Genetic and Epigenetic Indices of Spermatogonial Stem Cells In Vitro and In Vivo Groups

Background: Epigenetic changes, especially at the level of DNA methylation, play a role in the behavior of stem cells. It due to their ability to change, compared with genetic changes, is more suitable targets for new treatments. Although various pro-tocols have been developed to human spermatogenesis; an op-timal protocol is still needed to satisfy scientists. In this study, we examined epigenetic and genetic changes of various cultural systems according to their efficiency in proliferation and purifi-cation of human spermatogonial stem cells.Materials and Methods: Obstructive azoospermia patients-derived spermatogonial cells were cultured in five different groups under special conditions for 2 weeks, and then the cells were divided into two groups. Equal number of cell population in each group was enrichened by MACS for GFR- α1 antibody and the other part was not enrichened. Gene specific methyla-tion and quantitative genes expression of pluripotency (Nanog, C-Myc, and Oct-4) and specific germ cell (Integrin α6, Integ-rin β1, PLZF) genes in each stage were evaluated by MSP and quantitative PCR. To reveal functionality, spermatogonial cells from the selected group were transplanted to recipient azoo spermia mouse model. The immunohistochemical, morphologi-cal and genetic were evaluated.Results: Expression of germ specific genes in testicular cell suspension and after purification were significantly increased (P≤0.05). Nanog and C-Myc expression level were significantly decreased in this group (P≤0.05). The best culture system was the culture of the testicular suspension. There was no significant difference in the expression of Oct-4 among testicular cell sus-pension and other groups (P> 0.05). Gene specific methylation pattern of examined genes did not show any changes during culture period. The use of MACs for purification of human stem cells was also effective at about 69%.Conclusion: An examination of the epigenetic pattern showed that the specific methylation of the evaluated genes at this stage remained constant throughout the entire culture system over time and the culture conditions did not change the methylation pattern. Our results confirmed that culture of testicular cell sus-pension and selection of spermatogonial cells could be effective ways for purification and enrichment of the functional human spermatogonial cells and studied culture systems improved spermatogenesis in vitro.

Keywords:

Azoospermia , Genetic and Epigenetic , Human Sper-matogonial Cells