Coculture of supporting cell and spermatogonia in testes of neonatal kunming mice and the effect of Sertoli cell

Objective To isolate and purify Sertoli cells in testes of 7-8 days postnatal mice and to investigate the influence of Sertoli cells on spermatogonia cultured in vitro by the Co-culture of spermatogonia and Sertoli cell. Methods\The techniques of combined enzymatic digestion,discontinuous percoll gradients centrifugation were used prepare testicular cells suspension. Sertoli cells identified by Sudan IV staining,treated by mitomycin wereserved as feeder cells to coculture spermatogonia with the Sertoli cells layer. Compared to the spermatogonia directly cultured,the size of spermatogonia clones and spermatogonia survival time were observed and recorded under inverted phase contrast microscope,the survival quantity of spermatogonia was determined by MTT assay,and the apoptosis of the spermatogenic cells was detected by Annexin Ⅴ-FITC/PI staining,observed under laser confocal scanning microscope. Difference of the size of spermatogonia clones,survival time,proliferation rate and apoptosis rate were compared between both groups. Results Sertoli cells purity can reach to 95% and above after being identified by Sudan Ⅳ staining. Compared with the control group,the size of spermatoginia clones is larger,spermatogonia survival time is larger and survival quantity is higher,the apoptosis rate is lower. Conclusion Sertoli cells can support the spermatogonia survival and proliferation during the Co-culture in vitro.