Mitochondrial homeostasis via Pink1 control alleviates nickel nanoparticle-induced reproductive damage in mouse spermatocyte GC-2 cells

Nickel nanoparticles (Ni NPs), increasingly utilized in environmental energy, electronics, and biomedical fields, have raised growing concern due to their emerging reproductive toxicity. Our prior investigations revealed that Ni NPs trigger oxidative stress and apoptosis in male germ cells, contributing to compromised fertility in rodent models. Given the central role of mitochondria in cellular homeostasis, and mitophagy in maintaining mitochondrial integrity, we hypothesized that mitochondrial stress may mediate Ni NPs-induced spermatocyte injury. In the present study, we employed GC-2 mouse spermatocyte cells to elucidate the role of PINK1/Parkin-mediated mitophagy in Ni NPs-induced reproductive toxicity. Ni NPs exposure (25-100 μg/mL) led to significant reductions in cell viability and mitochondrial membrane potential (MMP), along with increases in apoptosis, reactive oxygen species (ROS) accumulation, and ATP depletion. Western blot analyses demonstrated elevated expression of mitophagy- and apoptosis-related proteins including PINK1, Parkin, LC3BII, P62, BAX, Caspase9, and Caspase3, with concurrent downregulation of LC3BI and the anti-apoptotic protein BCL2. Pharmacological inhibition of mitophagy using cyclosporin A (CsA, 3 μM) partially restored mitochondrial function, suppressed apoptosis, and ameliorated overall cytotoxicity. Moreover, lentivirus-mediated knockdown of Pink1 further reversed Ni NPs-induced cellular damage by reducing ROS levels, recovering ATP production and MMP, and downregulating mitophagy and apoptosis-related markers. These findings identify aberrant mitophagy as a critical driver of Ni NPs-induced reproductive toxicity and highlight PINK1 as a potential molecular target for therapeutic intervention. This study offers novel mechanistic insights into Ni NPs-induced male reproductive damage, with implications for occupational health and nanoparticle safety assessment.