Melatonin exists in porcine follicular fluid and improves in vitro maturation and parthenogenetic development of porcine oocytes

Abstract: This study focused on the effect of melatonin on in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Melatonin was measured in porcine follicular fluid of follicles of different sizes in the same ovary. Melatonin exists in follicular fluid, and the concentration is approximately 10^?11 m . Its concentration decreased as the diameter of follicle increased, which suggests an effect of melatonin on oocyte maturation. Therefore, immature oocytes were cultured in vitro in maturation medium supplemented with melatonin (10^?11, 10^?9, 10^?7, 10^?5 and 10^?3 m ) or without melatonin. The oocytes at maturation stage were collected and activated. The parthenogenetic embryos were cultured and observed in medium supplemented with or without melatonin. Fresh immature oocytes without melatonin treatment were used as control. When only maturation medium was supplemented with 10^?9 m melatonin, the cleavage rate, blastocyst rate and the cell number of blastocyst (70 ± 4.5%, 28 ± 2.4% and 50 ± 6.5%) were significantly higher (P < 0.05) than that of controls; when only culture medium was supplemented with melatonin, the highest cleavage rate, blastocyst rate and the cell number of blastocyst was observed at 10^?7 m melatonin, which were significantly higher than that of controls (P < 0.05). The best results (cleavage rates 79 ± 8.4%, blastocyst rates 35 ± 6.7%) were obtained when both the maturation and culture medium were supplemented with 10^?9 m melatonin respectively (P < 0.05). In conclusion, exogenous melatonin at the proper concentration may improve the in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Further research is needed to identify the effect of melatonin on in vitro and in vivo oocyte maturation and embryo development in porcine.     

Essential role of PTPN11 mutation in enhanced haematopoietic differentiation potential of induced pluripotent stem cells of juvenile myelomonocytic leukaemia

We established mutated and non-mutated induced pluripotent stem cell (iPSC) clones from a patient with PTPN11 (c.226G>A)-mutated juvenile myelomonocytic leukaemia (JMML). Both types of iPSCs fulfilled the quality criteria. Mutated iPSC colonies generated significantly more CD34⁺ and CD34⁺CD45⁺ cells compared to non-mutated iPSC colonies in a culture coated with irradiated AGM-S3 cells to which four growth factors were added sequentially or simultaneously. The haematopoietic differentiation potential of non-mutated JMML iPSC colonies was similar to or lower than that of iPSC colonies from a healthy individual. The PTPN11 mutation coexisted with the OSBP2 c.389C>T mutation. Zinc-finger nuclease-mediated homologous recombination revealed that correction of PTPN11 mutation in iPSCs with PTPN11 and OSBP2 mutations resulted in reduced CD34⁺ cell generation to a level similar to that obtained with JMML iPSC colonies with the wild-type of both genes, and interestingly, to that obtained with normal iPSC colonies. Transduction of the PTPN11 mutation into JMML iPSCs with the wild-type of both genes increased CD34⁺ cell production to a level comparable to that obtained with JMML iPSC colonies harbouring the two genetic mutations. Thus, PTPN11 mutation may be the most essential abnormality to confer an aberrant haematopoietic differentiation potential in this disorder.     

Nociceptive responses in melatonin MT2 receptor knockout mice compared to MT1 and double MT1/MT2 receptor knockout mice

Melatonin, a neurohormone that binds to two G protein-coupled receptors MT₁ and MT_2, is involved in pain regulation, but the distinct role of each receptor has yet to be defined. We characterized the nociceptive responses of mice with genetic inactivation of melatonin MT₁ (MT₁^−/−), or MT₂ (MT₂^−/−), or both MT₁/MT₂ (MT₁^−/−/MT₂^−/−) receptors in the hot plate test (HPT), and the formalin test (FT). In HPT and FT, MT₁^−/− display no differences compared to their wild-type littermates (CTL), whereas both MT₂^−/− and MT₁^−/−/MT₂^−/− mice showed a reduced thermal sensitivity and a decreased tonic nocifensive behavior during phase 2 of the FT in the light phase. The MT₂ partial agonist UCM924 induced an antinociceptive effect in MT₁^−/− but not in MT₂^−/− and MT₁^−/−/MT₂^−/− mice. Also, the competitive opioid antagonist naloxone had no effects in CTL, whereas it induced a decrease of nociceptive thresholds in MT₂^−/− mice. Our results show that the genetic inactivation of melatonin MT₂, but not MT₁ receptors, produces a distinct effect on nociceptive threshold, suggesting that the melatonin MT₂ receptor subtype is selectively involved in the regulation of pain responses.     

Melatonin influences proliferation and differentiation of rat dental papilla cells in vitro and dentine formation in vivo by altering mitochondrial activity

Melatonin mediates a variety of biological processes ranging from the control of circadian rhythms to immune regulation. Melatonin also influences bone formation and osteointegration of dental implants. However, the effects of melatonin on dentine formation have not been examined. This study investigated the effects of melatonin on the proliferation and differentiation of rat dental papilla cells (rDPCs) in vitro and dentine formation in vivo. We found that melatonin (0, 10^?12, 10^?10,10^?8 m ) induced a dose‐dependent reduction in rDPCs proliferation, increased alkaline phosphatase (ALP) activity, the expression of dentine sialoprotein (DSP), and mineralized matrix formation in vitro. In vivo melatonin (50 mg/kg, BW, i.p.) inhibited dentine formation. Melatonin (10^?8 m ) suppressed the activity of complex I and IV in the basal medium (OS?) and enhanced the activity of complex I and complex IV in osteogenic medium (OS+). These results demonstrate that melatonin suppresses the proliferation and promotes differentiation of rDPCs, the mechanisms of which may be related to activity of mitochondrial complex I and complex IV.     

Melatonin and its derivatives cyclic 3-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine and 6-methoxymelatonin reduce oxidative DNA damage induced by Fenton reagents

Abstract: Free radicals are generated in vivo and they oxidatively damage DNA because of their high reactivities. In the last several years, hundreds of publications have confirmed that melatonin is a potent endogenous free radical scavenger. Some of the metabolites produced as a result of these scavenging actions have been identified using pure chemical systems. This is the case with both N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK), identified as a product of the scavenging reaction of H₂O₂ by melatonin, and cyclic 3‐hydroxymelatonin (C‐3‐OHM) which results when melatonin detoxifies two hydroxyl radicals (?OH). In the present in vitro study, we investigated the potential of two different derivatives of melatonin to scavenger free radicals. One of these derivatives is C‐3‐OHM, while the other is 6‐methoxymelatonin (6‐MthM). We also examined the effect of two solvents, i.e., methanol and acetonitrile, in this model system. As an endpoint, using high‐performance liquid chromatography we measured the formation of 8‐hydroxy‐2′‐deoxyguanosine (8‐OH‐dG) in purified calf thymus DNA treated with the Fenton reagents, chromium(III) [Cr(III)] plus H₂O₂, in the presence and in the absence of these molecules. The 8‐OH‐dG is considered a biomarker of oxidative DNA damage. Increasing concentrations of Cr(III) (as CrCl₃) and H₂O₂ was earlier found to induce progressively greater levels of 8‐OH‐dG in isolated calf thymus DNA because of the generation of ?OH via the Fenton‐type reaction. We found that C‐3‐OHM reduces ?OH‐mediated damage in a dose‐dependent manner, with an IC₅₀ = 5.0 ± 0.2 μm ; melatonin has an IC₅₀ = 3.6 ± 0.1 μm . These values differ statistically significantly with P < 0.05. In these studies, AFMK had an IC₅₀ = 17.8 ± 0.7 μm (P < 0.01). The 6‐MthM also reduced DNA damage in a dose‐dependent manner, with an IC₅₀ = 4.2 ± 0.2 μm ; this value does not differ from the ICs for melatonin and C‐3‐OHM. We propose a hypothetical reaction pathway in which a mole of C‐3‐OHM scavenges 2 mol of ?OH yielding AFMK as a final product. As AFMK is also a free radical scavenger, the action of melatonin as a free radical scavenger is a sequence of scavenging reactions in which the products are themselves scavengers, resulting in a cascade of protective reactions.     

Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain

The involvement of melatonin in mammalian brain pathophysiology has received growing interest, but information about the anatomical distribution of its two G‐protein‐coupled receptors, MT₁ and MT₂, remains elusive. In this study, using specific antibodies, we examined the precise distribution of both melatonin receptors immunoreactivity across the adult rat brain using light, confocal, and electron microscopy. Our results demonstrate a selective MT₁ and MT₂ localization on neuronal cell bodies and dendrites in numerous regions of the rat telencephalon, diencephalon, and mesencephalon. Confocal and ultrastructural examination confirmed the somatodendritic nature of MT₁ and MT₂ receptors, both being localized on neuronal membranes. Overall, striking differences were observed in the anatomical distribution pattern of MT₁ and MT₂ proteins, and the labeling often appeared complementary in regions displaying both receptors. Somadendrites labeled for MT₁ were observed for instance in the retrosplenial cortex, the dentate gyrus of the hippocampus, the islands of Calleja, the medial habenula, the suprachiasmatic nucleus, the superior colliculus, the substantia nigra pars compacta, the dorsal raphe nucleus, and the pars tuberalis of the pituitary gland. Somadendrites endowed with MT₂ receptors were mostly observed in the CA3 field of the hippocampus, the reticular thalamic nucleus, the supraoptic nucleus, the inferior colliculus, the substantia nigra pars reticulata, and the ventrolateral periaqueductal gray. Together, these data provide the first detailed neurocytological mapping of melatonin receptors in the adult rat brain, an essential prerequisite for a better understanding of melatonin distinct receptor function and neurophysiology.     

Protein interactome mining defines melatonin MT1 receptors as integral component of presynaptic protein complexes of neurons

In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT₁ and MT₂ receptors, belonging to the G protein‐coupled receptor (GPCR) super‐family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep, and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT₁ and MT₂ receptors, which comprises 378 individual proteins. Among the proteins interacting with MT₁, but not with MT₂, we identified several presynaptic proteins, suggesting a potential role of MT₁ in neurotransmission. Presynaptic localization of MT₁ receptors in the hypothalamus, striatum, and cortex was confirmed by subcellular fractionation experiments and immunofluorescence microscopy. MT₁ physically interacts with the voltage‐gated calcium channel Ca_v2.2 and inhibits Ca_v2.2‐promoted Ca²⁺ entry in an agonist‐independent manner. In conclusion, we show that MT₁ is part of the presynaptic protein network and negatively regulates Ca_v2.2 activity, providing a first hint for potential synaptic functions of MT₁.     

Beneficial effects of melatonin on in vitro bovine embryonic development are mediated by melatonin receptor 1

In the current study, a fundamental question, that is, the mechanisms related to the beneficial effects of melatonin on mammalian embryonic development, was addressed. To examine the potential beneficial effects of melatonin on bovine embryonic development, different concentrations of melatonin (10^?11, 10^?9, 10^?7, 10^?5, 10^?3 m ) were incubated with fertilized embryos. Melatonin in the range of 10^?11 to 10^?5 m significantly promoted embryonic development both in early culture medium (CR1aa +3 mg/mL BSA) and in later culture medium (CR1aa + 6%FBS). The most effective concentrations applied in the current studies were 10^?9 and 10^?7 m . Using quantitative real‐time PCR with immunofluorescence and Western blot assays, the expression of melatonin receptor MT1 and MT2 genes was identified in bovine embryos. Further studies indicate that the beneficial effects of melatonin on bovine embryo development were mediated by the MT1 receptor. This is based on the facts that luzindole, a nonselective MT1 and MT2 antagonist, blocked the effect on melatonin‐induced embryo development, while 4‐P‐PDOT, a selective MT2 antagonist, had little effect. Mechanistic explorations uncovered that melatonin application during bovine embryonic development significantly up‐regulated the expression of antioxidative (Gpx4, SOD1, bcl‐2) and developmentally important genes (SLC2A1, DNMT1A, and DSC2) while down‐regulating expression of pro‐apoptotic genes (P53, BAX, and Caspase‐3). The results obtained from the current studies provide new information regarding the mechanisms by which melatonin promotes bovine embryonic development under both in vitro and in vivo conditions.     

Molecular and cellular pharmacological properties of 5‐methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT): a nonspecific MT3 ligand

Abstract: 5‐Methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT) has been initially described as a ligand at non MT₁, non MT₂ melatonin binding site (MT3) selective versus MT₁ and MT₂, two membrane melatonin receptors. MCA‐NAT activity has been reported by others in different models, in vivo, particularly in the intra‐ocular pressure (IOP) models in rabbits and monkeys. Its activity was systematically linked to either MT3 or to a new, yet unknown, melatonin receptor. In this article, the melatonin receptor pharmacology of MCA‐NAT is described. MCA‐NAT has micromolar range affinities at the melatonin receptors MT₁ and MT₂, while in functional studies, MCA‐NAT proved to be a powerful MT₁/MT₂ partial agonist in the sub‐micromolar range. These data strongly suggest that MCA‐NAT actions might be mediated by these receptors in vivo. Finally, as described by others, we show that MCA‐NAT is unable to elicit any type of receptor‐like functional responses from Chinese hamster ovary cells over‐expressing quinone reductase 2, the MT3.     

Melatonin improves reprogramming efficiency and proliferation of bovine‐induced pluripotent stem cells

Melatonin can modulate neural stem cell (NSC) functions such as proliferation and differentiation into NSC‐derived pluripotent stem cells (N‐iPS) in brain tissue, but the effect and mechanism underlying this are unclear. Thus, we studied how primary cultured bovine NSCs isolated from the retinal neural layer could transform into N‐iPS cell. NSCs were exposed to 0.01, 0.1, 1, 10, or 100 μm melatonin, and cell viability studies indicated that 10 μm melatonin can significantly increase cell viability and promote cell proliferation in NSCs in vitro. Thus, 10 μm melatonin was used to study miR‐302/367‐mediated cell reprogramming of NSCs. We noted that this concentration of melatonin increased reprogramming efficiency of N‐iPS cell generation from primary cultured bovine NSCs and that this was mediated by downregulation of apoptosis‐related genes p53 and p21. Then, N‐iPS cells were treated with 1, 10, 100, or 500 μm melatonin, and N‐iPS (M‐N‐iPS) cell proliferation was measured. We noted that 100 μm melatonin increased proliferation of N‐iPS cells via increased phosphorylation of intracellular ERK1/2 via activation of its pathway in M‐N‐iPS via melatonin receptors 1 (MT1). Finally, we verified that N‐iPS cells and M‐N‐iPS cells are similar to typical embryonic stem cells including the expression of pluripotency markers (Oct4 and Nanog), the ability to form teratomas in vivo, and the capacity to differentiate into all three embryonic germ layers.     

Melatonin prevents ultraviolet radiation‐induced alterations in plasma membrane potential and intracellular pH in human keratinocytes

Melatonin exhibits protective effects against ultraviolet radiation (UVR) via modulation of proinflammatory mediators and its free radical scavenging capacity. To date, several reports presented protective mechanisms of this agent against UVR‐induced alterations in mitochondria and nuclei. This investigation evaluates the potent preventing action of melatonin regarding early‐stage UVR‐mediated perturbations in plasma membrane potential (mbΔψ) and intracellular (cytosolic) pH (pH _i) analyzed by flow cytometry. Experiments were carried out in a dose‐ and time‐dependent manner using human keratinocytes [HaCaT and normal human epidermal keratinocytes (NHEK)]. First investigations, which used viability/cytotoxicity assays, showed the gradual mortality with increasing UVR doses and cultivation time. Pre‐incubation with melatonin (10^?3 m ) prior to UVR exposure reduced lactate dehydrogenase release by 30% (HaCaT) and 28% (NHEK) at the dose of 50 mJ/cm² after 48 hr (P < 0.001). Furthermore, UVR caused hyperpolarization of mbΔψ immediately (0 hr) after irradiation (25 or 50 mJ/cm²). At the dose of 50 mJ/cm², cells cultivated for 48 hr manifested a marked increase in mbΔψ by 112% (HaCaT) and 123% (NHEK). The presence of melatonin significantly protected the cells by 12% (HaCaT) and 14% (NHEK) (P < 0.001). Simultaneously, 50 mJ/cm² induced dramatic acidification reaching after 24 hr the level of 6.40 (without melatonin), 6.56 (with melatonin) for HaCaT and 6.11 (without melatonin), 6.43 (with melatonin) for NHEK. The results presented provide information about the protective mechanisms of melatonin itself on one hand and, combined with data reported so far, confirm the potent antiapoptotic action of melatonin.     

Melatonin‐mediated inhibition of Purkinje neuron P‐type Ca2+ channels in vitro induces neuronal hyperexcitability through the phosphatidylinositol 3‐kinase‐dependent protein kinase C delta pathway

Although melatonin receptors are widely expressed in the mammalian central nervous system and peripheral tissues, there are limited data regarding the functions of melatonin in cerebellar Purkinje cells. Here, we identified a novel functional role of melatonin in modulating P‐type Ca²⁺ channels and action‐potential firing in rat Purkinje neurons. Melatonin at 0.1 μm reversibly decreased peak currents (I_Ba) by 32.9%. This effect was melatonin receptor 1 (MT_R1) dependent and was associated with a hyperpolarizing shift in the voltage dependence of inactivation. Pertussis toxin pretreatment, intracellular application of QEHA peptide, and a selective antibody raised against the G_β subunit prevented the inhibitory effects of melatonin. Pretreatment with phosphatidylinositol 3‐kinase (PI3K) inhibitors abolished the melatonin‐induced decrease in I_Ba. Surprisingly, melatonin responses were not regulated by Akt, a common downstream target of PI3K. Melatonin treatment significantly increased protein kinase C (PKC) activity 2.1‐fold. Antagonists of PKC, but not of protein kinase A, abolished the melatonin‐induced decrease in I_Ba. Melatonin application increased the membrane abundance of PKC_δ, and PKC_δ inhibition (either pharmacologically or genetically) abolished the melatonin‐induced I_Ba response. Functionally, melatonin increased spontaneous action‐potential firing by 53.0%; knockdown of MT_R1 and blockade of P‐type channels abolished this effect. Thus, our results suggest that melatonin inhibits P‐type channels through MT_R1 activation, which is coupled sequentially to the βγ subunits of G_i/o‐protein and to downstream PI3K‐dependent PKC_δ signaling. This likely contributes to its physiological functions, including spontaneous firing of cerebellar Purkinje neurons.     

Association of disease‐predisposition polymorphisms of the melatonin receptors and sunshine duration in the global human populations

Abstract: Melatonin is predominantly involved in signaling circadian and seasonal rhythms, and its synthesis is regulated by the environmental light/dark cycle. The selection pressure by geographically different environmental light/dark cycles, which is predominantly determined by sunshine duration, on the global distribution of genetic polymorphisms in the melatonin pathway is not well understood. Recent genetic association studies identified various disease‐predisposition polymorphisms in this pathway. We investigated the correlations between the prevalence of these clinically important single nucleotide polymorphisms (SNPs) and sunshine duration among worldwide human populations from twelve regions in the CEPH‐HGDP database rs4753426, a recently reported predisposition SNP for type 2 diabetes in the promoter of the MT₂ melatonin receptor gene (MTNR1B), which was not included in the CEPH‐HGDP genotyping array, was additionally genotyped. This SNP showed a marginally significant correlation in 760 CEPH‐HGDP DNA samples (r = ?0.5346, P = 0.0733), and it showed the most prominent association among the candidate melatonin pathway SNPs examined. To control for population structure, which may lead to a false positive correlation, we genotyped this SNP in a replication set of 1792 subjects from China. The correlation was confirmed among Chinese populations (r = ?0.8694, P = 0.0002), and was also statistically significant after correction of other climatic and geographical covariants in multiple regression analysis (β = ?0.907, P = 1.94 × 10^?5). Taken together, it suggests that the human melatonin signaling pathway, particularly MT₂ melatonin receptor may have undergone a selective pressure in response to global variation in sunshine duration.