Effect of melatonin and nitric oxide on capacitation and apoptotic changes induced by epidermal growth factor in ram sperm

Apart from the canonical cAMP-PKA pathway, ram sperm capacitation can be achieved by the MAPK ERK1/2 signalling cascade, activated by epidermal growth factor (EGF).This study aims to investigate the effect of melatonin and nitric oxide (NO·) on capacitation and apoptotic-like changes in EGF-capacitated ram spermatozoa.In vitro capacitation was induced by EGF in the absence or presence of melatonin (100pM or 1μM). Also, a NO· precursor, L-arginine, or a NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), were added to capacitation media to study the interaction of NO· and melatonin during EGF-capacitation. Sperm functionality parameters (motility, viability, capacitation state), apoptotic markers (caspase activation and DNA damage), NO· levels, and phosphorylated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (assessed by Western blot), were evaluated in swim-up and capacitated samples with EGF.NO· levels and the apoptotic-related markers were raised after EGF incubation. Melatonin had a bimodal role on sperm EGF-capacitation, preventing it at high concentration and promoting acrosome reaction at low concentration, but neither of the two concentrations prevented the increase in apoptotic-like markers or NO· levels. However, melatonin at 1μM prevented the activation of JNK.NO· metabolism does not seem to modulate the apoptosis-like events in ram spermatozoa. Melatonin at 1μM prevents ram sperm capacitation induced by EGF independently from nitric oxide metabolism, and it could be exerted by limiting the JNK mitogen-activated protein kinase (MAPK) activation.This study improvesour understanding of the biochemical mechanisms involved in sperm capacitation, and ultimately, fertility.