The Role Of JNK Pathway-mediated Autophagosome Accumulation In Cadmium Chloride-induced Apoptosis Of Mouse GC-2 Spd Spermatocytes

Objective:To study the effect of autophagy in cadmium chloride-induced apoptosis of mouse spermatocytes（GC-2 spd）cells and explore the underlying molecular mechanisms.Methods:GC-2 spd cells were used as an in vitro model for studying reproductive toxicity induced by cadmium exposure.The cells were treated with different concentrations of Cd Cl₂（0,5 and 10μM）for 24 h.Hoechst 33342 and MDC staining were performed to explore the formation of autophagosomes and apoptotic bodies.The expression levels of autophagy-related proteins LC3,P62,Beclin-1,Atg5,Atg7,Atg13 and apoptosis-related proteins Bax,Bcl-2,Caspase-9,Cleaved Caspase-3were examined by Western blot.Autophagy inhibitor 3-MA（60μM）and apoptotic inhibitor z VAD-fmk（50 n M）were added to cell culture in the presence/absence of Cd Cl₂（10μM）to treat GC-2 spd cells for 24h.The relationship between autophagy and apoptosis of GC-2 spd cells induced by Cd Cl₂was analyzed.Lysosomal inhibitor CQ（5μM）were added to the cell culture with/without Cd Cl₂（10μM）to treat the cells for 24h and the expression of lysosomes and LC3 were detected by immunofluorescence.The protein levels of LC3,P62 and Cleaved Caspase-3 were also measured by Western blot.The JNK inhibitor SP600125（80n M）was used to treat GC-2 spd cells for 24h.Hoechst33342 and MDC staining were performed to examine autophagy and apoptosis in the cells.The expression levels of JNK/c-Jun signaling pathway proteins,autophagy-related proteins and apoptosis-related proteins were detected by Western blot,and the effect of JNK/c-Jun pathway on autophagy and apoptosis was analyzed.Results:Compared with the control group,autophagosomes aggregated and the number of apoptotic cells increased after exposure of GC-2 spd cells to Cd Cl₂for 24h.Western blot showed that the levels of LC3II/LC3I,P62,Beclin-1,Atg5,Atg7,Atg13,Bax,Caspase-9 and Cleaved Caspase-3 proteins were increased while the level of Bcl-2significantly decreased in the Cd Cl₂-treated group（P<0.05）.Addition of 3-MA significantly inhibited expression of LC3II/LC3I,P62,Beclin-1,Atg5,Atg7 and Atg13compared with the Cd Cl₂-treated group（P<0.05）,while z VAD-fmk had no significant effect on the expression of these proteins（P>0.05）.In addition,3MA and z VAD-fmk inhibited the expression of the pro-apoptotic protein Caspase-9 and Cleaved Caspase-3.Immunofluorescence staining showed that colocalization of lysosomes and LC3 were disturbed after Cd Cl₂and CQ treatment.Western blot showed that the expression of LC3-II,p62 and Cleaved Caspase-3 was significantly increased in the Cd Cl₂and CQ-treated group compared with the CQ group（P<0.05）.Compared with Cd Cl₂-treated group,SP600125 inhibited p-JNK,p-c-Jun,LC3II/LC3I,P62,Beclin-1,Agt5,Atg7,Atg13,Bax,Caspase-9 and Cleaved Caspase-3 expression and promoted Bcl-2expression（P<0.05）,but had no significant effect on the expression of c-Jun（P>0.05）Conclusion:Cadmium chloride induces apoptosis of mouse spermatocyte cells by inhibiting autophagosome-lysosomal fusion and prompting abnormal aggregation of autophagosomes.