Abstract

Cadmium (Cd) is a widespread environmental toxicant that impairs male reproductive health, though its testicular toxicity mechanisms remain incompletely defined. Combining single-cell RNA sequencing with functional assays, we identified a novel intercellular communication pathway mediated by Sertoli cell-derived extracellular vesicles (EVs) in Cd-induced testicular injury. In mice, Cd exposure caused testicular atrophy, spermatogenesis disruption, and fibrosis. Multi-omics analyses revealed activation of multiple programmed cell death pathways, including apoptosis, necroptosis, pyroptosis, and ferroptosis. Single-cell RNA sequencing (scRNA-seq) demonstrated testicular cellular remodeling featuring Sertoli cell depletion and fibroblast expansion. Mechanistically, Cd triggered multi-modal programmed cell death (PCD) in Sertoli cells, promoting EV release enriched with damage-associated molecular patterns (DAMPs) and mitochondrial components. These EVs were internalized by testicular macrophages, activating the TLR4/NF-κB pathway and inducing a pro-inflammatory phenotype. Consequently, activated macrophages stimulated fibroblast-mediated fibrosis via TGF-β/Smad signaling. These findings elucidate a Sertoli cell-EV-macrophage-fibroblast axis in Cd-induced testicular damage, offering new insights into environmental toxicant-induced male infertility and potential therapeutic targets.