Proliferation And Differentiation Effects Of Different Culture Methods And Cell Factors On Mouse Spermatogenic Cells In Vitro

Objective: To investigate the proliferation and differentiation effects for mouse spermatogenic cells between seminiferous tubule segments culture and mixed cells coculture in vitro, and try to establish an efficient and stable method for getting more haploid sperm cells close to mature stage. Methods: Spermatogenic cells of 7-8 days mouse were separated and cultured by the methods of seminiferous tubule segments culture and mixed cells coculture, the survival rates, proliferation and differentiation rates in two culture methods were evaluated by morphological observation, viability testing, pachytene-specific phosphoprotein gene(P19) and haploid sperm cell-specific transition protein gene(TP1) detecting and ploidy analysis of cells. Results: In seminiferous tubules fragment culture, round spermatids could be observed after 10-12 days culture, the haploid peak and haploid sperm cell-specific gene (TP1) could be detected after 10 days culture, a small amount of sperm cells with flagella or long-shaped sperms could be observed after 13-14 days culture. In the coculture of mixed cells, round spermatids could be observed after 5-7 days culture and the haploid peak and haploid sperm cell-specific gene (TP1) could be detected after 10 days culture, a small number of sperm cells with flagella or elongating spermatids could be observed after 8-10 days culture. Cell numebers, survival rates, rates of haploid sperm cells in coculture of mixed cells were significantly higher than that in culture of seminiferous tubule fragments (P<0.05). In both culture methods, the cells numbers and survival rates decreased gradually following the culturing time prolonged, and most of the germ cells and sertoli cells began to shed and apoptosis up to the 3th week and the 4th week inculture of seminiferous tubule fragments and coculture of mixed cells respectively. Conclusion: Both seminiferous tubules fragment culture and mixed cells coculture could obtain haploid sperm cells. Compared with seminiferous tubules fragment culture, coculture of mixed cells with higher cells survival rate could obtain more haploid sperm cells and earlier. Objective: To search the optimal concentrations of epidermal growth factor (EGF) and stem cell factor (SCF) as well as the they suitable compatibile concentrations during the process of spermatogenic cells coculture in vitro. Methods: Different concentrations of EGF and SCF (5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml and 100ng/ml) were added into basal mediums during the process of mouse spermatogenic cells coculture in vitro, and interaction experiments of EGF and SCF were performed simultaneously. The growth behaviors of spermatogenic cells, cell numbers, viability were observed, pachytene-specific phosphoprotein gene(P19) and haploid sperm cell-specific transition protein gene (TP1) and ploidy analysis of cells were performed during the process of cells coculture. Results: Spermatogenic cells began to multiplicate into corps or chains and connected to each other closely after 2-4 days, espacially when EGF concentration is 20ng/ml and SCF concentration is 40ng/ml. Round spermatids were found after 5-7 days, sperm cells with flagella or elongating spermatids were observed after 8-10 days, and most of the germ cells and sertoli cells began to shed and apoptosis up to the 4th week. Both EGF and SCF could increase cell numbers and survival rates with its maximum in 20 ng/ml EGF or 40ng/ml SCF, meanwhile, 40 ng/ml SCF could decrease the P19/TP1 and increase the rate of haploid sperm on the 7th day of spermatogenic cells coculture in vitro. Combinating EGF with SCF during the process of spermatogenic cells cocultrue, it was found that there are synergism between two cytokine with optimal EGF concentrations in 12 ng/ml, and SCF showed a progressive linear trend. Conclusion: Both EGF and SCF could increase spermatogenic cells proliferation in certain concentrations, besides that SCF could promote cells differentiation. There are synergism between two cytokines in increasing spermatogenic cells proliferation.