Expression And Regulation Mechanisms Of HSP32 Gene In Heat Stress Mouse Testes

In this paper,the effects of various type heat stresses on testes histology,testicular enzyme activities,serum testosterone,testicular cells cycle and apoptosis,HSP32 and Caspase-3 gene expression in mice were detected.Then Sertoli cells were cultured,and HSP32 and Caspase-3 gene expression and apoptosis of Sertoli cells after heat treatment were determined.The effects of HSP32 specific siRNA and HSP32 inducer on heat induced Sertoli cells apotosis were determined.The objective of this study was to reveal the protective mechanism of HSP32,and to provide some references for identifying the micro-environment of seminiferous tubules under stress condition.1 Effects of chronic moderate and acute transient heat stress on testes histology,testicular antioxidant capacity,serum testosteroneThe objective of this study was to determine whether exposure to chronic moderate and acute transient heat would lead to the change of testicular oxidative balance,histology and serum testosterone.Chronic moderate heat exposure:Fourty-eight 3 weeks old mouse were randomly divided into two groups.Animals were kept artificial climate incubator under moderate temperatures(39?)and 60%relative humidity for 1.5h/day up to 9 weeks old or left at room temperature.Acute transient heat exposure:Fourty-eight 9 weeks old mouse were randomly divided into two groups.Animals were kept artificial climate incubator under acute temperatures(43 ?)and 60%relative humidity for 30min,then recovery for 3h,6h and 12h at room temperature.Antioxidant enzyme activities and malondialdehyde(MDA)in testis were determined.The changes of testicular histology were detected and serum testosterone was quantified by RIA.The results showed that testicular superoxide dismutase(SOD)and GSH-Px significantly decreased(P<0.05),while testicular MDA significantly increased(P<0.05)by 1 week after moderate heat exposure.After acute heat exposure,there was no difference between control and acute heat exposure group on SOD and GSH-Px activities,while testicular MDA significantly increased(P<0.01).Pathological lesions were observed by histopathological examination after moderate heat exposure 1 and 3 weeks and acute heat exposure 6 and 12 hours.After chronic heat exposure,serum testosterone level decreased,but there was no significant difference.After acute heat exposure,serum testosterone level decreased(P<0.01).In conclusion,chronic heat exposure can induce the hyperthermal adaptation of mice and the influence of heat exposure in aucte heat exposure is graver than in moderate heat exposure.2 Effects of chronic moderate and acute transient heat stress on testicular cell cycle,apoptosis,HSP32 and Caspase-3 expressionHeat exposure is known to be a stressor in testis.In response to heat stress,cells respond by inducing a set of heat shock proteins that play important roles in repair and protective mechanisms.The purpose of this study was to investigate the expression pattern of heat stress protein 32(HSP32),testicular cell cycle,apoptosis after chronic and acute heat exposure,while Caspase-3 expression and activity were determined.The testicular cell apoptosis and cell cycle in mice were examined by flow cytometric(FCM).The expression of Hsp32 and caspase-3 were verified after chronic and acute heat treatment by RT-PCR and western blot.Chronic and acute heat exposure induced the change of cell cycle.Chronic heat exposure for 3 consecutive weeks induced HSP32 and Caspase-3 expression increased.During whole chronic heat treatment stage,the level of HSP32 expression was always high(P<0.05),but the level of Caspase-3 protein expression and Caspase-3 activity had no significant different after heat treatment 6 week.Acute heat exposure for 30min induced the expression of Hsp32 and Caspase-3 increased.These results indicate that exposure of mice to chronic and acute heat stress causes the protein level of HSP32 increased,it maybe act as a cytoprotective mechanism and protect the mouse testicular function through down-regulation the expression of Caspase-3 in heat stress mice.3 Effects of chronic moderate and acute transient heat stress on Sertoli cells apoptosis,HSP32 and Caspase-3 expressionHSP32 protein was present in the Sertoli cells and absent in the germ cells.Sertoli cells are the only somatic cells in the seminiferous epithelium;germ cells depend on Sertoli cells for structural and nutritional support.Any factors that impair Sertoli cells may cause ill effects on micro-environment of spermatogenesis.To examined the expression changes of HSP32 in Sertoli cells,apoptotic rate and Caspase-3 expression after moderate or acute heat exposure,we evaluated HSP32 and Caspase-3 mRNA and protein expression by RT-PCR and western blot,apoptosis by annexin V binding and caspase-3 activation by a colorimetric assay.There was no significant difference in caspase-3 protein expression and caspase-3 activity after moderate heat exposure;however,the mRNA and protein levels of HSP32 were significantly higher(P<0.01)compared to the control group.Tansient,acute heat exposure significantly affected the mRNA and protein expression of HSP32 and caspase-3 in the Sertoli cells.However,caspase-3 activity was significantly higher(P<0.01).These results suggest that HSP32 may have a protective function in heat-stressed Sertoli cells.4 Effect of blocked and activated HSP32 gene expression on hyperthermal inducedapoptosis and regulation pathwayThe aim of our investigations was to identify the possible roles of HSP32 in hyperthermia stress Sertoli cells and to examine whether HSP32-produced CO is involving in the regulation mechanism.An HSP32 anti-sense oligonucleotide blocked HSP32 expression and HSP32 activator hemin were used,we evaluated apoptosis by annexin V binding and caspase-3 activation by a colorimetric assay.In addition,we analyzed the hsp32-derived CO contents in cultured media and cyclic guanosine monophosphate(cGMP)production by ELISA.The results showed that the specific siRNA of HSP32 induced Sertoli cell apoptosis,while the specific siRNA of HSP32 inhanced hyperthermal induced Sertoli cell apoptosis.Preincubation with hemin suppressed Sertoli cell apoptosis induced by hyperthermia treatment.Hyperthermia and hemin increase HSP32 gene expression and the production of CO which,in turn,stimulates the generation of cGMP.Taken together,our results suggest that HSP32 is a protective factor in heat-stressed Sertoli cells,and that CO generated from HSP32 is involved in the apoptotic pathway mediated by caspase-3.HSP32 is a protective factor in the Sertoli cells,it may play a role in suppressing apoptotic signals.To further confirm the anti-apoptotic role of hsp32,we first used activator and hsp32-specific siRNAs in the Sertoli cells.