The Effects Of Endoplasmic Reticulum Stress On Arsenic-Inhibited Testosterone Synthesis In Mouse Leydig Cells

Objective Arsenic（As）is a toxic metalloid,exists ubiquitously in the environment,and induces major global public health problems because of its carcinogenicity.In recent years,more and more studies have shown that As exposure is related to adult male infertility.Reductions of testosterone and disruptions of steroidogenesis may be major causes of inorganic As-evoked reproductive dysfunctions in males.However,the molecular mechanism of As-inhibited testosterone synthesis remains unclear.The main purpose of this study was to explore the effects of inorganic As on testosterone biosynthesis in TM3 cells,and to investigate the role of endoplasmic reticulum stress（ER stress）on As-evoked testosterone synthesis inhibition in mouse testicular leydig cells.Methods（1）In order to investigate the effect of arsenic on testosterone synthesis in TM3 cells,TM3 cells were exposed to 5μM NaAsO₂.After 2,4 and 8 hours of exposure,collected supernatants and cells.The cell supernatants were used to detect testosterone,the cells were used to detect testosterone synthetic key enzymes on mRNA and protein levels.（2）In order to investigate whether arsenic could induce endoplasmic reticulum stress in TM3 cells,TM3 cells were exposed to 5μM NaAsO₂.After 2,4 and 8 hours of exposure,the cells were collected for detecting ER stress-related gene and protein expressions.（3）To elucidate whether chemical chaperone PBA could alleviate As-induced endoplasmic reticulum stress in TM3 cells,TM3 cells were pretreated with PBA,then incubated with 5μM NaAsO₂ for 8h,then collected cells.The cells were used to detect endoplasmic reticulum stress on mRNA and protein levels.（4）To further elucidate whether arsenic-inhibited testosterone synthesis was associated with endoplasmic reticulum stress,TM3 cells were pretreated with PBA,then incubated with 5μM NaAsO₂ for 8h,collected supernatants and cells.The cell supernatants were used to detect testosterone,and the cells were used to detect testosterone synthetic key enzymes expressions.Results（1）As expected,As significantly decreased testosterone level in the culture supernatant fluid（P<0.01）.Compared with the control group,NaAsO₂ markedly reduced the testosterone biosynthetic enzymes mRNA expression levels of St AR,P450SCC,3β-HSD and 17β-HSD（P<0.01）.Meanwhile,the testosterone biosynthetic enzymes protein expression levels of StAR,P450_SCC,3β-HSD and P450_17αwere also down-regulated in As-treated groups（P<0.05,P<0.01）.（2）Compared with the the control group,NaAsO₂ induced the protein expression level of GRP78 and also markedly induced the mRNA expression level of GRP94（P<0.05,P<0.01）,showing that the ATF6 signaling pathway could be activated by NaAsO₂.NaAsO₂ significantly increased TM3 cells PERK and eIF2αphosphorylation（P<0.01）;and also significantly upregulated the protein expression level of CHOP in a time-dependent manner（P<0.01）.In addition,NaAsO₂ also markedly induced the mRNA expression level of CHOP and ATF4（P<0.01）,indicating that the PERK signaling pathway could be activated by NaAsO₂.Additional experiment showed that,NaAsO₂ significantly increased TM3 cells IRE1αand JNK phosphorylation（P<0.05,P<0.01）,which indicated that the IRE1 signaling pathway could be activated by NaAsO₂.（3）TM3 cells were pretreated with PBA,then incubated with 5μM NaAsO₂ for 8h.Compared with NaAsO₂ group,PBA could decrease the mRNA expression levels of ATF4 and CHOP induced by NaAsO₂（P<0.01）.Moreover,PBA could decrease the phosphorylation levels of PERK,e IF2αand IRE1αinduced by NaAsO₂（P<0.01）,which indicated that PBA could attenuate As-induced ER stress in TM3 cells.（4）TM3 cells were pretreated with PBA,then incubated with 5μM NaAsO₂ for 8h.Compared with NaAsO₂ group,PBA could increase the level of testosterone in supernatants（P<0.01）.PBA could increase the mRNA expression levels of STAR,P450_scc and 17β-HSD inhibited by NaAsO₂（P<0.05,P<0.01）,and up-regulated the protein expression levels of STAR and 3β-HSD inhibited by NaAsO₂（P<0.01）.Conclusions（1）Arsenic could inhibit the synthesis and secretion of testosterone in testicular leydig cells.Arsenic decreased the mRNA and protein expressions of testosterone synthetic key enzyme may contribute to arsenic-inhibited testosterone synthesis in testicular leydig cells（2）Arsenic increased the levels of endoplasmic reticulum stress in testicular leydig cells,and PBA could up-regulate the testosterone biosynthetic key enzymes mRNA and protein expression levels and increase testosterone level inhibited by As.Taken together,these results suggest that ER stress signaling pathway may play an important role in arsenic-inhibited testosterone synthesis.