New evidence of melatonin receptor contribution to ram sperm functionality

Abstract

The present study analysed the involvement of melatonin, acting via its receptors (MT₁ and MT₂), in ram sperm functionality. Indirect immunofluorescence assays revealed no changes in the distribution or intensity of MT₁ receptors, whereas different subpopulations were established for MT₂ receptors in control, in vitro capacitated and acrosome-reacted ram spermatozoa. Chlortetracycline staining revealed the following correlations between the pattern of staining for MT₂ receptors in: (1) non-capacitated (NC) sperm rate and the proportion of spermatozoa with equal immunostaining intensity in the acrosome and post-acrosome (r=0.59, P<0.001); (2) in capacitated (C) sperm rate and the proportion of spermatozoa with stronger reactivity in the acrosome (r=0.60, P<0.001); and (3) in acrosome-reacted (AR) sperm rate and the proportion of spermatozoa with more intense staining on the post-acrosome (r=0.67, P<0.001). Incubation of swim-up-selected samples with either 1μM melatonin or MT₁ and MT₂ receptor agonists (2-phenylmelatonin 1µM and 8-Methoxy-2-propionamidotetralin (8M-PDOT) 1µM and 10nM) at 39°C and 5% CO₂ for 3h resulted in a higher proportion of the NC pattern compared with the control group (P<0.05), whereas treatment with MT₁ and MT₂ receptor antagonists (luzindole 1µM and 4-phenyl-2-propionamidotetralin (4P-PDOT) 1µM and 10nM) decreased the proportion of spermatozoa exhibiting the NC pattern (P<0.001) concomitant with an increase in those exhibiting the C pattern (P<0.01). In conclusion, melatonin exerts a modulating effect on ram sperm functionality, primarily via activation of the MT₂ receptor.