Difenoconazole impairs male reproductive function by inducing spermatogenic ferroptosis via the Wnt/β-catenin/NCOA4-mediated ferritinophagy axis.

Difenoconazole (DFZ), a widely used triazole fungicide, may pose a potential risk to male reproductive health, although its mechanistic basis in mammals remains incompletely understood. This study assessed DFZ-induced testicular injury through in vivo mouse models and in vitro GC-1 spermatogonia and GC-2 spermatocyte cell systems, conducting transcriptomic, metabolomic, and functional analyses concurrently. Chronic DFZ exposure was associated with dose-dependent testicular atrophy, disorganization of seminiferous tubule architecture, reduced sperm count, increased sperm malformation, and decreased serum testosterone levels, along with detectable DFZ accumulation in serum. Multi-omics profiling revealed coordinated molecular alterations in testicular tissue and germ cells, with ferroptosis-related pathways and lipid metabolic reprogramming being prominently enriched. In testicular tissue and germ cells, DFZ induced features consistent with ferroptosis, including depletion of GSH, accumulation of MDA and Fe2+, inactivation of GPX4, and mitochondrial damage. These changes were alleviated by Fer-1 and DFO, suggesting that ferroptosis contributes to DFZ-induced cytotoxicity. Moreover, GPX4 knockdown further aggravated DFZ-induced ferroptotic injury, supporting a central role for GPX4 in this process. Mechanistically, DFZ appeared to activate autophagy and NCOA4-mediated ferritinophagy, which may promote iron overload and lipid peroxidation. Wnt/β-catenin signaling activation reduced NCOA4 upregulation and ferroptotic damage, but this protective effect was nullified by NCOA4 silencing, suggesting NCOA4 operates downstream of Wnt/β-catenin signaling. These findings reveal that DFZ triggers spermatogenic ferroptosis through Wnt/β-catenin-NCOA4-mediated ferritinophagy, offering insights into triazole fungicide-induced male reproductive toxicity and highlighting potential targets for environmental infertility interventions.