Copper (Cu) is a necessary micronutrient at lower concentration, while excessive Cu exposure or Cu homeostasis disorders can lead to toxicity. The mechanism of male reproductive toxicity induced by Cu is still unknown. This study aims to investigate whether autophagy plays an important role in copper-induced spermatogenesis disorder in vivo and vitro. The present study showed that copper sulfate (CuSO₄) might significantly promote autophagy level in the testis and mouse-derived spermatogonia cell line GC-1 spg cells. Concurrently, CuSO₄ could induce autophagy via AMPK-mTOR pathway that downregulated p-mTOR/mTOR and subsequently upregulated p-AMPKα/AMPKα as well as p-ULK1/ULK1. In the meanwhile, CuSO₄ treatment could also increase expression levels of the autophagy-related proteins. Then, the role of oxidative stress in CuSO₄-induced autophagy was investigated. The findings demonstrated that oxidative stress inhibitor (NAC) attenuated CuSO₄-induced autophagy in vivo and vitro, reversing the activation for AMPK-mTOR pathway. Additionally, the study also investigated how autophagy worked under the spermatogenesis disorder induced by CuSO₄. Inhibition of autophagy could decrease cell viability, and enhance the ROS accumulation and apoptosis in the GC-1 cells, meanwhile, the spermatogenesis disorder, oxidative stress and histopathological changes were increased in the testis. Furthermore, co-treatment with the apoptosis inhibitor (Z-VAD-FMK) could decrease the spermatogenesis disorder but not influence autophagy. Besides, the crosslink between autophagy and ferroptosis were also measured, the data showed that inhibition of autophagy could suppress CuSO₄-induced ferroptosis in in vivo and vitro. Altogether, abovementioned results indicated that CuSO₄ induced autophagy via oxidative stress-dependent AMPK-mTOR pathway in the GC-1 cells and testis, and autophagy activation possibly led to the generation of protection mechanism through oxidative damage and apoptosis inhibition, however, autophagy also aggravate CuSO₄ toxicology through promoting ferroptosis. Overall, autophagy plays a positive role for attenuating CuSO₄-induced testicular damage and spermatogenesis disorder. Our study provides a possible targeted therapy for Cu overload-induced reproduction toxicology.

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