Systematic mRNA interactome analysis reconceptualizes translational quiescence in bovine sperm.

Ejaculated mammalian sperm must undergo a series of biochemical changes called capacitation to gain fertilizing competence. Mounting evidence on protein synthesis during capacitation contradicts the widely accepted dogma of translational quiescence in sperm. However, mechanisms regulating mRNA translation in sperm is ambiguous, necessitating elucidation and understanding its role in enabling fertilizing competence. Here, we perform proteome analysis from bovine sperm and identify proteins involved in translation, encompassing initiation and elongation factors, ribosomal proteins, tRNA synthetase, ligase, and RNA-binding proteins (RBPs) involved in mRNA export, degradation, and binding. We further explore the mRNA-binding activity of RBPs during capacitation, identifying 48 RBPs; 13 and 8 RBPs were exclusive to fresh-uncapacitated and capacitation groups, respectively, with an overlap of 27 RBPs present in both groups. Interestingly, cytoskeletal proteins and metabolic enzymes associate differentially with mRNAs during capacitation. Since phosphorylation is a known regulatory mechanism dynamically modulating RBPs' interactions with mRNAs, we performed sperm phosphoproteome analysis, revealing few RBPs to phosphorylate during capacitation. These observations suggest that RNA-binding functions of these proteins are coupled with capacitation-associated phosphorylation events, enabling concomitant protein synthesis and fertilizing competence in sperm. These findings will assist in elucidating translational regulation of mRNA in sperm and advancing our knowledge in regulation of male fertility.