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. • Adolescent exposure to AgNPs induces testicular toxicity and impairs fertility. • AgNPs alter the testicular metabolic landscape and reprogram lipid metabolism. • AgNPs specifically reduce spermatogonial stem cells and disrupt Sertoli cell homeostasis. • Mechanistic dissection links to mitochondrial dysfunction, and PINK1/Parkin-mediated mitophagy activation.