Research Of Specific Expression Of Nanos2 Gene In Buffalo Spermatogonial Stem Cells

Buffalo spermatogonial stem cells(SSCs)are a kind of stem cell not only can proceed self-renewal but also activate meiosis to produce haploid gametes that carry genetic material.It plays an important role in the process of buffalo reproductive physiology.The studies of buffalo spermatogonial stem cells are hopefully to solve the fecundity problem of buffalo,also are more favorable for us to take deep analysis of buffalo spermatogenesis and to produce transgenic animals using spermatogonial stem cells.In the process of studying spermatogonial stem cells,finding a gene that can be used as a specific marker of buffalo spermatogonial stem cells is the basis for our subsequent experiments.Nanos2 belongs to the Nanos gene family and is an important RNA binding protein gene.It can regulate and control the development and self-renewal of male germline stem cells in mice.There is no related research on that gene in buffalo.In this research project,we expect to confirm a specific marker gene of buffalo spermatogonial stem cells through with the detection of gene Nanos2 expression in the buffalo spermatogonial stem cells,thus to do some in-depth study with the related regulation and signal pathway.Chapter one:get the full length of the Nanos 2 sequence of buffalo by using the method of rapid amplification of cDNA(RACE).Then through the bioinformatics analysis,we found that buffalo Nanos2 was highly conserved with other common mammalian species.Chapter two:detecte the mRNA expression level of Nanos2 in buffalo by using the method of qRT-PCR,and we found that although Nanos2 was expressed among multiple tissues of buffalo,the mRNA expression level in testis tissues was the highest.The mRNA expression level of Nanos2 in the buffal testis decreased as they grow older,and it was higher in both fetal and adolescent tissues and decreased in adult testes significantly.After cell sorting,the sorting-out spermatogonial stem cells were taken gene expression detection using qRT-PCR,and found the expression level of Nanos2 in buffalo spermatogonial stem cells was significantly higher than that in other types cells in buffalo testes.We then used the immunohistochemical analysis found that the expression of NANOS2 in puberty testis was similar with PGP9.5,a typical spermatogonial stem cell marker.On the contrary,the expression of NANOS2 was lower in adult testes and was limited to elongated sperm cells.The size of the NANOS2 protein was about 37 kDa which detected by WesternBlot experiments.Chapter three:We are tried to explore the in vitro structural reorganization of buffalo testis using the experimental vehicle of immunodeficient nude mice,and hope to construct a functional model for spermatogonial stem cells research in vitro.The feasibility of tissue remodeling in xenografts of nude mice was verified by preliminary trial,which provided the basis for subsequent experiments of effects of Nanos2 regulatory mechanisms on spermatogonial stem cells in buffalo male reproductive physiological process.The above study has proved that Nanos2 is a molecular marker of early stage spermatogonial stem cells,and may be similar to its expression pattern in mice,was an regulatory gene which plays a key role in the development and self-renewal of buffalo spermatogonial stem cells.