The Role Of Endoplasmic Reticulum Stress-activated PERK/eIF2α Signal Pathway On Glyphosate-inhibited Testosterone Synthesis In TM3 Cells

Objective: Glyphosate is the most widely used herbicide in the world.In recent years,many studies have demonstrated that glyphosate exposure was related to male reproductive dysfunction such as abnormal development of male reproductive organs and decline in semen quality.Inhibition of testosterone secretion might be one of the important inducements of male reproductive dysfunction.However,the molecular mechanism of glyphosate-induced testosterone synthesis disorders is still unclear.In the present study,the effects of glyphosate on testosterone secretion and the possible role of endoplasmic reticulum stress and downstream PERK/eIF2α signal pathway in the process were investigated in TM3 mouse Leydig cells.Methods:(1)After TM3 cells were exposed to glyphosate at different concentrations from 0.01 to 2000 mg/L for 24 h,the effect of glyphosate exposure on cell viability was detected by CCK8 method.The culture medium of cells was collected and the concentration of testosterone in the medium was determined by enzyme-linked immunosorbent assay(ELISA).(2)TM3 cells were treated with 5 mg/L glyphosate for different durations(1,3,6,12 or 24 h),and then the proteins were extracted using RIPA lysing buffer.The protein expression levels of steroid hormone synthases,endoplasmic reticulum(ER)stress and PERK/eIF2α signal pathway-related proteins were detected by western blotting.(3)To analyze the role of ER stress in glyphosate-induced testosterone synthesis disorders,TM3 cells were pretreated with PBA and exposed to 5 mg/L glyphosate for 24 h.The expression levels of steroid hormone synthases St AR and CYP17A1 were analyzed by western blotting and immunofluorescence analysis,the concentration of testosterone in the culture medium was detected by ELISA.(4)In order to further analyze whether the PERK/eIF2α signal pathways is involved in testosterone synthesis disorders induced by glyphosate,TM3 cells were pretreated with GSK2606414 and exposed to 5 mg/L glyphosate for 24 h.The expression levels of St AR and CYP17A1 were analyzed by western blotting and immunofluorescence analysis,and the concentration of testosterone in the culture medium was detected by ELISA.Results:(1)Glyphosate at concentrations below 200 mg/L had no effect on cell viability,while glyphosate above 0.5 mg/L could inhibit the testosterone secretion in TM3 cells.(2)Treatment TM3 cells with glyphosate at 5 mg/L not only reduced the protein levels of testosterone synthase St AR and CYP17A1,but also increased the protein level of ER stress molecule Bip and promoted the phosphorylation of PERK and eIF2α.The difference between sham group and exposure group was statistically significant.(3)Pretreatment cells with PBA,an inhibitor of ER stress,alleviated glyphosate-induced increase in Bip,p-PERK and p-eIF2α protein levels,meanwhile significantly rescuing glyphosate-inhibited expressions both of St AR and CYP17A1 and restored the testosterone secretion inhibited by glyphosate.(4)When pretreatment with GSK2606414,a PERK inhibitor,glyphosate-induced phosphorylation of PERK and eIF2α were reduced.In addition,the decrease both of St AR and CYP17A1 protein expression induced by glyphosate were completely blocked,and the glyphosate-inhibited testosterone secretion was also effectively rescued.Conclusions: Based on the experimental results,we draw the following conclusions:(1)Glyphosate can inhibit testosterone synthesis via decreasing the expression of steroid hormone synthase St AR and CYP17A1.(2)Glyphosate can induce ER stress and activate downstream PERK/eIF2α signal pathways.(3)Glyphosate-induced testosterone synthesis disorders depends on the ER stress-mediated the activation of PERK/eIF2α signal pathway in Leydig cells.