Adolescent exposure to environmental silver nanoparticles induces spermatogenic impairment and Sertoli cell dysfunction: Integrative single-cell transcriptomics, metabolomics and mechanistic dissection

The widespread application of silver nanoparticles (AgNPs) raises increasing concerns over their male reproductive toxicity, yet a systematic, single-cell-resolved mechanistic understanding remains lacking. Here, we employed single-cell transcriptomics, metabolomics and functional experiments to explore AgNPs-triggered cellular remodeling of testes. Pubertal rats were orally exposed to AgNPs for 35 consecutive days. The particles traversed the blood-testis barrier (BTB), accumulated within the gonad, and ultimately impaired fertility. Metabolomics revealed that AgNPs altered metabolic milieu and reprogramed lipid metabolism. Then, high-resolution single-cell transcriptomic profiling identified ten distinct testicular cell populations, with spermatogenic cells and Sertoli cells exhibiting heightened susceptibility to AgNPs. Pseudotime trajectory analysis further revealed a selective reduction in undifferentiated spermatogonial stem cells. Functional and molecular pathway explorations were further conducted using C18-4, GC-2, and TM4 cell lines, as well as human primary Sertoli cells. Integrated analysis of in vitro and sequencing data reciprocally demonstrated the cell-type-specific toxicity: suppressed proliferation and elevated apoptosis in spermatogonia; intensified oxidative stress and apoptosis in spermatocytes; impaired spermatid differentiation; and disrupted growth factor synthesis and BTB integrity in Sertoli cells. Mechanically, these toxic effects were driven by AgNPs-induced mitochondrial dysfunction, glutathione metabolism dysregulation, and activation of the PINK1/Parkin-mediated mitophagy pathway, thereby disturbing Sertoli cell homeostasis and ultimately impaired the nutritional support and microenvironmental maintenance essential for spermatogenesis. These findings provide a comprehensive understanding of AgNPs-induced testicular toxicity, delineate cellular vulnerabilities and molecular mechanisms, and promise potential therapeutic countermeasures against nanoparticle-induced male infertility.