Hydrogen Sulfide as a Mediator of Protein Persulfidation and Motility Regulation in Mammalian Sperm

Aims: Hydrogen sulfide (H 2 S) is a signaling molecule implicated in diverse physiological processes, yet its role in sperm function remains poorly understood. This study characterizes protein persulfidation, a post-translational modification mediated by H 2 S in boar spermatozoa, tracks its distribution during sperm maturation, and assesses whether H 2 S influences sperm motility during capacitation. Methods: Persulfidation was localized using fluorescent labeling and confirmed by Western blotting across epididymal and post-ejaculatory stages. LC-MS/MS identified persulfidated proteins, which were subsequently assigned to their biological roles. In functional assays, the H 2 S biosynthesis inhibitor aminooxyacetic acid (AOAA) was applied at graded concentrations during sperm capacitation. These assays included computer-assisted sperm analysis (CASA) with k-means clustering, electrochemical measurement of H 2 S, and flow cytometry to assess mitochondrial membrane potential. Results: Persulfidation was consistently detected in spermatozoa, predominantly in the midpiece, and persisted throughout maturation. Proteomic analysis revealed 36 persulfidated proteins, including regulators of motility, energy metabolism, and antioxidant defense. AOAA exposure induced time- and dose-dependent changes: early inhibition of H 2 S synthesis reduced motility, whereas later recovery of H 2 S levels correlated with better preservation of motility. Cluster analysis uncovered shifts in motile subpopulations, notably prolonged maintenance of hyperactivated-like sperm under AOAA treatment. Conclusion: This study provides the first comprehensive evidence that persulfidation is a functionally relevant modification in boar spermatozoa. H 2 S appears to modulate motility during capacitation, either through persulfidation of proteins or via ion channels and other signaling pathways. These findings highlight H 2 S signaling as a novel regulatory axis in male reproduction and suggest new avenues for fertility research. Antioxid. Redox Signal. 00, 000–000.