Zinc Protects against Heat Stress–Induced Apoptosis via the Inhibition of Endoplasmic Reticulum Stress in TM3 Leydig Cells

Heat stress (HS)-induced apoptosis in Leydig cells is mediated by various molecular mechanisms, including endoplasmic reticulum (ER) stress. Zinc, an inorganic mineral element, exhibits several cytoprotective properties, but its potential protective action against Leydig cell apoptosis and the related molecular mechanisms has not been fully elucidated. In this study, we evaluated the effects of zinc sulfate, a predominant chemical form of zinc, exerted on cell viability, apoptosis, and testosterone production in HS-treated TM3 Leydig cells and investigated the underlying signaling pathways. HS treatment inhibited cell viability and induced apoptosis, which was accompanied by the induction of the activity of caspase 3, an executioner of apoptosis, involved in the expression of pro-apoptotic protein B cell lymphoma 2-associated X protein (Bax), and in the reduction of the expression of anti-apoptotic protein B cell lymphoma 2 (Bcl-2), thereby activating ER stress marker protein expression (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP)). However, zinc sulfate led to the attenuation of deleterious effects, including increases in apoptosis, caspase-3 activity, Bax, GRP78, and CHOP expression, and decreases in cell viability and Bcl-2 protein expression in cells treated with HS or thapsigargin (an ER stress activator). Furthermore, 4-phenylbutyric acid (an ER stress inhibitor) treatment markedly alleviated the HS-induced adverse effects in cells exposed to HS, which was similar to zinc sulfate. Additionally, zinc sulfate supplementation in the culture medium effectively restored the HS-induced decrease in testosterone levels in HS-treated cells. In summary, these findings indicate that HS triggers apoptosis in TM3 Leydig cells via the ER stress pathway and that zinc confers protection against these detrimental effects. This study provides new insights into the benefits of using zinc against HS-induced apoptosis and cell injury.