(240) Hydrogen Sulfide as a Modulator of Boar Sperm Physiology: Its Potential Role in Sperm Motility Regulation and Protein Persulfidation

Zuzana Pilsova¹; Aneta Pilsova¹; Ondrej Simonik²; Pavla Postlerova^1,2; Marketa Sedmikova¹

1. Department of Veterinary Sciences, Faculty of Agrobiology, Food, and Natural Resources, Czech University of Life Sciences Prague, Czech Republic
2. Laboratory of Reproductive Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Vestec, Czech Republic

Abstract Text:

Hydrogen sulfide (H₂S) is an endogenously produced gaseous signaling molecule synthesized by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). While its role in various cellular processes has been well established, its function in sperm physiology remains largely unexplored. H₂S modulates cellular functions through different signaling pathways, including post-translational protein modifications, particularly persulfidation (S-sulfhydration). This study aimed to confirm the presence of persulfidated proteins in boar spermatozoa, examine their distribution during sperm maturation, and evaluate the potential role of H₂S in sperm motility regulation. Persulfidation was detected in spermatozoa from the caput, corpus, and cauda epididymis and in ejaculated, capacitated, and acrosome-reacted sperm using indirect immunofluorescence and Western blot. An intense signal was observed in the late stages of spermatogenesis within the testes. In contrast, no specific signal of persulfidation was detected in earlier stages, such as spermatocytes, suggesting that this post-translational modification occurs predominantly in the final phases of sperm development in the testes. The signal of persulfidation persisted across all stages of sperm maturation, indicating a potential regulatory role in sperm function. Further, we investigated the contribution of H₂S to the regulation of sperm motility as a key factor in fertilization success. Sperm motility was evaluated using a Computer-Assisted Sperm Analysis (CASA) system after exposure to aminooxyacetic acid (AOAA), an inhibitor of H₂S-producing enzymes. Spermatozoa were incubated under capacitation conditions with AOAA at concentrations ranging from 2 to 100 μM for 180 minutes, during which motility was assessed at multiple time points. In parallel, we evaluated mitochondrial activity using flow cytometry to determine whether AOAA-induced motility reduction is associated with changes in mitochondrial function using JC-1 probe. Additional sperm functional parameters were assessed to investigate the effect of AOAA on sperm motility. Acrosome integrity and sperm viability were evaluated using fluorescent staining, revealing that AOAA treatment had no detectable impact on either parameter, suggesting that the observed motility reduction was not due to compromised cell survival. The exact mechanism by which H₂S influences sperm motility remains unknown but may involve modulation of ion channels, promotion of ATP production in mitochondria, or activation of signaling pathways. These findings suggest that H₂S is an essential regulator of sperm physiology and may play a key role in proper sperm maturation, including capacitation, hyperactivation, acrosome reaction, and overall fertilization potential.

Acknowledgment: This work was supported by the Internal Grant Agency of the Czech University of Life Sciences in Prague (SV24-21-21230), by the Ministry of Education, Youth, and Sports of the Czech Republic under the INTER-EXCELLENCE II program, subprogram INTER-ACTION (LUAUS25072), and the institutional support of Institute of Biotechnology (RVO: 86652036).