부산대학교 도서관

Context: 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). Aims: This study aims to investigate the effect of melatonin and nitric oxide (NO·) on capacitation and apoptotic-like changes in EGF-capacitated ram spermatozoa. Methods: In vitro capacitation was induced by EGF in the absence or presence of melatonin (100 pM or 1 μM). 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. Key results: 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 1 μM prevented the activation of JNK. Conclusions: NO· metabolism does not seem to modulate the apoptosis-like events in ram spermatozoa. Melatonin at 1 μM 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. Implications: This study improves our understanding of the biochemical mechanisms involved in sperm capacitation, and ultimately, fertility. In our previous works, we demonstrated the implication of an alternative pathway to achieve ram sperm capacitation, which involves epidermal growth factor. Previously, we described that melatonin is able to modulate ram sperm in vitro capacitation achieved through the canonical cAMP/PKA pathway. Here we found that melatonin also prevents ram sperm capacitation induced by EGF by limiting the JNK MAPK activation. This mechanism isindependent from nitric oxide metabolism. [ABSTRACT FROM AUTHOR]