Ergothioneine rescues obesity-induced testicular dysfunction via dual restoration of steroidogenesis and mitochondrial redox homeostasis.

BACKGROUND: Although obesity is closely linked to reduced male fertility, the specific testicular metabolic and redox mechanisms driving impaired spermatogenesis remain elusive. METHODS: Using a high-fat diet (HFD) mouse model, combined with multi-omics profiling, cellular assays, and ex vivo human testis cultures, we show that chronic HFD feeding progressively disrupts sperm quality, seminiferous architecture, and steroidogenic capacity. RESULTS: Despite unchanged testis weight, HFD significantly reduced sperm density by 21.6% and motility by 44.9%. Transcriptomic and metabolomic analyses revealed a marked suppression of oxidative phosphorylation and depletion of steroidogenic intermediates. Notably, ergothioneine (ET) was identified as the only metabolite consistently 8 reduced across time-course analyses, highlighting its potential as a testis-intrinsic biomarker of cumulative redox stress. ET supplementation (100 mg/kg/day) markedly restored seminiferous epithelial organization and increased the expression of spermatogenic markers. Functionally, ET alleviated the intracellular oxidative burden by reducing lipid peroxidation (TBARS levels decreased by 1.5-fold), and restoring antioxidant enzyme activities. ET enhanced mitochondrial stability, preserving mitochondrial membrane potential (ΔΨm) and reducing mitochondrial superoxide (O(2)(• -)) overproduction. Mechanistically, ET reactivated the canonical PKA-CREB-StAR signaling cascade in Leydig cells, reinstating androgen biosynthesis (in vivo DHT increased 1.3-fold, P < 0.01). Finally, ex vivo human testis cultures confirmed that ET attenuated oxidative stress indicators (reducing fluorescence intensity by 2.1-fold) and enhanced testosterone release by 1.4-fold. CONCLUSION: These findings establish progressive ET depletion as a hallmark of obesity-induced testicular dysfunction and demonstrate that ET supplementation restores steroidogenesis and mitochondrial redox homeostasis, providing a robust mechanistic basis for antioxidant-guided interventions in male infertility.