MT1 mRNA Expression Research Articles

Dexamethasone reduces cisplatin-induced hair cell damage by inducing cisplatin resistance through metallothionein-2.

Hair cell damage is a common side effect caused by the anticancer drug cisplatin (CDDP), which reduces patient quality of life. One CDDP resistance mechanism that occurs in recurrent cancers is heavy metal detoxification by metallothionein-2 (mt2). Here, we show that in zebrafish larvae, dexamethasone (DEX) reduces CDDP-induced hair cell damage by enhancing mt2 expression. Transgenic zebrafish (cldn: gfp; atoh1: rfp) that express green and red fluorescent proteins in neuromasts and hair cells, respectively, were used. The zebrafish were pretreated with DEX at 52h post-fertilization (hpf) for 8h, followed by CDDP treatment for 12h. The lateral line hair cells of CDDP-treated zebrafish at 72hpf were observed by fluorescence microscopy. Reporting odds ratio (ROR) analysis using an adverse event database indicated an association between a decrease in CDDP-induced ototoxicity and DEX as an antiemetic treatment for cancer chemotherapy. Pretreatment with DEX protected 72hpf zebrafish hair cells from CDDP-induced damage. The expression of mt2 mRNA was significantly increased by the combination of 10µM DEX with CDDP. Gene editing of mt2 reversed the protective effect of DEX against CDDP-induced damage in hair cells. DEX protects hair cells from CDDP-induced damage through increased mt2 expression, which is a resistance mechanism for platinum-based anticancer drugs.

Effect of initial periodontal therapy on metallothionein levels in smokers and non-smokers with periodontitis.

The aim of this study was to determine the effect of non-surgical periodontal therapy (NSPT) on mRNA expression of metallothionein (MT) and its levels in serum, saliva and gingival crevicular fluid (GCF) of smokers (S) and non-smokers (NS) with periodontitis (P).A total of 100 participants were included: 48 periodontally healthy (PH) subjects (24 S [PH + S] and 24 NS [PH + NS]) and 52 patients with P (27 S [P + S] and 25 NS [P + NS]). Clinical parameters were recorded, and biofluids (serum, saliva and GCF) and gingival tissue samples were obtained at baseline in all groups and 3months after NSPT in P groups. MT levels in biofluids were determined by ELISA. In gingival tissues, MT-mRNA expression was quantified using real-time PCR. mRNA expression of MT and its levels in biofluids were significantly higher in P + S compared to other groups, and the differences between P + NS and PH + S were non-significant. A significant decrease was observed for MT levels in biofluids, and MT-mRNA expression in periodontitis patients after NSPT. In conclusion, smoking and periodontitis are associated with higher MT expression which decreases after NSPT. MT as an oxidative stress biomarker and its therapeutic role in periodontitis should be investigated in future studies.Clinical trial registration: The study was prospectively registered at Clinical Trials Registry-India (ctri.nic.in) as CTRI/2018/08/015427 on August 23, 2018.

The potential therapeutic effect of melatonin in oxaliplatin combination therapy against chemoresistant colorectal cancer cells.

Oxaliplatin is one of the main therapeutics in colorectal cancer (CRC) chemotherapy. However, in light of multidrug resistance (MDR) phenotype development, the efficacy of oxaliplatin has decreased. This study aimed to assess the potential therapeutic effect of melatonin in oxaliplatin combination therapy for drug-resistant colorectal cancer cells. Initially, the oxaliplatin-resistant cell line was created of LS174T (LS174T/DR) by using the oxaliplatin IC50 concentration and resting cycles. MTT assays and flow cytometry were applied for assessing cell viability and apoptotic cells. The mRNA expression level of Bax, Bcl2, MT1, MT2, and ABCB1 as well as protein levels of ABCB1, Bcl2, BAX were measured by the qRT-PCR and western blot techniques respectively. P-gp activity was assessed by Rho123 staining. The IC50 concentration of oxaliplatin in resistant cells was increased from 500.7 ± 0.2 nM to 7119 ± 0.1 nM. Bcl2, MT1, MT2, and ABCB1 mRNA plus protein expression levels of Bcl2 and ABCB1 were significantly reduced in resistant cells, along with a marked increase in Bax mRNA and protein levels compared to parental cells. Rho 123 staining revealed a marked reduction in P-gp activities in the combination-treated group compared to the oxaliplatin-treated group. The results of cytotoxicity assays, MTT, and flow cytometry revealed that the combination of melatonin and oxaliplatin exerts synergistic effects on induction of oxaliplatin's cytotoxicity in CRC. Our research suggests that combining the treatments of melatonin and oxaliplatin may be considered as a new approach to overcoming oxaliplatin resistance in CRC patients.

Toxicological effects of copper on bioaccumulation and mRNA expression of antioxidant, immune, and apoptosis-related genes in Chinese striped-necked turtle (Mauremys sinensis).

Heavy metals are among the most ubiquitous environmental pollutants of recent decades. Copper is commonly used to control algal blooms or macrophyte and waste infestations, its ambient concentration has increased significantly, indicating possible environmental risk. To investigate the effects of copper exposure on bioaccumulation, antioxidant defense, immune response, and apoptosis in the Chinese Striped-necked Turtle Mauremys sinensis, three experimental groups, control (0.0mg/L), Cu2 (2mg/L) and Cu4 (4mg/L) were designed, and sampled at 14 and 28days. Results showed that copper accumulates in different organs depending on the concentration and exposure time, Liver > Kidney > Gut > Heart > Brain > Muscle and the time order was 28days > 14days. The liver enzymes AST, ALT, and ALP decreased when the turtles were exposed to copper stress, while the contents of bilirubin TBIL, DBIL, IBIL, and LDH showed a significant upward trend. Similarly, the mRNA expression level of acetylcholinesterase AChE in the brain was significantly downregulated upon copper exposure. An upward trend was noticed in the liver Metallothionein MT mRNA expression levels compared to the control group. The mRNA expression levels of antioxidant enzymes CAT, SOD, MnSOD, and GSH-PX1 in the liver increased initially and then significantly decreased. Furthermore, the relative mRNA expression levels of inflammatory cytokines IL-1β, IL-8, TNF-α, and IFN-γ involved in inflammatory response significantly upregulated. Copper significantly increased the hepatic mRNA transcription of heat shock proteins HSP70 and HSP90 at different exposure durations. In addition, the relative mRNA levels of caspase3, caspase8, and caspase9 related to the caspase-dependent apoptotic pathway significantly increased under copper stress. These results explain that copper toxicity causes bioaccumulation, promotes oxidative stress, obstructs immunity, and induces inflammation and apoptosis by altering their gene expression levels in M. sinensis.

Procyanidin B2 alleviates uterine toxicity induced by cadmium exposure in rats: The effect of oxidative stress, inflammation, and gut microbiota

Environmental exposure to hazardous materials causes enormous socioeconomic problems due to its deleterious impacts on human beings, agriculture and animal husbandry. As an important hazardous material, cadmium can promote uterine oxidative stress and inflammation, leading to reproductive toxicity. Antioxidants have been reported to attenuate the reproductive toxicity associated with cadmium exposure. In this study, we investigated the potential protective effect of procyanidin oligosaccharide B2 (PC-B2) and gut microbiota on uterine toxicity induced by cadmium exposure in rats. The results showed that the expression levels of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were reduced in utero. Proinflammatory cytokines (including tumor necrosis factor-α, interleukin-1β and interleukin-6), the NLRP3 inflammasome, Caspase-1 and pro-IL-1β were all involved in inflammatory-mediated uterine injury. PC-B2 prevented CdCl2-induced oxidative stress and inflammation in uterine tissue by increasing antioxidant enzymes and reducing proinflammatory cytokines. Additionally, PC-B2 significantly reduced cadmium deposition in the uterus, possibly through its significant increase in MT1, MT2, and MT3 mRNA expression. Interestingly, PC-B2 protected the uterus from CdCl2 damage by increasing the abundance of intestinal microbiota, promoting beneficial microbiota, and inhibiting harmful microbiota. This study provides novel mechanistic insights into the toxicity of environmental cadmium exposure and indicates that PC-B2 could be used in the prevention of cadmium exposure-induced uterine toxicity.

Micro-polystyrene plastic and benzo[α]pyrene exposure affects the endocrine system and causes physiological stress in Carassius auratus

Microplastics, owing to their hydrophobic properties and the various chemicals used in their production, can act as carriers of persistent organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs). In this study, we exposed the goldfish Carassius auratus to benzo[α]pyrene (BaP, 10 μg/L), a representative PAH, and micro-polystyrene plastic (MP; 10 and 100 beads/L), of size 1.0 μm, as a single or complex environmental stressor, and evaluated the stress response and the resulting DNA damage. The expression of CRH and ACTH mRNA in the pituitary gland and hypothalamus, of the hypothalamus–pituitary–interrenal (HPI) axis, increased significantly after 6 h of exposure. Plasma cortisol levels showed a similar trend to the expression of stress-regulating genes along the HPI axis, and a significant increase was observed in the combined exposure groups (BaP + LMP [low-concentration MP] and BaP + HMP [high-concentration MP]) compared to those in the single exposure group. H2O2 concentration and CYP1A1 and MT mRNA expression levels in the liver were significantly higher in the combined exposure groups compared with in the single exposure groups. In situ hybridization revealed a similar pattern of MT mRNA expression, and many signals were observed in the BaP + HMP group. Furthermore, the BaP + HMP group showed more DNA damage, and the degree of DNA damage increased with exposure time for all experimental groups, except for the control group. Therefore, exposure to BaP and MP alone can induce stress in goldfish; however, when a combination of both substances is provided, their synergistic effect leads to increased stress and DNA damage. MP was confirmed to be a more serious stress-inducing factor in goldfish than BaP, based on the expression levels of stress-regulating genes along the HPI axis.

Seasonal Variation of Melatonin Concentration and mRNA Expression of Melatonin-Related Genes in Developing Ovarian Follicles of Mares Kept under Natural Photoperiods in the Southern Hemisphere.

This study investigated the seasonal variations in mRNA expression of FSH (Fshr), LH (Lhr) receptors, melatonin (Mt1 and Mt2) receptors, melatonin-synthetizing enzymes (Asmt and Aanat) and melatonin concentration in developing follicles from mares raised in natural photoperiods. For one year, ultrasonographic follicular aspiration procedures were performed monthly, and small (<20 mm), medium (20 to 35 mm) and large (>35 mm) follicles were recovered from five mares. One day before monthly sample collections, an exploratory ultrasonography conducted to record the number and the size of all follicles larger than 15 mm. The total number of large follicles were higher during the spring/summer (8.2 ± 1.9) than during autumn/winter (3.0 ± 0.5). Compared to autumn/winter seasons, there was an increase of Fshr and Aanat mRNA expressions in small, medium and large follicles, an increase of Lhr and Asmt mRNA expressions in medium and large follicles and an increase of Mt1 and Mt2 mRNA expressions in small and large follicles during spring/summer. The melatonin levels in follicular fluid were also higher during the spring/summer seasons. The present data show that melatonin locally upregulates the mRNA expression of Mt1 and Mt2 receptors and melatonin-forming enzymes in mare developing follicles during reproductive seasons.

Dexamethasone leads to Zn2+ accumulation and increased unbound Zn2+ in C2C12 muscle and 3T3-L1 adipose cells.

Skeletal muscle atrophy is associated with increases in circulating glucocorticoid levels and insulin resistance. Zinc accumulates in atrophic muscle, but the relationship between atrophy, insulin resistance, and Zn2+ homeostasis remains unclear. In this study, the effect of the glucocorticoid dexamethasone (DEX) on insulin and Zn2+ homeostasis was explored. Treatment of differentiated C2C12 skeletal myotubes and 3T3-L1 adipocytes with DEX significantly increased mRNA expression of the metal-binding proteins Mt1 and 2 and altered energy storage as shown by the increased size of lipid droplets in 3T3-L1 cells. In C2C12 cells the total cellular Zn2+ was higher after DEX treatment, and in both C2C12 and 3T3-L1 adipocytes, free unbound Zn2+ was increased. Insulin treatment led to a gradual increase in free Zn2+ in C2C12 cells, and no significant change in DEX-treatedcells such that concentrations were similar 10 min after insulin treatment. These data demonstrate that DEX disturbs Zn2+ homeostasis in muscle and fat cells. Further study of the molecular pathways involved to identify novel therapeutic targets for treatment of skeletal muscle atrophy is warranted.

Role of melatonin receptor 1B gene polymorphism and its effect on the regulation of glucose transport in gestational diabetes mellitus.

Melatonin receptor 1B (MT2, encoded by the MTNR1B gene), a high-affinity receptor for melatonin, is associated with glucose homeostasis including glucose uptake and transport. The rs10830963 variant in the MTNR1B gene is linked to glucose metabolism disorders including gestational diabetes mellitus (GDM); however, the relationship between MT2-mediated melatonin signaling and a high birth weight of GDM infants from maternal glucose abnormality remains poorly understood. This article aims to investigate the relationship between rs10830963 variants and GDM development, as well as the effects of MT2 receptor on glucose uptake and transport in trophoblasts. TaqMan-MGB (minor groove binder) probe quantitative real-time polymerase chain reaction (qPCR) assays were used for rs10930963 genotyping. MT2 expression in the placenta of GDM and normal pregnant women was detected by immunofluorescence, western blot, and qPCR. The relationship between MT2 and glucose transporters (GLUTs) or peroxisome proliferator-activated receptor γ (PPARγ) was established by western blot, and glucose consumption of trophoblasts was measured by a glucose assay kit. The results showed that the genotype and allele frequencies of rs10830963 were significantly different between GDM and normal pregnant women (P<0.05). The fasting, 1-h and 2-h plasma glucose levels of G-allele carriers were significantly higher than those of C-allele carriers (P<0.05). Besides, the protein and messenger RNA (mRNA) expression of MT2 in the placenta of GDM was significantly higher than that of normal pregnant women (P<0.05). Melatonin could stimulate glucose uptake and GLUT4 and PPARγ protein expression in trophoblasts, which could be attenuated by MT2 receptor knockdown. In conclusion, the rs10830963 variant was associated with an increased risk of GDM. The MT2 receptor is essential for melatonin to raise glucose uptake and transport, which may be mediated by PPARγ.

Toxic effects of polyethylene microplastics on transcriptional changes, biochemical response, and oxidative stress in common carp (Cyprinus carpio)

Aquatic ecosystems have become a place for accumulating microplastics (MPs). MPs can directly or indirectly damage organisms. Although studies of the toxicity of MPs, there are insufficient literature reports on the effects of MPs on freshwater aquatic life. Therefore, this study aimed to evaluate the effect of MPs toxicity on Cyprinus carpio. In this study, biochemical parameters, oxidative biomarkers, and gene expression were assayed in fish exposed to 0, 175, 350, 700, and 1400 μg L−1 of MPs for 30 days. MPs were detected in the liver and intestine of fish using FTIR-analysis. Mt1, Ces2, and P450 mRNA expression were enhanced in the hepatocytes of fish exposed to MPs, while Mt2 gene expression was significantly decreased. After exposure to MPs, MDA and carbonyl protein levels were higher than those of the reference group. The antioxidant capacity and glycogen contents in the hepatocytes significantly declined. MPs significantly inhibited glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), and catalase (CAT) activities. However, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities increased. MPs decreased the total protein, globulin levels, and butyrylcholinesterase (BChE) activity in blood. In contrast, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and creatine phosphokinase (CPK) activities increased in treated-fish with MPs. Glucose, creatinine, cholesterol and triglyceride concentrations in fish exposed to MPs were significantly higher than that of the reference group. Consequently, MPs exposure could disrupt biochemical homeostasis, oxidative stress and alter the expression of genes involved in detoxification.

Pineal melatonin deprivation alters the mRNA expression of melatonin and steroidogenic-related receptor genes in rat oviduct and uterus during the estrus stage of estrous cycle

The effects of pinealectomy and melatonin replacement therapy on the mRNA expression of melatonin (Mt1, Mt2 and Rora), steroidogenic (Era - Estrogen a, Erb - Estrogen b, Ar - Androgen, Pgr - Progesterone, Lhr - LH and Oxr - Oxytocin receptors) -related receptors and melatonin-synthetic enzyme genes (Tph1, Asmt and Aanat) in oviduct and uterus of Wistar rats were studied. Tissues were collected from sham-pinealectomized (Control), pinealectomized (Pinx) and Pinx plus melatonin- supplemented (Pinx-Mel) females during the estrus stage of both the light (ZT6) and dark (ZT18) phases of the light-dark cycle. In oviduct, Pinx altered the mRNA expression of Mt1, Mt2, Rora, Era, Pgr, Oxr, Asmt and Aanat genes. In uterus, Pinx altered the mRNA expression of Mt1, Mt2, Rora, Lhr, Pgr, Oxr and Tph1 genes. Melatonin treatment partially or completely restored the Mt2, Rora, Era, Oxr, Asmt and Aanat mRNA expressions in oviduct and the Mt1, Mt2 and Rora mRNA expressions in uterus. These alterations varied according to the phase of light-dark cycle. The results suggest that pineal melatonin regulates the daily mRNA expression of melatonin and steroidogenic-related receptors in the rat oviduct and uterus during the estrus stage of estrous cycle. Melatonin also regulates the daily mRNA expression of Asmt and Aanat in oviduct and Tph1 in uterus.

MT1 and MT2 melatonin receptors play opposite roles in brain cancer progression.

Primary brain tumors remain among the deadliest of all cancers. Glioma grade IV (glioblastoma), the most common and malignant type of brain cancer, is associated with a 5-year survival rate of < 5%. Melatonin has been widely reported as an anticancer molecule, and we have recently demonstrated that the ability of gliomas to synthesize and accumulate this indolamine in the surrounding microenvironment negatively correlates with tumor malignancy. However, our understanding of the specific effects mediated through the activation of melatonin membrane receptors remains limited. Thus, here we investigated the specific roles of MT1 and MT2 in gliomas and medulloblastomas. Using the MT2 antagonist DH97, we showed that MT1 activation has a negative impact on the proliferation of human glioma and medulloblastoma cell lines, while MT2 activation has an opposite effect. Accordingly, gliomas have a decreased mRNA expression of MT1 (also known as MTNR1A) and an increased mRNA expression of MT2 (also known as MTNR1B) compared to the normal brain cortex. The MT1/MT2 expression ratio negatively correlates with the expression of cell cycle-related genes and is a positive prognostic factor in gliomas. Notably, we showed that functional selective drugs that simultaneously activate MT1 and inhibit MT2 exert robust anti-tumor effects in vitro and in vivo, downregulating the expression of cell cycle and energy metabolism genes in glioma stem-like cells. Overall, we provided the first evidence regarding the differential roles of MT1 and MT2 in brain tumor progression, highlighting their relevance as druggable targets. KEY MESSAGES: • MT1 impairs while MT2 promotes the proliferation of glioma and medulloblastoma cell lines. • Gliomas have a decreased expression of MT1 and an increased expression of MT2 compared to normal brain cortex. • Tumors with a high MT1/MT2 expression ratio have significantly better survival rates. • Functional selective drugs that simultaneously activate MT1 and inhibit MT2 downregulate the expression of cell cycle and energy metabolism genes in glioma stem-like cells and exert robust anti-tumor effects in vivo.

Zinc Homeostasis Alters Zinc Transporter Protein Expression in Vascular Endothelial and Smooth Muscle Cells.

Zinc is an important essential micronutrient with anti-oxidative and anti-inflammatory properties in humans. The role of zinc in signalling has been characterized in the nervous, endocrine, gastrointestinal, renal and reproductive systems. Relatively little is known regarding its role in the vascular system, but the role of zinc homeostasis in augmenting vascular health and vasorelaxation is emerging. Zinc transport proteins are integral to the protective function of zinc, but knowledge of their expression in vascular endothelial and smooth muscle cells is lacking. Human coronary artery endothelial cells and pulmonary artery smooth muscle cells were assessed for gene expression (RT-PCR) of SLC39A (ZIP), SLC30A (ZnT) and metallothionein (MT) families of Zn transporters and storage proteins. Protein expression (fluorescence confocal microscopy) was then analysed for the proteins of interest that changed mRNA expression: ZIP2, ZIP12, ZnT1, ZnT2 and MT1/2. Endothelial and smooth muscle cell mRNA expression of ZnT1, ZnT2 and MT1 was significantly downregulated by low and high Zn conditions, while ZIP2 and ZIP12 expression was induced by Zn depletion with the Zn chelator, TPEN. Changes in gene expression were consistent with protein expression levels for ZIP2, ZIP12 and MT1, where ZIP2 was localized to intracellular bodies and ZIP12 to lamellipodia. Vascular endothelial and smooth muscle cells actively regulate specific Zn transport and metallothionein gene and protein expressions to achieve Zn homeostasis.

Expression of Melatonin Receptor 1 in Rat Mesenteric Artery and Perivascular Adipose Tissue and Vasoactive Action of Melatonin

Melatonin is released by the pineal gland and can modulate cardiovascular system function via the G protein-coupled melatonin receptors MT1 and MT2. Most vessels are surrounded by perivascular adipose tissue (PVAT), which affects their contractility. The aim of our study was to evaluate mRNA and protein expression of MT1 and MT2 in the mesenteric artery (MA) and associated PVAT of male rats by RT-PCR and Western blot. Receptor localization was further studied by immunofluorescence microscopy. Effects of melatonin on neurogenic contractions were explored in isolated superior MA ex vivo by measurement of isometric contractile tension. MT1, but not MT2, was present in MA, and MT1 was localized mainly in vascular smooth muscle. Moreover, we proved the presence of MT1, but not MT2 receptors, in MA-associated PVAT. In isolated superior MA with intact PVAT, neuro-adrenergic contractile responses were significantly smaller when compared to arteries with removed PVAT. Pre-treatment with melatonin of PVAT-stripped arterial rings enhanced neurogenic contractions, while the potentiating effect of melatonin was not detected in preparations with preserved PVAT. We hypothesize that melatonin can stimulate the release of PVAT-derived relaxing factor(s) via MT1, which can override the direct pro-contractile effect of melatonin on vascular smooth muscle. Our results suggest that melatonin is involved in the control of vascular tone in a complex way, which is vessel specific and can reflect a sum of action on different layers of the vessel wall and surrounding PVAT.

5-Azacytidine upregulates melatonin MT1 receptor expression in rat C6 glioma cells: oncostatic implications.

The multiple physiological effects of the indoleamine melatonin, are mediated primarily by its two G protein-coupled MT1 and MT2 receptors. Treatment with histone deacetylase (HDAC) inhibitors, including valproic acid (VPA) and trichostatin A, upregulates melatonin receptors in cultured cells and the rat brain. VPA increases histone H3 acetylation of the MT1 gene promoter in rat C6 glioma cells, indicating that this epigenetic mechanism is involved in upregulation of MT1 expression. Since HDAC inhibitors can alter DNA methylation, the possible involvement of this other epigenetic mechanism, in the regulation of MT1 expression, was examined. RT-qPCR and western blotting studies confirmed that treatment with the DNA demethylating agent, 5-azacytidine (AZA; 10 or 20µM) for 24 or 48h, suppressed DNA methyltransferase 1 mRNA and protein expression in C6 cells. Subsequent treatment with AZA (1-25µM) for 24h, revealed a significant concentration-dependent upregulation of MT1 mRNA expression. Moreover, a combination of 5µM AZA plus 3mM VPA caused a synergistic upregulation of the MT1 receptor, which exceeded the sum of the independent effects of these drugs. These results show that DNA methylation plays a role in the regulation of the MT1 receptor, consistent with the established effects of this major epigenetic mechanism on gene transcription. Combinatorial epigenetic regulation of melatonin receptor expression could provide novel strategies for enhancing the oncostatic, neuroprotective and other therapeutic benefits of this pleiotropic indoleamine and its receptor agonists.

Expression of melatonin receptors and CD4 in the ovine thymus, lymph node, spleen and liver during early pregnancy.

As a pineal gland hormone, melatonin acts through its receptors to modulate the immune system. The immune system is composed of primary and secondary organs, and immune organs are adapted to the presence of the fetal alloantigen during pregnancy. However, it is unclear whether melatonin affects maternal immune organs during early pregnancy in sheep. In this study, the ovine thymus, lymph node, spleen and liver were sampled at day 16 of the oestrous cycle, and at days 13, 16 and 25 of pregnancy. The expression of melatonin receptor 1A (MT1), melatonin receptor 1B (MT2) and cluster of differentiation 4 (CD4) was detected by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry experiments. Our results showed that during early pregnancy there was an upregulation of MT1 mRNA and protein in the thymus, lymph node and liver, and there was a downregulation in the spleen. The expression of MT2 mRNA and protein was increased in the thymus but decreased in the spleen and liver, and there was no significant change in the lymph node during early pregnancy. CD4 protein was upregulated in the thymus, lymph node and liver, but there were no significant changes in the spleen during early pregnancy. In conclusion, early pregnancy induces tissue-specific expression of MT1, MT2 and CD4, which may be due to the different functions of the thymus, lymph node, spleen and liver. Further, melatonin is involved in immune regulation of the maternal thymus, lymph node, spleen and liver during early pregnancy in sheep.

The Absence of Pineal Melatonin Abolishes the Daily Rhythm of Tph1 (Tryptophan Hydroxylase 1), Asmt (Acetylserotonin O-Methyltransferase), and Aanat (Aralkylamine N-Acetyltransferase) mRNA Expressions in Rat Testes.

This study examined the effects of pinealectomy in Wistar rats and melatonin replacement therapy on the daily mRNA expression of melatonin (Tph1, Aanat, Asmt, Mt1, Mt2, and Rorα), and steroidogenic (Star, 17βhsd3, and Lhr) related genes as well as clock genes (Rev-erbα, Bmal1, Per1, Per2, Cry1, and Cry2) in testes. The testes of control animals express the Tph1, Aanat, and Asmt and Per2 genes with 24-h rhythms in mRNA, reaching the maximal values during the dark phase. Pinealectomy abolished and melatonin treatment restored the 24-h rhythmicity. Daytime differences in mRNA expression were significant for Star, Lhr, Mt1, Mt2, Rorα, Rev-erbα, Bmal1, Cry1, and Cry2 genes in testes of control rats. Conversely, 17βhsd3 and Per1 mRNA expression did not show a daytime difference in testes of control animals. Pinealectomy abolished the peak time of Mt1 and Mt2 mRNA expression, phase shifted the peak time of Star, Rorα, Rev-erbα, Bmal1, and Cry2 mRNA expression, downregulated the 24-h Lhr mRNA expression, and inverted the peak time of Per1, Per2, and Cry1 mRNA expression to the light phase. The melatonin replacement therapy completely restored the control levels of Lhr, Rev-erbα, and Per1 mRNA expression patterns, partially restored the daily control of Star, Mt2, Rorα, Bmal1, Cry1, and Cry2 mRNA expression but did not re-establish the daily control of Mt1 mRNA expression. This suggests that the daily mRNA expression of these genes is probably driven by pineal melatonin and melatonin treatment restores (partially or completely) the daily control of gene expression patterns.

Growth, development and survival of European sea bass ( Dicentrarchus labrax ) larvae cultured under different light spectra and intensities

We assessed the effects of light intensity and spectrum on the growth, development and survival of Dicentrarchus labrax larvae from 30 to 96 days after hatching. Twelve lighting regimes were applied using 0.3, 1 and 2 W/m2 full spectrum white, blue, red and green light. By day 66 of exposure, growth performance was significantly better in larvae reared under green or red light compared with white or blue light (p < 0.05), while it was significantly better at 2.0 W/m2 compared with 1.0 or 0.3 W/m2 (p < 0.05). Larvae reared under white or blue light had significantly more deformities (p < 0.05) than did larvae reared under light of other spectra, while larvae reared under 0.3 W/m2 light intensity had a significantly higher proportion of jaw malformations than reared under 1.0 and 2.0 W/m2 (p ˂ 0.05). Dicentrarchus labrax larvae subjected to green light showed significantly lower survival compared with the other light spectra (p < 0.05). Moreover, light significantly affected brain aanat1a, aanatt1b, mellc, mt1, mt2, gh, tsh and crf mRNA expression. Therefore, the red and green light groups (or under 2.0 W/m2) could improve the growth performance and enhance secretion of melatonin, gh and tsh, and reduce the proportion of jaw malformations.

Profile of melatonin and its receptors and synthesizing enzymes in cumulus\u2013oocyte complexes of the developing sheep antral follicle\u2014a potential estradiol-mediated mechanism

BackgroundMelatonin is an amine hormone that plays an important role in regulating mammalian reproduction. This study aimed to investigate the expression pattern of melatonin synthesis enzymes AANAT and HIOMT and melatonin receptors MT1 and MT2 in sheep cumulus–oocyte complexes (COCs) as well as the change of melatonin level in follicular fluid (FF) during antral follicle development. In this research, we also study the effect of β-estradiol (E2) on MT1 and MT2 expression as well as melatonin synthesis in COCs so as to lay the foundation for further exploration of the regulation mechanism of melatonin synthesis in the ovary.MethodsCOCs and FF were collected from different size (large follicles (diameter ≥ 5 mm), medium follicles (diameter 2–5 mm), and small follicles (diameter ≤ 2 mm)) of antral follicles in sheep ovaries. To assess whether E2 regulates melatonin synthase and its receptors expression in sheep COCs and whether it is mediated through estrogen receptor (ER) pathway. The collected COCs were cultured in vitro for 24 h and then treat with 1 μM E2 and/or 1 μM ICI182780 (non-selective ER antagonist). The expression of AANAT, HIOMT, MT1 and MT2 mRNA and protein were determined by qRT-PCR and western blot. The melatonin level was determined by ELISA.ResultsThe expression of AANAT, HIOMT, MT1 and MT2 were significantly higher expression in the COCs of small follicles than in those of large follicles (P < 0.05). However, the melatonin level was significantly higher in large follicle FF than in small follicle FF (P < 0.05). Further, the expression of AANAT, HIOMT, MT1, and MT2 and melatonin production were decreased by E2 treatment (P < 0.05), but when ICI182780 was added, the expression of AANAT, HIOMT, MT1, and MT2 and melatonin production recovered (P < 0.05).ConclusionsWe suggest that sheep COCs can synthesize melatonin, but this ability is decreased with increasing follicle diameter. Furthermore, E2 play an important role in regulated the expression of MT1 and MT2 as well as melatonin synthesis in sheep COCs through the ER pathway.

Melatonin suppresses TLR9-triggered proinflammatory cytokine production in macrophages by inhibiting ERK1/2 and AKT activation

Toll-like receptor (TLR) signaling plays major roles in innate immune response in macrophages. Melatonin regulates TLR3- and TLR4-mediated innate immune responses in macrophages. However, it remains unknown whether melatonin regulates TLR9-mediated innate immune responses in macrophages. Here we demonstrated that melatonin suppressed TLR9 ligand-induced proinflammatory cytokines mRNA and protein production in peritoneal macrophages without interrupting the viability of peritoneal macrophages. Using a melatonin membrane receptors MT1/MT2 antagonist luzindole, we found that MT1 and MT2 were dispensable for melatonin’s inhibitory effects on TLR9-mediated proinflammatory cytokines production, even though melatonin upregulated mRNA expression of MT1 and MT2 in macrophages. Furthermore, melatonin did not affect mRNA expressions of TLR9 and MyD88 but attenuated TLR9 ligand-induced ERK1/2 and AKT phosphorylation without affecting p38 and NF-κB p65 phosphorylation. Also, melatonin inhibited TLR9-mediated proinflammatory cytokines production in vivo. Taken together, our results demonstrate that melatonin suppresses TLR9-triggered proinflammatory cytokines production in macrophages via melatonin membrane receptor-independent manners and probably through inhibiting ERK1/2 and AKT activation, which further elucidates the roles of melatonin in regulating TLR-mediated innate immune responses in macrophages.