New evidence of melatonin receptor contribution to ram sperm functionality.

The present study analysed the involvement of melatonin, acting via its receptors (MT and MT), in ram sperm functionality. Indirect immunofluorescence assays revealed no changes in the distribution or intensity of MT receptors, whereas different subpopulations were established for MT receptors in control, in vitro capacitated and acrosome-reacted ram spermatozoa. Chlortetracycline staining revealed the following correlations between the pattern of staining for MT 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 MT and MT receptor agonists (2-phenylmelatonin 1µM and 8-Methoxy-2-propionamidotetralin (8M-PDOT) 1µM and 10nM) at 39°C and 5% CO for 3h resulted in a higher proportion of the NC pattern compared with the control group (P<0.05), whereas treatment with MT and MT 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 MT receptor.