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

Although obesity is closely linked to reduced male fertility, the specific testicular metabolic and redox mechanisms driving impaired spermatogenesis remain elusive. 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. 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 (O2• -) 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. 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. Preventive ET supplementation restores StAR-mediated steroid hormone transport, stabilizes mitochondrial/redox homeostasis, and rescues steroidogenic function. • Chronic HFD triggers intrinsic redox-metabolic reprogramming in the testis, disrupting steroidogenic homeostasis. • Integrate metabolomics and RNA-seq to reveal mitochondrial dysfunction and oxidative stress in testes. • Identify a progressive decline of L-ergothioneine with HFD, nominating a diet-derived redox biomarker. • ET rescues steroidogenesis via reactivating the PKA-CREB-StAR pathway in Leydig cells.