberty and adult'buffalo testicular cell suspensions.The results showed that the expression of Nanos2 in pre-pubertal testicular cell suspension was significantly higher than that in adult buffalo.The buffalo testicular cell suspension was enriched in SSCs cells by gelatin and collagen rat tail.The results showed that the number of PGP9.5 positive cells in the enriched cells was significantly higher than that in the undifferentiated adherent group,and the differential adherent PGP9.5 was positive.The number of cells is 2.3 times that of undifferentiated adherent cells.3.The effects of melatonin and CHIR-99021(CHIR)on the in vitro proliferation of SSCs were investigated.Add different concentrations of melatonin or CHIR in the IMDM culture system,SSCs were passaged after 4 days of culture,and after 4 days of culture,cells were collected.QRT-PCR analysis of SSCs-specific genes and germline related genes(PLZF,DDX4,NANOS2),differentiation-related genes(DMRT1,REC8,DAZL),oxidation-related genes(CAT,SOD3)and cell proliferation-related genes(CCND1,CCNE1).The results showed that the expression levels of PLZF,DDX4 and SOD3 in SSCs of 10-6 M Melatonin treatment group were significantly higher than those of the control group.DMRT1 and REC8 were significantly down-regulated;CCND1 gene expression was not significantly different in each group.There was no significant difference in the number of DDX4 and PGP9.5 positive cells between the 10 6 M Melatonin treatment group and the control group.The expression of PLZF in SSCs treated with 10 ?M CHIR was significantly higher than that in other groups.Compared with the control group,the expression levels of NANOS2,CCND1 and CCNE1 were significantly up-regulated;DAZL,DMRT1,CAT and SOD3 were significantly down-regulated.The number of DDX4 and PGP9.5 positive cells in the 10 ?M CHIR treatment group was significantly higher than that in the control group,and the number of positive cells in the 10 ?M CHIR treatment group was increased by 1.7 times.