(215) Localization of H2S-Producing Enzymes CBS and CSE in Reproductive Tissues and Spermatozoa at Different Stages of Maturation

Aneta Pilsova¹; Zuzana Pilsova¹; Pavel Vejl²; Barbora Klusackova¹; Pavla Postlerova¹; Marketa Sedmikova¹

1. Department of Veterinary Sciences, Faculty of Agrobiology, Food, and Natural Resources, Czech University of Life Sciences Prague, Czech Republic
2. Department of Genetics and Breeding, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Czech Republic

Abstract Text:

Hydrogen sulfide (H₂S), traditionally regarded as a toxic gas, has been recognized as a crucial signaling molecule with diverse biological functions, particularly in male reproductive physiology. H₂S modulates various signaling pathways, regulates ion channels, and post-translationally modifies proteins via persulfidation. This study investigates the expression and localization of two major H₂S-producing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), in boar reproductive tissues and spermatozoa, aiming to elucidate their role in essential sperm maturation processes, including spermatogenesis, epididymal maturation, capacitation, and acrosome reaction. We detected mRNA of CBS and CSE in multiple reproductive tissues, including the testis, epididymis (caput, corpus, cauda), and seminal vesicles, using qRT-PCR and ddPCR techniques. Protein localization via indirect immunofluorescence and Western blot analysis confirmed the presence of both enzymes in spermatozoa across different maturation stages (spermatids, epididymal sperm, ejaculated, capacitated, and acrosome-reacted sperm). The highest signal was observed in the post-acrosomal region, midpiece, and flagellum, indicating their potential role in sperm motility. CBS and CSE were particularly absent in the early spermatogenic stages, suggesting their transfer from Sertoli cells during late spermatogenic development. However, the presence of CBS and CSE in epididymal, ejaculated, capacitated, and acrosome-reacted spermatozoa indicates that these enzymes may play a role in subsequent post-testicular maturation processes. Further, Western blot analysis confirmed the presence of CBS and CSE in reproductive fluids, including epididymal fluid and seminal plasma. Interestingly, CBS was also detected in seminal vesicle fluid, whereas CSE was absent, suggesting enzyme-specific roles in reproductive tissues. Our findings indicate that H₂S, through CBS and CSE, plays a pivotal role in sperm physiology, probably by influencing motility via signaling pathways, ion channels, or post-translational modifications, such as protein persulfidation, further highlighting its complex regulatory function in male fertility. Understanding the distribution of H₂S-producing enzymes and their role in the male reproductive tract provides valuable insights into the molecular mechanisms governing sperm development and function. These findings extend our knowledge of the regulatory processes modulating sperm function and underscore the potential significance of H₂S in male fertility, paving the way for future research into its precise role in sperm physiology and reproductive medicine.

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