Effects Of Epidermal Growth Factor On Type A Spermatogonial Stem Cells In Vitro

Objectives: To study the stimulating effects of epidermal growth factor (EGF) on the self-renew and/or proliferation of the type A Spermatogonial stem cells and its mechanisms. Furthermore,to explore the effect of EGF on the type A Spermatogonial stem cells is whether or not induced by epidermal growth factor receptor(EGFR).Finally,to establish the culture system of spermatogonial stem cells in vitro and to offer the therapy technique for the male sterility. Methods: Type A spermatogonial stem cells were isolated from 9 day-old SD rat testes by discontinuous Percoll gradient centrifugation. The cell viability was determined by Trypan blue dye exclusion test.Anti c-kit antibody was applied to identify spermatogonial stem cells. The percentage of spermatogonial stem cells were determined by the cells shape through Hematoxylin-eosin coloration.The spermatogonial stem cells were cultured with EGF at different concentrations in vitro and the quantity of live cells was detected by MTT assay at different time. EGF and AG1478(EGF receptor inhibitor),PD98059(mitogen-activated protein kinase inhibitor, MAPK),AG490(janus kinase inhibitor, JAK-STAT) and BAPTA(intracellular Ca²⁺ chelator) were added respectively to the medium and the percentage of the live cells was determined by MTT assay after incubation. Results: The cell viability of stermatogonial stem cells in the medium was 88.73%±0.85%. 90.48%±1.78% of the cells which we harvested was spermatogonial stem cells. EGF significantly enhanced the number and percentage of cell survival in a dose-dependent and time-dependent manner (P<0.01).AG1478 could reduce the effects of EGF significantly(P﹤0.5).Compared with EGF group, the percentage of cell survival in PD98059 group,AG490 group and BAPTA group reduced markedly(P﹤0.01).Conclusions: EGF could promote spermatogonial stem cells proliferation obviously. It suggests that EGF play a role in the regulation of survival and /or proliferation of spermatogonial stem cells. Activation of epidermal growth factor receptor may play a very important role in EGF induced the spermatogonial stem cells proliferation. The effect of EGF on the spermatogonial stem cells may be mediated by the activations of intracellular MAPK system and/or STAT system. In addition, intracellular Ca2+ may also play a role.