The Effects And Mechanisms Of IGF-I On Type A Spermatogonial Stem Cells In Vitro

Objective: To study the stimulating effects of insulin-like growth factor-I(IGF-I) on the self-renew and/or proliferation of the type A spermatogonial stem cells and its mechanisms. Furthermore, to explore the proper environment of survival of spermatogonial stem cells in vitro. Finally, to establish the culture system of spermatogonial stem cells in vitro. Methods: Type A spermatogonial stem cells were isolated from 10-day-old SD rat testes by discontinuous Percoll gradient centrifugation. The percentage of spermatogonial stem cells expressing c-Kit protein was determined by immunohistochemistry, and cell viability in the medium was determined by trypan blue dye exclusion test. The primary spermatogonial stem cells were cultured with IGF-I at different concentrations in vitro, and the quantity of live cells was detected by MTT assay at the 72th hours. IGF-I, PD98059 ( mitogen-activated protein kinase inhibitor, MAPK), SB203580 (janus kinase inhibitor, JAK-STAT) and BisindolymaleimideⅢ (protein kinase C inhibitor, PKC) were added respectively to the medium of spermatogonial stem cells, and the percentage of cell survival in the medium was determined by MTT after incubation. Results: 92.38% ±1.57% of the type A spermatogonial stem cells which we had harvested was c-kit positive by immunohistochemistry. IGF-I significantly enhanced the number and percentage of cell survival (Ｐ<0.01). Compared with IGF-I group, the percentage of cell survival in PD98059 group reduced markedly(Ｐ<0.01), but no significant differences in SB203580 group and BisindolymaleimideⅢ group. Conclusion: IGF-I could promote spermatogonial stem cells proliferation obviously. It suggests that IGF-I play a role in the regulation of survival and/or proliferation of spermatogonial stem cells. We suppose that the regulative effect of IGF-I on spermatogonial stem cells is mediated by activation of intracellular MAPK system, but not STAT or PKC system.