Altered MT1 and MT2 melatonin receptors expression in the hippocampus of pilocarpine-induced epileptic rats.

Clinical and experimental findings show that melatonin may be used as an adjuvant to the treatment of epilepsy-related complications by alleviates sleep disturbances, circadian alterations and attenuates seizures alone or in combination with AEDs. In addition, it has been observed that there is a circadian component on seizures, which cause changes in circadian system and in melatonin production. Nevertheless, the dynamic changes of the melatoninergic system, especially with regard to its membrane receptors (MT1 and MT2) in the natural course of TLE remain largely unknown. The aim of this study was to evaluate the 24-hour profile of MT1 and MT2 mRNA and protein expression in the hippocampus of rats submitted to the pilocarpine-induced epilepsy model analyzing the influence of the circadian rhythm in the expression pattern during the acute, silent, and chronic phases. Melatonin receptor MT1 and MT2 mRNA expression levels were increased in the hippocampus of rats few hours after SE, with MT1 returning to normal levels and MT2 reducing during the silent phase. During the chronic phase, mRNA expression levels of both receptors return to levels close to control, however, presenting a different daily profile, showing that there is a circadian change during the chronic phase. Also, during the acute and silent phase it was possible to verify MT1 label only in CA2 hippocampal region with an increased expression only in the dark period of the acute phase. The MT2 receptor was present in all hippocampal regions, however, it was reduced in the acute phase and it was found in astrocytes. In chronic animals, there is a reduction in the presence of both receptors especially in regions where there is a typical damage derived from epilepsy. Therefore, we conclude that SE induced by pilocarpine is able to change melatonin receptor MT1 and MT2 protein and mRNA expression levels in the hippocampus of rats few hours after SE as well as in silent and chronic phases.

Obtaining anti-type 1 melatonin receptor antibodies by immunization with melatonin receptor-expressing cells.

Antibodies (Abs) specific to cell-surface receptors are attractive tools for studying the physiological role of such receptors or for controlling their activity. We sought to obtain such antibodies against the type 1 receptor for melatonin (MT1). For this, we injected mice with CHO cells transfected with a plasmid encoding human MT1 (CHO-MT1-h), in the presence or absence of an adjuvant mixture containing Alum and CpG1018. As we previously observed that the immune response to a protein antigen is increased when it is coupled to a fusion protein, called ZZTat101, we also investigated if the association of ZZTat101 with CHO-MT1-h cells provides an immunogenic advantage. We measured similar levels of anti-CHO and anti-MT1-h Ab responses in animals injected with either CHO-MT1-h cells or ZZTat101/CHO-MT1-h cells, with or without adjuvant, indicating that neither the adjuvant mixture nor ZZTat101 increased the anti-cell immune response. Then, we investigated whether the antisera also recognized murine MT1 (MT1-m). Using cloned CHO cells transfected with a plasmid encoding MT1-m, we found that antisera raised against CHO-MT1-h cells also bound the mouse receptor. Altogether our studies indicate that immunizing approaches based on MT1-h-expressing CHO cells allow the production of polyclonal antibodies against MT1 receptors of different origins. This paves the way to preparation of MT1-specific monoclonal antibodies.

Melatonin regulates splenocytes proliferation via IP3-dependent intracellular Ca2+ release in seasonally breeding bird, Perdicula asiatica.

Melatonin plays an important role in the immune regulation of birds. Both endogenous and exogenous melatonin modulates lymphocyte proliferation via its specific membrane receptors, Mel(1a), Mel(1b) and Mel(1c), though the mechanisms behind this process are poorly understood. We investigated the differences in melatonin membrane receptor Mel(1a), Mel(1b) and Mel(1c) expression by western blot and reverse transcription reaction and the in vitro effect of melatonin on the intracellular Ca(2+) concentration ([Ca2+]i) in splenocytes of the Indian Jungle Bush Quail, Perdicula asiatica. We used a non-selective melatonin receptor antagonist for Mel(1a) and Mel(1b), luzindole, and the selective Mel(1b) blocker, 4P-PDOT to check the specific role of melatonin receptor on ([Ca2+]i). The expression of Mel(1a), Mel(1b) and Mel(1c) receptors mRNA and protein was upregulated by melatonin (10(-7) M) with a significant high rise in ([Ca2+]i), which was differentially blocked by supplementation of antagonist, luzindole (10(-7) M) and 4P-PDOT (10(-7) M). Furthermore, we noted in vitro effect of melatonin and 2-aminoethoxydiphenyl borate (2-APB), a cell-permeable antagonist of inositol 1, 4, 5-trisphosphate (IP3) receptor to check the rise in ([Ca2+]i) through the IP3 pathway. Significantly low ([Ca2+]i) was noted in melatonin and 2-APB pretreated splenocytes when compared with splenocytes where 2-APB was absent. Thus, our data suggest that melatonin through its membrane receptor induced the elevation of ([Ca2+]i) via IP(3)-dependent pathway for splenocyte proliferation in P. asiatica.

Evidence of melatonin synthesis in the cumulus oocyte complexes and its role in enhancing oocyte maturation in vitro in cattle.

Melatonin is a multifunctional molecule that mediates several circadian and seasonal reproductive processes. The exact role of melatonin in modulating reproduction, however, is not fully understood-especially its effects on the ovarian follicles and oocytes. This study was conducted to investigate the expressions of the ASMT and melatonin-receptor MTNR1A and MTNR1B genes in bovine oocytes and their cumulus cells, as well as the effects of melatonin on oocyte nuclear and cytoplasmic maturation in vitro. Cumulus-oocyte complexes (COCs) from abattoir ovaries were cultured in TCM-199 supplemented with melatonin at concentrations of 0, 10, 50, and 100 ng/ml. The expression of ASMT, MTNR1A, and MTNR1B genes was evaluated by RT-PCR. Moreover, the effects of melatonin on cumulus cell expansion, nuclear maturation, mitochondrial characteristics and COCs steroidogenesis were investigated. Furthermore, the level of reactive oxygen species (ROS) was evaluated in denuded oocytes. Our study revealed that ASMT and MTNR1A genes were expressed in COCs, while the MTNR1B gene was expressed only in oocytes. Additionally, melatonin supplementation at 10 and 50 ng/ml to in vitro maturation medium significantly enhanced oocyte nuclear maturation, cumulus cell expansion and altered the mitochondrial distribution patterns, but had no effects on oocyte mitochondrial activity and COCs steroidogenesis. Melatonin-treated oocytes had a significantly lower level of ROS than controls. The presence of melatonin receptors in COCs and its promoting effects on oocyte nuclear and cytoplasmic events, indicate the potentially important roles of this hormone in regulating bovine oocyte maturation. Moreover, the presence of ASMT transcript in COCs suggests the possible involvement of these cells in melatonin biosynthesis.

Expression of functional melatonin MT(1) receptors in equine luteal cells: in vitro effects of melatonin on progesterone secretion.

In the present study, we analysed the molecular mechanism(s) by which melatonin directly affects ovarian function in the mare. In Experiment 1, follicles and corpora lutea (CL) were collected from slaughterhouse ovaries and analysed for melatonin (MT(1)) receptor mRNA and protein. In Experiment 2, CL were collected from slaughterhouse ovaries and cultured in Dulbecco's modified Eagle's medium-F12 medium (control medium) supplemented with 50 ng mL(-1) equine chorionic gonadotrophin (eCG), 1 nM-1 µM melatonin, 1 µM forskolin or 1 µM luzindole. Explants were cultured for 3 h in the presence of these drugs. Conditioned media were analysed for progesterone production; luteal cells were analysed for cholesterol side-chain cleavage enzyme (P450scc), a steroidogenic enzyme that converts cholesterol into pregnenolone. Both MT(1) receptor mRNA and protein were expressed in follicles and CL. Melatonin inhibited both the eCG- and forskolin-stimulated production of progesterone, as well as the forskolin-stimulated expression of P450scc, in equine luteal cells and the effect was dose-dependent. The inhibitory effect of melatonin was blocked by luzindole, a non-selective melatonin MT(1) and MT(2) receptor antagonist. The data support the presence of functional melatonin receptors in luteal cells and a regulatory role for melatonin in the endocrine function of the equine CL.

Effects of melatonin on in vitro maturation of porcine oocyte and expression of melatonin receptor RNA in cumulus and granulosa cells.

Melatonin is a multifunctional molecule that mediates several circadian and seasonal processes in animal reproduction. Melatonin and its metabolites are antioxidants and free radical scavengers. We investigated the effects of melatonin on porcine oocyte maturation and embryo development. We then investigated the local expression of the melatonin receptor 1 (MT1) gene in cumulus cells, granulosa cells, and the oocytes with the reverse transcription-polymerase chain reaction (RT-PCR) method. We further evaluated the antioxidant effects [reactive oxygen species (ROS) levels in cumulus-oocytes complexes] of melatonin supplementation during in vitro maturation (IVM). Compared with control, melatonin supplementation (10 ng/mL) during IVM resulted in a greater proportion of oocytes extruding the polar body (75.6% versus 84.6%). Significantly greater proportion of parthenogenetically activated oocytes developed to blastocysts when the in vitro medium was supplemented with melatonin; however, cleavage frequency and blastocyst cell number were not affected by the treatment. RT-PCR analysis revealed the expression of MT1 gene in cumulus and granulosa cells but not in oocytes. Melatonin-treated oocytes had significantly lower levels of ROS than did control (untreated) oocytes. We conclude that exogenous melatonin has beneficial effects on nuclear and cytoplasmic maturation during porcine IVM. Some of the observed effects may be mediated by receptor binding and while others may have been receptor independent, e.g., direct free radical scavenging.

[Circadian changes of the density of melatonin receptors 1A in the neurons of the suprachiasmatic nuclei of the rat hypothalamus under conditions of diverse functional activiity of the pineal gland].

An immunohistochemical study of the density of melatonin receptors 1A in the neurons of the rat suprachiasmatic nuclei with diverse functional activity of the pineal gland has been carried out. The density of melatonin receptors 1A under conditions of the physiological function of the pineal gland was characterized by clear-cut diurnal variations. Simultaneously, a dysfunction of the gland results in their marked disturbance. In case of a hypofunction of the pineal body the density of the structures was reliably lower than in case of hyperfunction. It has been demonstrated that in case of a suppressed activity of the pineal body the maximum number of melatonin receptors 1A in the neurons of the hypothalamic suprachiasmatic nuclei shifts from 02.00 a.m. to 02.00 p.m. and constitutes 0.35+/-0.012 conventional units (c.u.) of density, whereas a larger index is noticed at 20 hours making up 0.43+/-0.015 c.u. of density when the gland is activated.

Maternal melatonin effects on clock gene expression in a nonhuman primate fetus.

In the adult mammal the circadian system, which allows predictive adaptation to daily environmental changes, comprises peripheral oscillators in most tissues, commanded by the suprachiasmatic nucleus (SCN) of the hypothalamus. The external environment of the fetus is provided by its mother. In primates, maternal melatonin is a candidate to entrain fetal circadian rhythms, including the SCN rhythms of metabolic activity. We found in the 90% of gestation capuchin monkey fetus expression of the clock genes Bmal-1, Per-2, Cry-2, and Clock in the SCN, adrenal, pituitary, brown fat, and pineal. Bmal-1, Per-2, and the melatonin 1 receptor (MT1) showed a robust oscillatory expression in SCN and adrenal gland, whereas a circadian rhythm of dehydroepiandrosterone sulphate was found in plasma. Maternal melatonin suppression changed the expression of Bmal-1, Per-2, and MT1 in the fetal SCN. These effects were reversed by maternal melatonin replacement. In contrast, neither maternal melatonin suppression nor its replacement had effects on the expression of Per-2 and Bmal-1 or MT1 in the fetal adrenal gland or the circadian rhythm of fetal plasma dehydroepiandrosterone sulphate. Our data suggest that maternal melatonin is a Zeitgeber for the fetal SCN but probably not for the adrenal gland.