Melatonin regulates rabbit sperm motility and kinematics via the MT1/PKC signaling pathway.

OBJECTIVE: Melatonin, a highly conserved molecule, plays an essential role in various physiological functions. Research suggests that incorporating melatonin into semen extender enhances livestock sperm viability. However, the effect of melatonin on rabbit sperm and the molecular mechanisms underlying melatonin-regulated rabbit sperm motility and kinematics remain unclear. This study aimed to reveal the molecular mechanism by which melatonin regulates rabbit sperm motility and kinematics. METHODS: This study investigated the expression and localization of melatonin-related proteins in rabbit testis and epididymis. Rabbit sperm was incubated at different concentrations of melatonin for 60 min or 90 min at 37°C, followed by an evaluation of sperm motility parameters using IVOS II computer assisted sperm analyzer system (CASA). Then we examined the integrity of the sperm plasma membrane, mitochondrial membrane potentials and intracellular reactive oxygen species. Furthermore, melatonin receptor antagonists were added to the extender and investigated the involvement of the melatonin receptors in the regulation of sperm motility parameters. We carried out phosphoproteomics analysis and verified regulation of rabbit sperm kinematics by melatonin via the inhibition of potential signaling pathway. RESULTS: Melatonin-related proteins were expressed and localized in testes and epididymis of rabbits. Melatonin (5 mM) markedly increased rabbit sperm motility, kinematics, and overall sperm quality. Melatonin regulated rabbit sperm motility and kinematics via the MT1 receptor. Phosphoproteomics combined with GPS 5.0 software revealed the potential signaling pathway by which melatonin regulates sperm kinematics. Moreover, the inhibition of protein kinase C (PKC) markedly reduced rabbit sperm kinematics, whereas the inhibition of ERK1/2, p38 MAPK, PKG and JNKs using kinase inhibitors did not result in obvious changes in sperm kinematics. In addition, inhibition of the MT1 receptor significantly weakened PKC activity, suggesting that PKC is downstream of MT1. CONCLUSION: We conclude that melatonin regulates rabbit sperm kinematics through the MT1/PKC signaling pathway.