Identification and immunolocalisation of melatonin MT1 and MT2 receptors in Rasa Aragonesa ram spermatozoa

The reproductive seasonality of sheep suggests that melatonin receptors may be present in ram spermatozoa. The present study confirms the presence of melatonin MT1 and MT2 receptors. The MT1 receptor was detected using immunocytochemistry, with four sperm subpopulations identified based on the following labelling patterns: (1) one small subpopulation with labelling over the entire head and tail; (2) one of two main subpopulations that exhibited reactivity at the equatorial, post-acrosomal, neck and tail regions; (3) another main subpopulation with equatorial and tail labelling only; and (4) a subpopulation in which staining was detected only in the tail. Immunocytochemistry revealed the presence of the melatonin MT2 receptor, with intense staining on the acrosome, post-acrosomal region and neck and tail regions of all cells, but not in the equatorial region. Western blot identification of ram protein extracts revealed a 39-kDa band compatible with both MT1 and MT2 receptors, a 75-kDa band compatible with MT1/MT2 heterodimerisation, a 32-kDa band compatible with MT1 receptor activation and a double band of 45-55 kDa that is compatible with MT2 receptor homodimerisation or heterodimerisation with other G-proteins. In conclusion, we provide evidence of the presence of MT1 and MT2 receptors in ram spermatozoa, although the biochemical pathway triggered by these receptors and their function in terms of fertility remains to be elucidated.