Autophagy In Sertoli Cell Antagonizes Cadmium-induced Testicular Germ Cell Apoptosis

Objective:The aim of the study is to explore the influence of Sertoli cell autophagy on Cd-induced male mouse germ cell apoptosis.Methods:In the present study,the wild type and transgenic animal models are used to explore the effect of Sertoli cell autophagy on Cd-induced male mouse germ cell apoptosis.Animal experiments include four compartments.Experiment 1,8 weeks male C57BL/6 mice are intraperitoneal injected with Cd（2 mg/kg）.At 0,2,8,24 h,mouse serum and testes are collected for the concentration of Cd,autophagy and apoptosis measurements.Experiment 2,8 weeks male C57BL/6 mice are intraperitoneal injected with 3-Methyladenine（autophagy inhibitor,3-MA,20 mg/kg）for 1 h before Cd treatment.At 0,8,24 h,mouse testes are collected for the assessments of autophagy and apoptosis.Experiment 3,8 weeks male C57BL/6 mice are intraperitoneal injected with Rapamycin（autophagy inducer,Rap,1 mg/kg）for 1h before Cd treatment.At 0,8,24 h,mouse testes are collected for the assessments of autophagy and apoptosis.Experiment 4,8 weeks male Amh-Cre+/Atg5^flox/flox mice are intraperitoneal injected with Cd（2 mg/kg）.At 0,2,8,24 h,mouse testes are collected for the assessments of autophagy and apoptosis.Results:TUNEL staining displays Cd remarkably elevates the mouse testicular TUNEL positive germ cell ratio.Meanwhile,immunoblot results reveal that Cd exposure significantly elevates the testicular protein levels of C-casp 3 and C-PARP.These results suggest that Cd induces the testicular germ cell apoptosis.Additionally,immunoblot suggests that the protein levels of testicular ATG5,ATG7,LC3B-II/LC3B-I and p62 have been increased after Cd exposure.Double immunofluorescence staining also reveals the Sertoli cell LC3 positive puncta have been increased upon Cd exposure.These results suggest that Cd activates the mouse testicular cell autophagy contains Sertoli cell autophagy.Further study shows that autophagy inhibitor 3-MA pretreatment significantly reduces the protein levels of testicular LCB-II/LC3B-I and p62 compares to Cd group.Importantly,3-MA pretreatment remarkably elevates testicular C-casp 3 protein level and the ratio of TUNEL positive germ cell compares to Cd group.These results suggest that inhibition of autophagy aggravates Cd-induced male mouse germ cell apoptosis.Inversely,autophagy inducer Rap pretreatment notably elevates the protein level of testicular LCB-II/LC3B-I,and decreases the testicular p62 protein level compares to Cd group.Importantly,Rap pretreatment remarkably reduces testicular C-casp 3protein level and the ratio of TUNEL positive germ cell compares to Cd group.These results suggest that activation of autophagy inhibits Cd-induced male mouse germ cell apoptosis.Most importantly,compares to the wild type+Cd group,deficiency of Sertoli cell autophagy significantly aggravates Cd-elevated testicular C-casp 3 protein level and the ratio of TUNEL positive germ cell,remarkably reduces the Cd-elevated testicular protein levels of ATG5,LC3B-II/LC3B-I,and increases the Cd-elevated testicular p62 protein level.Meanwhile,double immunofluorescence staining displays that deficiency of Sertoli cell autophagy notably reduces the number of Sertoli cell LC3 positive puncta.Taken together,deficiency of Sertoli cell autophagy aggravates Cd-induced germ cell apoptosis in male mice.Conclusion:Sertoli cell autophagy antagonizes Cd-induced germ cell apoptosis in male mice.