Catalase in Unexpected Places: Revisiting HO Detoxification Pathways in Stallion Spermatozoa.

Oxidative stress plays a critical role in regulating sperm function, yet species-specific antioxidant mechanisms remain poorly understood. This study compared hydrogen peroxide (HO) tolerance in horse and human sperm and investigated the roles of catalase and glutathione peroxidase (GPx) in horses. A HO dose-response assay (0-2000 µM) showed that horse sperm were significantly more resistant to oxidative damage, with an IC for progressive motility over 14-fold higher than that of human sperm (391.6 µM vs. 27.3 µM). Horse sperm also accumulated more intracellular HO without loss of motility or viability. DNA damage assays (Halo and SCSA) revealed HO-induced fragmentation in human but not horse sperm. Enzyme inhibition experiments in horse sperm using 3-amino-1,2,4-triazole (catalase inhibitor) and (1S,3R)-RSL3 (GPx inhibitor) at 250 µM HO showed that catalase inhibition severely impaired motility and increased intracellular HO > 100-fold, while GPx inhibition had a milder effect (~5-fold increase). Immunocytochemistry localized catalase to the sperm head, particularly the post-acrosomal region, challenging the notion that sperm lack peroxisomes. The dependence of horse sperm on oxidative phosphorylation may drive the need for enhanced antioxidant defenses. These findings reveal species-specific oxidative stress adaptations and highlight catalase as a key antioxidant in equine sperm.