Induction of autophagy via the ROS-dependent AMPK-mTOR pathway protects copper-induced spermatogenesis disorder

Abstract

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 (CuSO ₄) might significantly promote autophagy level in the testis and mouse-derived spermatogonia cell line GC-1 spg cells. Concurrently, CuSO ₄ 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, CuSO ₄ treatment could also increase expression levels of the autophagy-related proteins. Then, the role of oxidative stress in CuSO ₄-induced autophagy was investigated. The findings demonstrated that oxidative stress inhibitor (NAC) attenuated CuSO ₄-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 CuSO ₄. 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 CuSO ₄-induced ferroptosis in in vivo and vitro. Altogether, abovementioned results indicated that CuSO ₄ 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 CuSO ₄ toxicology through promoting ferroptosis. Overall, autophagy plays a positive role for attenuating CuSO ₄-induced testicular damage and spermatogenesis disorder. Our study provides a possible targeted therapy for Cu overload-induced reproduction toxicology.

Graphical abstract

Highlights

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  CuSO ₄-treatment could induce autophagy through AMPK-mTOR pathway.

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  Oxidative stress is an essential inducer of CuSO ₄-induced autophagy.

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  Autophagy inhibits oxidative damage and apoptosis in the testis of CuSO ₄-treatment mice.

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  Autophagy promotes ferroptosis in the testis of CuSO ₄-treatment mice.

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  Autophagy plays a protective role in copper-induced spermatogenesis disorder.