Lactylated Proteomic Analysis Reveals Functional Implications of Lysine Lactylation In Asthenozoospermia

The mechanism underlying asthenozoospermia in male infertility has been a prominent topic in reproductive medicine research. Human sperm function is modified by various protein post-translational modifications (PTMs). Among these, lactylation modification, a relatively novel PTM, has not been previously reported in the context of the male reproductive system. Comparative analyses between asthenozoospermic and normal sperm have revealed a significant down-regulation in the level of lysine lactylation (Kla) in proteins from asthenozoospermic sperm. Based on proteomic studies of protein Kla, 220 lactylated proteins were identified in sperm. Bioinformatics results showed that these lactylated proteins were highly enriched in the glycolytic pathway. Phosphoglycerate kinase 2 (PGK2), a key glycolytic enzyme and testis-specific protein, has been found to have 10 lactylated sites (K6, K11, K31, K41, K141, K192, K220, K272, K322, and K353). In asthenozoospermic sperm, both the lactylation level of PGK2 and its enzyme activity were reduced, while exogenous supplementation with PGK2 downstream products ameliorated sperm motility dysfunction. Mutation experiments at the K220 site confirmed that PGK2 (K220) lactylation affects glycolysis by regulating its enzyme activity. This study provides the first evidence of the regulatory role of proteins lactylation in sperm function.