Synthesis of functional enzymes involved in glutathione production during linear motility in boar sperm

Sperm cells are highly susceptible to oxidative stress, which decreases their motility and fertility. However, glutathione (GSH) plays a critical role in protecting sperm cells from oxidative damage, a common byproduct of mitochondrial oxidative phosphorylation. On the other hand, GSH biosynthesis in sperm is limited by the availability of cysteine (Cys), which is inherently unstable and found at low concentrations in boar seminal plasma. In somatic cells, Cys can be produced through the transsulfuration pathway, catalyzed by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). In this study, we report that a group of enzymes involved in GSH synthesis is present in boar sperm. Notably, CBS and CTH protein levels increase during incubation, suggesting active regulation of their synthesis. This increase is inhibited by cycloheximide (CHX), indicating that ongoing protein synthesis is necessary for maintaining these levels. Our study also identified the presence of translation factors, such as eukaryotic initiation factor 4E (eIF4E), and their activation through phosphorylation of the ERK1/2-RSK-eIF4E pathway during incubation. Additionally, we found that CBS mRNA transcripts with short poly(A) tails are present in boar sperm, and polyadenylation of these short-tailed mRNAs occurs during incubation to enhance their translation. The use of cordycepin, a polyadenylation inhibitor, significantly reduced the translation of CBS, leading to decreased GSH synthesis and impaired sperm motility. However, the addition of cysteine counteracted the inhibitory effects of cordycepin, underscoring the essential role of cysteine in maintaining GSH levels. These findings provide new insights into the post-transcriptional regulation of GSH synthesis in sperm and suggest potential strategies for enhancing sperm preservation and fertility by targeting polyadenylation and translation mechanisms.