Effect of melatonin on ram sperm capacitation induced by epidermal growth factor

• Published in: Animal reproduction science 2022-12, Vol.247, p.107115, Article 107115
• Main Authors: Miguel-Jiménez, Sara, Carvajal-Serna, Melissa, Peña-Delgado, Victoria, Casao, Adriana, Pérez-Pe, Rosaura
• Format: Article
• Language: English
• ISSN: 0378-4320; 1873-2232
• DOI: 10.1016/j.anireprosci.2022.107115

Apart from the canonical cAMP-PKA pathway, ram sperm capacitation could be achieved by the MAPK ERK1/2 signalling cascade, activated by the epidermal growth factor binding to its receptor (EGF/EGFR) (Luna et al., Biol Reprod, 96(4), 2017). Certain MAP kinases implied in sperm capacitation, such as c-Jun N-terminal (JNK) and p38, are also related to apoptosis. Melatonin modulates ram sperm capacitation, and it is involved in apoptosis-related pathways in some somatic cells. The objective of this study was to investigate the effects of melatonin on capacitation and apoptosis when ram sperm capacitation is induced by EGF. Semen from nine Rasa Aragonesa rams was swim-up- selected and subjected to in vitro capacitation with EGF 100nM during 3h in the absence or presence of melatonin (100 pM or 1µM). Sperm functionality parameters (motility, membrane integrity, capacitation state), apoptotic markers (phosphatidylserine (PS) translocation, caspase activation, DNA damage), and phosphorylated JNK and p38 (active forms, assessed by western blot), were evaluated in swim-up and capacitated samples. Four replicates were performed, and data were analysed by Chi-squared test (sperm quality values) and ANOVA (western blot densitometry). There was an increase in the percentage of capacitated and acrosome-reacted spermatozoa after in vitro capacitation with EGF. Samples incubated with melatonin 1µM had a significantly higher rate of non-capacitated spermatozoa, whereas melatonin at 100 pM raised acrosome-reacted spermatozoa compared to samples without hormone. The percentage of live spermatozoa without apoptotic markers (PS translocation, active caspases and damaged DNA) decreased after in vitro capacitation, and the presence of melatonin did not cause any significant effect at the tested concentrations. The active form of JNK, but not that of p-38, increased after EGF-induced capacitation. Melatonin 1µM partially prevented the phosphorylation of JNK during capacitation, as there were no differences compared to the swim-up sample. It can be concluded that melatonin at 1µM prevents ram sperm capacitation induced by EGF, avoiding JNK activation. However, it cannot be attributed an anti-apoptotic role under these capacitation conditions. Grants: AGL-2017- 83799-R, DGA A07-17R and S M-J has a grant from Aragón Government.