Studies On In Vitro Culture Of Spermatogenic Cells

In present studies, the different cell-separation methods, the concentrations of testosterone(T) which were supplemented to the culture medium, the various conditions by cocultured with Sertoli cells, the evaluation methods in their developmental stages and early stages spermatids maturing to later stages were investigated by cultured dissociated spermatogenic cells from rat. Based on this experiments, human spermatogenic cells were further cultured in order to open new ways for the treatment of patients with azoospermia and help them to give birth to their own normal progenies. The studies were divided into two phases, the first phase was to culture spermatogenic cells in vitro by means of animal experiments, the second was to culture the spermatogenic cells from the testicular tissue of patients.The first phase consists of five experiments.In the first experiment,three kinds of cell-separation methods, namely, grind-percoll method, single enzyme-grind-percoll method, and combination of enzymatic digestion were used to obtain dissociated spermatogenic cells from rats testicular tissues. The total cell number, the isolating time and the cell livability were evaluated in this experiment. The results demonstrated that the combination of enzymatic digestion obtained the largest cell number and highest percentage of living cells and consumed the shortest time among the three kinds of methods(P<0.05). The highest percentage motility of the cells isolated by combination of enzymatic digestion was obtained after in vitro culture 48h(P<0.05), but the cell death rate was the highest of three kinds of methods, yet no statistically significant difference was observed in it(P > 0.05).The different concentrations of hormone which was supplemented to the culture medium with T were studied during the spermatogenic cells culture in vitro in the second experiment.When the culture medium was supplemented with 0ng/ml, 340ng/ml, 1700ng/ml, and 3400ng/ml testosterone, namely, T0 T1 T2 T3 group respectively and with 50IU/L FSH in each group in order to evaluate the spermatogenic cells growth action, percentage motility and their differentiationfrom spermatocytes to spermatids. The results showed that, there were no distinct effect on the percentage motility of spermatogenic cells at different concentrations of T, but the largest number of spermatids differentiated from spermatocytes was obtained at concentrations of 1700 ng/ml T(P<0.01).Three conditions of spermatogenic cells coculture with Sertoli cells in vitro were studied in the third experiment.In the first group, the rat spermatocytes were cultured in DMEM/F12 medium supplemented with other components, whereas the second group, Vero cells were added into DMEM/F12 medium, and the third group.the cells were cultured in modified human tubal fluid (mHTF) medium supplemented with other components.The study was to observe the cells developmental state, percentage motility in different time during coculture and the number of early phase spermatids differentiated from primary spermatocytes. The results showed that primary spermatocytes in all groups were able to differentiate into spermiogenic phase,but the first group was better than that of other two groups in term of the cells developmental state, percentage motility during culture and the number of early phase spermatids differentiated from primary spermatocytes (P< 0.01).Some matured to Sbl, Sb2 phase spermatids presented both in the second group and in the third group.The second group showed better effect compared with the third group (P<0.01).The first group showed no Sb2 spermatids appearance.5-bromo-2-deoxyuridine(BRDU)-labeled immunocytochemistry and flow cytometry (FCM) were used to detect spermatogenic cell differentiation in the fourth experiment. Rats were injected with BRDU 9 days before experiment, in order to labeled the spermatogenic cells'DNA in vivo.The BRDU-labeled secondary spermatocytes presented in cultures after 4~6 days of culture, and round spermatids presented in cultures after 8-12 days of culture.