Effect Of Hypoxia On Proliferation Of Sertoli Cell And The Expression Of Occludin In Vitro

Objective:TO explore the mechanism of infertility induced by hypoxia for investigating the effect of hypoxia on cell survival and the expression of occluding by primary rat sertoli cell culture.Methods:The testicles of Wistar rats,which were18-22days after birthday, were digested with two series enzymatic for building the primary rat sertoli cell system.The cell were identified by oil red O,hematoxylin-eosin staining and immunofluorescence methods.The cultured cells were randomly divided into oxygenous concentration groups,which were digested by trypsin:20%normoxic group,15%,10%,5%,1%hypoxia groups,each group was cultured for6h,12h,24h,48h and72h.The CCK-8assay was detected the proliferation of sertoli cell. The changes of cell morphology were observed by inverted microscope.Western blot was performed to measure the expression of occludin in each group,which was evaluated by difference analysis.Results:The growth cycle of the primary rat sertoli cell was about14days.Red lipid droplets in cytoplasm were performed in sertoli cell by oil red O,the cell was wide. The cells were monolayer by HE staining,two satellite bodies were founded in cell nucleus.The Fasl protein was expressed by immunofluorescence method.The purity of sertoli cell in vitro was more than95%.The cells were obviously retracted,the intercellular were increased by inverted microscope. The cytoplasmic contained more vacuoles and devour particles. The monolayer structure was destroyed,the apoptotic cells were increased.The cck-8assay:Compared with20%normoxic group,the proliferation of sertoli cell was gradually suppressed in15%and10%hypoxia groups,the survival of cells was declined in5%and1%hypoxia groups (P<0.01).Western blot:the occludin expression of hypoxia groups were not statistically significant in6h(P>0.05).They were gradually diminished with time and oxygen concentration after12h,the differences were statistically significant (P<0.01)Conclusion:Results suggest that hypoxia could suppress the proliferation of sertoli cell, reduce the expression of occludin.It can be inferred that hypoxia could badly affected blood testis barrier,which was partly constituted by sertoli cells.It disassembled the integrity of tight junctions,which were constituted between sertoli cells,so the regular microenvironment of spermatogenesis was destroyed in testicle,the fertility of male animals was reduced or vanished.