Rotenone-induced cell apoptosis via endoplasmic reticulum stress and PERK-eIF2α-CHOP signalling pathways in TM3 cells

Rotenone (ROT), a commonly used natural pesticide, has an unclear impact on reproductive toxicity. In this study, we randomly assigned 20 mice into four groups, each receiving ROT doses of 0, 2, 4, and 8 mg/kg/day over a period of 28 days. The findings revealed that ROT caused significant testicular damage, characterized by impaired spermatogenesis, reduced testosterone synthesis, and apoptosis of Leydig cells. Additionally, ROT disrupted the normal ultrastructure of the endoplasmic reticulum (ER) in testicular tissue, resulting in ER stress in Leydig cells. To investigate whether ROT-induced apoptosis in Leydig cells is linked to ER stress, we treated the mouse Leydig cell line (TM3 cells) with ROT at concentrations of 0, 250, 500, and 1000 nM. ROT decreased the viability of TM3 cells, induced cytotoxicity, and lowered testosterone levels in the culture supernatants. Moreover, ROT treatment triggered apoptosis in TM3 cells by activating ER stress and the PERK-eIF2α-CHOP signaling pathway. Pre-treating TM3 cells with the ER stress inhibitor 4-phenylbutyric acid (4-PBA) mitigated these effects, reducing apoptosis and maintaining testosterone levels. Further intervention with the PERK inhibitor GSK2606414 decreased both ROT-induced apoptosis and testosterone reduction by inhibiting PERK activity. In conclusion, ROT-induced male reproductive toxicity is primarily driven by apoptosis, with the activation of the PERK-eIF2α-CHOP signaling pathway, prompted by ER stress, playing a vital role in the apoptosis of Leydig cells induced by ROT.