Irisin Restores Testicular Metabolic Homeostasis and Spermatogenesis Against Chronic Unpredictable Stress in Rats: an Integrated 1H NMR Metabolomics and Molecular Study

Chronic unpredictable stress (CUS) is a significant contributor to male reproductive dysfunction; however, no effective clinical therapies are available, highlighting the need to explore novel preclinical interventions with translational potential. CUS disrupts testicular metabolic homeostasis, however the metabolic mechanisms underlying spermatogenic dysfunction remain poorly understood. Irisin is an exerkine with recognized metabolic functions and may counteract stress-induced testicular injury. In the present study, we investigated whether Irisin restores stress-induced metabolic remodeling in the testis using 1H NMR-based metabolomics together with molecular and histological analyses. The study demonstrated for the first time that Irisin (100 ng/kg/day, s.c., for 4 weeks) exerts protective effects in a CUS-induced adult male rat model. CUS elevated corticosterone, anxiety-like behaviour, decreased sperm count, viability, chromatin integrity, and circulating Irisin, luteinizing hormone, testosterone, and follicle-stimulating hormone, which were restored by Irisin. Irisin also restored testicular histoarchitecture, increased the Johnsen score and seminiferous tubule diameter, thereby improving spermatogenesis impaired by CUS. At the molecular level, Irisin suppressed NF-κB signaling, enhanced Nrf2/HO-1 antioxidant defense, and restored blood-testis barrier (BTB) integrity and ectoplasmic specialization (ES) proteins disrupted by CUS. Further, metabolomic profiling identified significant alterations in lactate, pyruvate, citrate, succinate, glutamate, betaine, glycerol, and choline, reflecting impaired glycolysis, tricarboxylic acid cycle activity, antioxidant metabolism, and membrane homeostasis in the testis. Pathway analysis further identified disruptions in alanine, aspartate, and glutamate; glycine, serine, and threonine; phenylalanine; and taurine and hypotaurine metabolism. Collectively, Irisin preserved testicular function by restoring metabolic homeostasis, redox balance, and BTB integrity under chronic stress.