The New And Specific Markers Of Spermatogonial Stem Cells Were Determined In Mice

There is an up trend of male infertility rates in recent years. The World Health Organization(WHO) showed the prevalence of infertility ranged from 10% to 15% in the world, and the prevalence of male infertility accounted for more than a half. The poor and the declined of human sperm are the main reason for male infertility. Now, with the science and technology development, environment pollution, heredity, toxicology, drug abuse and other varieties of factors have made the scarce species extinction and male reproductive capacity decline. The new researches have confirmed that male animals(including human)infertility can be caused by chemical pollution. The male has evolved into a weak ethnic groups, even at stake. Meanwhile,it is bound to bring incalculable consequences to human and wildlife. WHO predicted that the most serious three diseases are male infertility, cancer, cardiovascular disease in 21 st century. Sperm low-temperature freezing is the only method by reproduction fostering to maintain the male fertility, including the follicle intracytoplasmic sperm injection(ICSI) and injection technology of circular sperm. But the rate was so low, just about 25%. In aspermatism patients, non-obstructive aspermatism patients accounted for 60%, and it seems difficult to capture the mature sperm. Therefore, how to protect reproductive ability of male animals(including human) has become an important task in scientific area. Spermatogonia stem cell transplantation is an advanced technology developed in such a big background, which collects the Spermatogonia stem cell from appropriate male, and then injects into seminiferous tubule of receptor animals in order to produce sperm. With the deep study of this technology, Spermatogonia stem cells can be transplanted not only homogeneously but also heterogeneously. Furthermore, much higher success rate can be achieved by completing the culture system and improving the screening and transplantation method. The premise of the application of Spermatogonia stem cell transplantation is to use the specific markers to extract the stem cells from many cells and immigrate them from the tissue recipients. The technology of Spermatogonia stem cell transplantation provides a method to improve the production livestock, protect wildlife resources, breed transgenic animals and cure infertility. Up to now, more than 10 spermatogonia stem cell markers have been discovered, but all of them lack specificity. Therefore, the objective of this study is to find better spermatogonia stem cell specific markers and observe its dynamic expression in each age stage.Methods TO observe spermatagonial stem cells protein markers in each age stage of the dynamic change using ITRAQ protein mass spectrometry technology previously and discover new markers(P63,CD71,CD98,ALDH2,E-cadherin,PLZF). The research on the basis of Gonocyte Cells Inside the testicles of mice developed into spermatogonia stem cells after born six days with normal male C57 BL / 6 mice, According to the different age groups were randomly assigned as follows: 1- 6 days, 7-14 days, 15-21 days, 22-28 days, 29-35 days, 36-42 days, 43-49 days and more than 50 days after birth( totally 8 groups), six in each group. First, to detect the above protein expression inside the testicles of mice of all ages using immunohistochemical staining method, if the proteins expressed inside the testicle specifically, to detect the expression position of the new markers using the immunofluorescence double standard method with known SSCs markers(CD90)as control,using the new found markers in mice at various stages to determine whether the new markers expressed in SSCs,with fluorescent immunohistochemical double standard.TO detect the level of gene expression of the new markers in each stage in mice using the fluorescence quantitative-PCR technique, and the expression of protein levels of the new markers at the different stages of the mice with protein western blot test.Results Immunohistochemical staining found that protein P63 expressed in all ages in mice testis has no specificity, and expressed dispersedly in the seminiferous tubule; E-cadherin protein positively expressed positively in basal membrane of testicular cells in each age mice, and other locations of seminiferous tubule; protein CD98 expressed in all ages mice testis has no specificity, expressed dispersedly in the seminiferous tubule; CD71 surface protein also expressed in all ages mice testis has no specificity, expressed dispersedly in the seminiferous tubule; ALDH2 expressed among all ages cells in mice and positively expressed in mesenchymal cells in mice. These five protein were expressed with no specificity.PLZF with specificity expressed all ages cells in mice.Conclusion P63 protein, E- cadherin protein, CD98 protein, CD71 surface protein and ALDH2 protein were all expressed in testicular tissue in mice, but have no specificity of spermatagonial stem cells.PLZF protein with specificity expressed in the testis of mice.