Glyphosate is the most widely used herbicide in the world. In recent years, many studies have demonstrated that exposure to glyphosate-based herbicides (GHBs) was related to the decrease of serum testosterone and the decline in semen quality. 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 role of endoplasmic reticulum (ER) stress in the process were investigated in TM3 cells. The effects of glyphosate at different concentrations on the viability of TM3 cells were detected by CCK8 method. The effect of glyphosate exposure on testosterone secretion was determined by enzyme-linked immunosorbent assay (ELISA). The expression levels of testosterone synthases and ER stress-related proteins were detected by Western blot and Immunofluorescence stain. Results showed that exposure to glyphosate at concentrations below 200 mg/L had no effect on cell viability, while the glyphosate above 0.5 mg/L could inhibit the testosterone secretion in TM3 cells. Treatment TM3 cells with glyphosate at 5 mg/L not only reduced the protein levels of testosterone synthase StAR and CYP17A1, inhibited testosterone secretion, but also increased the protein level of ER stress molecule Bip and promoted the phosphorylation of PERK and eIF2α. Pretreatment cells with PBA, an inhibitor of ER stress, alleviated glyphosate-induced increase in Bip, p-PERK and p-eIF2α protein levels, meanwhile rescuing glyphosate-induced testosterone synthesis disorders. When pretreatment with GSK2606414, a PERK inhibitor, the glyphosate-induced phosphorylation of PERK and eIF2α was blocked, and the glyphosate-inhibited testosterone synthesis and secretion was also restored. Overall, our findings suggest that glyphosate can interfere with the expression of StAR and CYP17A1 and inhibit testosterone synthesis and secretion via ER stress-mediated the activation of PERK/eIF2α signaling pathway in Leydig cells.

草甘膦是世界上使用最广泛的除草剂。近年来，许多研究表明，草甘膦基除草剂（GHBs）的暴露与血清睾丸激素的下降和精液质量的下降有关。然而，草甘膦诱导的睾丸激素合成障碍的分子机制仍不清楚。在本研究中，在TM3细胞中研究了草甘膦对睾丸激素分泌的影响以及内质网（ER）应激在该过程中的作用。用CCK8法检测了不同浓度的草甘膦对TM3细胞活力的影响。草甘膦暴露对睾丸激素分泌的影响通过酶联免疫吸附测定（ELISA）确定。通过Western blot和免疫荧光染色检测睾丸激素合酶和ER应激相关蛋白的表达水平。结果表明，浓度低于200 mg / L的草甘膦暴露对细胞活力没有影响，而浓度高于0.5 mg / L的草甘膦则可以抑制TM3细胞中的睾丸激素分泌。用5 mg / L的草甘膦处理TM3细胞不仅降低了睾丸激素合酶StAR和CYP17A1的蛋白水平，抑制了睾丸激素的分泌，还增加了ER应激分子Bip的蛋白水平，并促进了PERK和eIF2α的磷酸化。用ER应激抑制剂PBA预处理的细胞减轻了草甘膦诱导的Bip，p-PERK和p-eIF2α蛋白水平的升高，同时挽救了草甘膦诱导的睾丸激素合成障碍。使用GSK2606414进行预处理时，作为PERK抑制剂，草甘膦诱导的PERK和eIF2α的磷酸化被阻断，草甘膦抑制的睾丸激素合成和分泌也得以恢复。总体而言，我们的发现表明草甘膦可通过ER应激介导的Leydig细胞激活PERK /eIF2α信号通路来干扰StAR和CYP17A1的表达并抑制睾丸激素的合成和分泌。