Abstract
Some melatonin functions in mammals are exerted through MT1 and MT2 receptors. However, there are no reports of their presence in the reproductive tract of the ram, a seasonal species. Thus, we have investigated their existence in the ram testis, epididymis, accessory glands and ductus deferens. Real-time polymerase chain reaction (qPCR) revealed higher levels of m-RNA for both receptors in the testis, ampulla, seminal vesicles, and vas deferens, than in the other organs of the reproductive tract (p < 0.05). Western blot analyses showed protein bands compatible with the MT1 in the testis and cauda epididymis, and for the MT2 in the cauda epididymis and deferent duct. Immunohistochemistry analyses revealed the presence of MT1 receptors in spermatogonias, spermatocytes, and spermatids, and MT2 receptors in the newly-formed spermatozoa in the testis, whereas both receptors were located in the epithelial cells of the ampulla, seminal vesicles, and ductus deferens. Indirect immunofluorescence showed significant differences in the immunolocation of both receptors in spermatozoa during their transit in the epididymis. In conclusion, it was demonstrated that melatonin receptors are present in the ram reproductive tract. These results open the way for new studies on the molecular mechanism of melatonin and the biological significance of its receptors.
Highlights
Melatonin is a multifunctional molecule, widely distributed among all taxa [1]
The analysis of the MT2 receptor distribution revealed that the predominant immunotype in the testis was type N (36.3% ± 1.2%, Figure 4b).This immunotype was increased in the caput epididymis (53.6% ± 6.1%; p < 0.001, when compared with the testis), mainly due to the high decrease in the percentage of non-stained spermatozoa, and decreased during sperm maturation
The analysis of the MT2 receptor distribution revealed that the predominant immunotype in the testis was type N (36.3% ± 1.2%, Figure 4b).This immunotype was increased in the caput epididymis (53.6% ± 6.1%; p < 0.001, when compared with the testis), mainly due to the high decrease in the percentage of non-stained spermatozoa, and decreased during sperm maturation (35.0% ± 14.3% and 15.0% ± 0.5% for the corpus and cauda epididymis, respectively; p < 0.001 for the corpus when compared with the cauda, and the cauda when compared with the testis and caput epididymis, respectively)
Summary
Melatonin is a multifunctional molecule, widely distributed among all taxa [1]. In unicellular organisms its main function seems to be protection against free radical damage [2], while in mammals it has an extensive range of functions, including the regulation of circadian rhythms, immunomodulation, cancer inhibition, gastrointestinal tract protection, cardiovascular regulation, and seasonal reproduction [3].Melatonin can exert many of these functions due to its antioxidant properties [4], but it can interact with cytosolic molecules, such as calmodulin [5] or tubulin [6]. Melatonin receptors are located in the suprachiasmatic nucleus to regulate the circadian pacemaker but are present in peripheral tissues, such as blood vessels for vasomotor control, or white and bone marrow cells for regulation of the immune system [9]. Melatonin receptors have been identified in the reproductive system. Melatonin receptors have been identified in rat [16] and hamster [17] testis by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry techniques, in the rat epididymis by means of melatonin receptor agonists [18], by 2-[125I] iodomelatonin, and in human prostate epithelial cells [19] and in the prostatic section of the ductus deferens [20]. There is no evidence of the presence of melatonin receptors in the other accessory glands of the male reproductive tract (ampulla, seminal vesicle, or bulbourethral glands). There are no reports of the presence of melatonin receptors in the ram reproductive tract despite the great importance of melatonin in sheep reproduction
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