Comparative Lactylomic Profiling Unveils Dynamic Reprogramming of Post-translational Lactylation Landscapes During Human Sperm Capacitation.

Lactylation, a novel post-translational modification involving lactate conjugation to lysine residues, bridges metabolic flux and epigenetic regulation and has emerged as a conserved regulatory mechanism across eukaryotes and prokaryotes, governing cell proliferation, immune responses, and metabolic reprogramming via a "Writer-Eraser" system. Despite the established roles of lactylation in energy metabolism, its function in male reproduction remains unexplored. This study investigates the lactylation modification dynamics during human sperm capacitation, a process requiring metabolic reprogramming for hyperactivated motility. Through 4D FastDIA-based quantitative dynamic profiling, we first conducted the comprehensive characterization of lysine lactylation modifications during sperm capacitation, systematically mapping differential lactylation patterns at the pathway, proteomic, and site-specific levels. We identified 2030 quantifiable lactylated proteins, revealing significant capacitation-dependent modulation: 115 proteins with 133 sites demonstrated upregulated lactylation, while 287 proteins containing 471 sites showed downregulation (p < 0.05, Fold Change >1.5). Notably, 30 proteins exhibited bidirectional regulatory patterns with coexisting upregulated and downregulated sites, and a predominant proportion of modified proteins (263 of 372) displayed single-site differential lactylation. A marked reduction in lactylation levels of mitochondrial respiratory chain proteins was observed after sperm capacitation, indicating heightened ATP requirements. Moreover, proteins with differential lactylation were enriched in biological processes including fertilization, sperm capacitation, β-oxidation, and microtubule dynamics. Strikingly, the presence of known lactylation regulatory enzymes including Alanine-tRNA ligase 1 (AARS1), Sirtuin (SIRT) 2/3, and Histone deacetylase (HDAC) 1/2/6 were detected in the sperm proteome, revealing a 1.6-fold upregulation of SIRT3, implicating its role in delactylation regulation during capacitation. Collectively, these findings delineate a lactylation-coordinated mechanism governing mitochondrial oxidative phosphorylation optimization and cytoskeletal architecture remodeling. This work substantiates a trinity regulatory mechanism integrating "metabolism-epigenetics-function" axes through lactylation circuitry, which may have implications for understanding male infertility.