Single-cell transcriptomics reveals the toxic mechanism and therapeutic target of ubiquitously distributed mycotoxin deoxynivalenol.

Deoxynivalenol (DON), a prevalent mycotoxin contaminating herbal medicines and daily diets, poses significant threats to male reproductive health, yet dissection of its testicular toxicity at single cell resolution remains unexplored. Herein, we integrated single-cell RNA sequencing (scRNA-seq) with functional analyses to characterize the cellular and molecular features of DON-induced testicular toxicity. The toxicity of DON was evaluated by morphology, organ index, sex hormones, sperm morphology and quantity, and H&E staining. scRNA-seq was used to dissect the cellular and molecular mechanisms of DON-induced testicular injury. Nuclear spreading immunostaining, Western blot, and supplement of inhibitor were used to confirm the toxic mechanism of DON. In sexually mature rats, chronic DON exposure caused dose-dependent testicular structural damage, hormonal imbalance, and impaired sperm quality. scRNA-seq of 70,000 testicular cells revealed DON-driven aberrant accumulation of early spermatocytes. Further analysis indicated defective meiotic progression at the leptotene stage, accompanied by suppressed homologous recombination-related genes including Brca2 and Rad51, and disrupted chromosome pairing. SCENIC regulatory network analysis suggested that KDM5A is functionally associated with leptotene spermatocyte maturation, and its activity was inhibited by DON. Notably, pharmacological activation of KDM5A using agonist D18 reversed the pathological decrease of BRCA2 and RAD51, partially rescued meiotic progression, and ameliorated sperm defects in DON-exposed models. Our study establishes the first single-cell atlas of DON-induced testicular injury and identifies KDM5A as a critical component of the regulatory network disrupted during mycotoxin-impaired spermatogenesis. These findings provide mechanistic insights into mycotoxin-related male infertility and highlight a potential therapeutic avenue for its mitigation.