Vitamin A effects on growth and proliferation of mouse spermatogonial stem cells during in vitro culture / 中国组织工程研究

ABSTRACT

BACKGROUND:Vitamin A has important effects on growth of spermatogonial stem cells.At present,we have not found an inductive substance of promoting growth and differentiation of spermatogonial stem cells during in vitro culture.OBJECTIVE:To investigate the effect of Vitamin A on growth and proliferation of mouse spermatogonial stem cells in vitro culture.METHODS:Bilateral testis of Kunming male mice aged 5-7 days were sterileiy collected.Spermatogonial stem cells were isolated and purified using adherence and noncontinuity Percoll density gradient centrifugation.Bilateral testis was obtained from Kunming male mice aged 12-15 days under sterile conditions.Sertoli cells were isolated and purified by using enzyme digestion method.Following adherence and polarization,Sertoli cells served as feeder layer.The spermatogonial stem cells were seeded on simple-layer Sertoli cells.We set two groups,In the experimental group,1 g/L Vitamin A was added in the DMEM/F12.In the control group,Vitamin A was not added.Growth and proliferation of spermatogonial stem cells were measured using enzyme linked immunosorbent assay.The cell cycle of spermatogonial stem cells was determined by flow cytometry.RESULTS AND CONCLUSION:At days 6,9,12 and 15 following coculture,absorbance value of spermatogonial stem cells in experimental group was faster than control group(P ＜ 0.05 or 0.01).With prolongation of coculture,quantity of chromosome in S phase in spermatogonial stem cells was increased,and then decreased in the experimental group.Another division cycle began.Compared with experimental group,quantity of chromosome in S phase in spermatogonial stem cells was slowly increased in the control group(P ＜ 0.05).During in vitro culture of mouse spermatogonial stem cells,Vitamin A can improve the proliferation and polarization of spermatogonial stem cells.