Characterization and maturational differences of melatonin binding sites in the masu salmon brain

To obtain a better understanding of the roles of melatonin in the mediation of photoperiodic signaling, we have examined the pharmacological characteristics, guanine nucleotide modulation, and maturational differences of melatonin binding sites in the brain of masu salmon Oncorhynchus masou by radioreceptor assay using 2-[125I]iodomelatonin as the radioligand. The specific binding of 2-[125I]iodomelatonin was rapid, stable, saturable, and reversible. Saturation experiments demonstrated that 2-[125I]iodomelatonin binds to a single class of receptor sites with an affinity constant (K(d)) of 6.3+/-0.5 pM and a total binding capacity (B(max)) of 15.18+/-0.22 fmol/mg protein in underyearling precocious males in July. Competition experiments revealed that the binding sites are highly specific for melatonin and related analogues. Treatment with guanosine 5(')-O-(3-thiotriphosphate) significantly reduced the specific binding, indicating that melatonin binding sites in the masu salmon brain are coupled to G protein. Significant differences were seen in B(max), but not K(d), among the fish groups differing in maturity. In the underyearling fish in July, the B(max) of precocious males and immature males was significantly higher than that of immature females. Then, the B(max) of precocious males decreased in October, when the fish spermiated. In the 2-year-old fish, B(max) was significantly higher in spermiating males than ovulated females. These results indicate that melatonin plays neuromodulatory roles in the central nervous system through specific receptors. Furthermore, gonadal maturation affects the density of melatonin binding sites in the masu salmon brain by an unknown mechanism.     

Characterization of melatonin binding sites in the brain and retina of the frog Rana perezi

The aim of this study was to characterize 2-[125I]iodomelatonin binding sites in the neural retina and central nervous system (telencephalon, diencephalon, and optic tectum) of the anuran amphibian Rana perezi. Saturation and kinetic studies and pharmacological characterization revealed the existence of a unique melatonin-binding site that belongs to the Mel 1 receptor subtype. The affinity of this site is similar in all tissues studied (Kd, 10.5-12.8 pM), but the density varied from diencephalon and optic tectum, which exhibit the highest density, to telencephalon with the lowest. Neural retina showed an intermediate receptor density. This melatonin-binding site fulfills the requirements of a real hormone receptor; the binding is saturable, reversible, and inhibited by different melatonin agonists and antagonists. The affinity order of ligands is: 2-phenyl-melatonin = 2-I-melatonin > 6-Cl-melatonin = melatoninz > luzindole. Additionally, specific binding is decreased by non-hydrolysable GTP analogue, sodium, and by pretreatment of membranes with pertussis toxin. All these results suggest the existence of a widely distributed and pharmacologically homogeneous melatonin receptor of the subfamily Mel 1 in the nervous system of Rana perezi coupled to a Gi/o protein.     

Circadian expression of Mel1a and PL-II genes in placenta: effects of melatonin on the PL-II gene expression in the rat placenta

In the mammal, melatonin regulates the seasonal and/or circadian rhythm of PRL levels. Since several members of the PRL gene family are expressed during late pregnancy, we investigated the relationship between the expression of placental lactogen (PL)-II-one member of the PRL family- and melatonin, as well as the placental expression of one of the receptors for melatonin, melatonin receptor 1a (Mel_1a⁾. Herein we provide the first demonstration that Mel_1a is not only expressed in the rat placenta, but that it is spatially and temporally regulated throughout late pregnancy. In situ hybridization and Northern blot analyses show that Mel_1a mRNA is localized in the rat placenta on gestational day 19, and is mainly restricted to the spongiotrophoblast and trophoblast giant cells. Interestingly, the junctional zone of the placenta at this time showed the strongest gene expression when the tissue was obtained at 16:00 h (daytime) and showed the least expression when it was obtained at 04:00 h (night-time). In contrast, the labyrinth zone showed the strongest expression in tissue obtained at night and showed the least expression in tissue obtained during the day. PL-II gene expression also exhibited a circadian rhythm but the direction of the fluctuation was exactly opposite to that of the Mel_1a gene, such that at night the junctional zone had the strongest expression, while the labyrinth zone had the weakest. In vitro treatment of placental tissue with an melatonin agonist, chloromelatonin, greatly decreased PL-II mRNA levels. That Mel_1a plays a regulatory role in the expression of PL-II in the late-pregnancy rat placenta is strongly suggested by the pattern of its own spatial and temporal expression.     

Cloning and retinal expression of melatonin receptors in the European sea bass, Dicentrarchus labrax

Melatonin contributes to synchronizing behaviors and physiological functions to daily and seasonal rhythm in fish. However, no coherent vision emerges because the effects vary with the species, sex, age, moment of the year or sexual cycle. And, scarce information is available concerning the melatonin receptors, which is crucial to our understanding of the role melatonin plays. We report here the full length cloning of three different melatonin receptor subtypes in the sea bass Dicentrarchus labrax, belonging, respectively, to the MT1, MT2 and Mel1c subtypes. MT1, the most abundantly expressed, was detected in the central nervous system, retina, and gills. MT2 was detected in the pituitary gland, blood cells and, to a lesser extend, in the optic tectum, diencephalon, liver and retina. Mel1c was mainly expressed in the skin; traces were found in the retina. The cellular sites of MT1 and MT2 expressions were investigated by in situ hybridization in the retina of pigmented and albino fish. The strongest signals were obtained with the MT1 riboprobes. Expression was seen in cells also known to express the enzymes of the melatonin biosynthesis, i.e., in the photoreceptor, inner nuclear and ganglion cell layers. MT1 receptor mRNAs were also abundant in the retinal pigment epithelium. The results are consistent with the idea that melatonin is an autocrine (neural retina) and paracrine (retinal pigment epithelium) regulator of retinal function. The molecular tools provided here will be of valuable interest to further investigate the targets and role of melatonin in nervous and peripheral tissues of fish.     

Melatonin receptors in brain areas and ocular tissues of the teleost Tinca tinca: characterization and effect of temperature

The aim of the present study was to characterize the central melatonin receptors in brain areas and ocular tissues of the teleost Tinca tinca. We investigated the temperature-dependence of 2-iodo-melatonin ([(125)I]Mel) binding in the optic tectum-tegmentum area and the neural retina. The binding of [(125)I]Mel showed a widespread distribution in brain and ocular tissues, with the highest density in the optic tectum-thalamus and the lowest in hindbrain. The [(125)I]Mel affinity was similar in all the studied tissues, and it was on the order of the low pM range. Saturation, kinetic and pharmacological studies showed the presence of a unique MT(1)-like melatonin binding site. In addition, the non-hydrolysable GTP analog, the GTPgammaS, and sodium cations induced a specific binding decrease in the optic tectum and neural retina, suggesting that such melatonin binding sites in the tench are coupled to G protein. Thus, these melatonin binding sites in optic tectum and neural retina fulfil the requirements of a real hormone receptor, the specific binding is rapid, saturable, and reversible, and is inhibited by GTP analogs. Additionally, a clear effect of temperature on such central melatonin receptors was found. Temperature did not modify the B(max) and K(d), but the kinetics of [(125)I]Mel binding resulted in a highly thermosensitive process in both tissues. Both association and dissociation rates (K(+1) and K(-1)) significantly increased with assay temperature (15-30 degrees C), but the K(d) constant (estimated as K(-1)/K(+1)) remained unaltered. In conclusion, this high thermal dependence of the melatonin binding to its receptors in the tench central nervous system supports the conclusion that temperature plays a key role in melatonin signal transduction in fish.     

Effects of pinealectomy and melatonin on gonadotropin-releasing hormone (GnRH) gene expression in the male rat brain

Melatonin, a pineal hormone, is known to be an important neurohormonal factor involved in the timing of reproductive events which occur seasonally in various mammalian species. In order to evaluate the influence of melatonin on neurons which are producing gonadotropin-releasing hormone (GnRH), we studied the effect of light-dark cycle as well as pinealectomy and melatonin administration on GnRH gene expression in the adult male rat medial preoptic area (MPOA) using quantitativein situ hybridization. The animals were kept under artificial light (light on 6:00 h–20:00 h). In animals which were sacrificed at 24:00 h (when endogenous melatonin levels are high), the hybridization signal was higher than that detected in animals sacrificed at 20:00 h (before the onset of darkness). Administration of melatonin during the light period (16:00 h) induced a 15% increase in the amount of GnRH mRNA after 4 h. Three weeks after pinealectomy mRNA levels were decreased by 35%. Injection of melatonin to pinealectomized rats 4 h before sacrifice increase the amount of GnRH mRNA, completely reversing the decrease in mRNA induced by pinealectomy. These results strongly suggest that melatonin produced by the pineal gland exerts a positive influence on GnRH neuronal activity in the male rat.     

Different expression pattern of hepcidin genes in the liver and pancreas of C57BL/6N and DBA/2N mice

BACKGROUND/AIMS: Male C57BL/6 and DBA/2 mice differ in their liver iron content. The aim of this study was to examine possible differences in the expression of hepcidin genes (Hamp and Hamp2) between the two strains. METHODS: Hepatic mRNAs were quantified by real-time PCR. RESULTS: Ferroportin1, transferrin receptor 2 and HAMP mRNA levels displayed no significant strain differences. However, HAMP2 mRNA levels were higher in DBA/2N mice. In both strains, HAMP2 mRNA content was sex-dependent, with higher values in female animals. Both hepatic HAMP and HAMP2 mRNA levels were elevated by iron overload, but treatment with lipopolysaccharide increased only HAMP mRNA. Lipopolysaccharide also elevated the amount of HAMP mRNA in the pancreas, while pancreatic HAMP2 mRNA levels were decreased. Sequence analysis of hepcidin amplicons from DBA/2N mice predicted an Asn-->Lys substitution at position 73 of the HAMP peptide and a Ser-->Phe substitution at position 76 of the HAMP2 peptide. CONCLUSIONS: Hepatic Hamp2 expression displays considerable strain- and sex-dependent variation. Lipopolysaccharide increases expression of Hamp both in the liver and pancreas, but Hamp2 does not respond to lipopolysaccharide treatment. The significance of the amino acid substitutions in hepcidin peptides in DBA/2N mice is at present unknown.     

Expression of GnRH genes is elevated in discrete brain loci of chum salmon before initiation of homing behavior and during spawning migration

Our previous studies suggested the importance of gonadotropin-releasing hormones (GnRHs) for initiation of spawning migration of chum salmon, although supporting evidence had been not available from oceanic fish. In farmed masu salmon, the amounts of salmon GnRH (sGnRH) mRNAs in the forebrain increased in the pre-pubertal stage from winter through spring, followed by a decrease toward summer. We thus hypothesized that gene expression for GnRHs in oceanic chum salmon changes similarly, and examined this hypothesis using brain samples from winter chum salmon in the Gulf of Alaska and summer fish in the Bering Sea. They were classified into sexually immature and maturing adults, which had maturing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GnRH mRNAs were determined by real-time PCRs. The amounts of sGnRH mRNA in the maturing winter adults were significantly larger than those in the maturing summer adults. The amounts of sGnRH and chicken GnRH mRNAs then peaked during upstream migration from the coast to the natal hatchery. Such changes were observed in various brain loci including the olfactory bulb, terminal nerve, ventral telencephalon, nucleus preopticus parvocellularis anterioris, nucleus preopticus magnocellularis and midbrain tegmentum. These results suggest that sGnRH neurons change their activity for gonadal maturation prior to initiation of homing behavior from the Bering Sea. The present study provides the first evidence to support a possible involvement of neuropeptides in the onset of spawning migration.     

急性坏死性胰腺炎胰腺组织中褪黑激素受体表达及褪黑激素干预作用

目的 探讨急性坏死性胰腺炎(ANP)胰腺组织中褪黑激素受体MT1表达及褪黑激素(MT)干预作用.方法 54只SD大鼠随机分为假手术组(SO组)、急性坏死性胰腺炎组(ANP组)、MT干预组(MT组),每组18只.应用胰胆管逆行注射牛磺胆酸钠的方法诱导ANP模型,MT组于诱导模型前30 min腹腔注射MT.术后4 h、8 h和12 h分批处死大鼠(每个时间点6只),以免疫组化和实时定量PCR分别检测胰腺组织中MT1蛋白及MT1 mRNA的表达;检测血清淀粉酶、胰腺组织中丙二醛(MDA)、超氧化物歧化酶(SOD)及TNFα的含量,并行胰腺病理检查.结果 (1)ANP组胰腺病理损伤呈进行性加重,MT组胰腺病理损伤较ANP组明显减轻(P＜0.05).(2)淀粉酶、MDA、TNFα水平在ANP组较S0组明显增高(P＜0.05或P＜0.01),而MT组较相应时间点ANP组显著降低[12 h,(2348.00±278.90)U/L比(3194.83±538.10)U/L,(2.255±0.472)μmol/L比(2.960±0.722)μmol/L,(102.929±29.399)ng/L比(378.544±183.454)ng/L,P＜0.05].SOD水平在ANP组各时点较SO组显著降低(P＜0.01),而MT组较ANP组显著升高[12 h,(11.448±1.594)U/L比(8.427±1.950)U/L,P＜0.05].(3)与SO组相比,MT1蛋白及MT1 mRNA表达在ANP组胰腺组织中明显下降(P＜0.05),且随ANP病变加重表达减弱,而MT组表达较ANP组明显增多.结论 MT干预可提高SOD活力,降低MDA、TNFα水平,故能显著减轻ANP时胰腺病理损伤,MT1在ANP发病机制中可能具有重要作用,MT对ANP中的干预作用可能与上调胰腺组织中MT1的表达有关.     

Diurnal and circadian regulation of a melatonin receptor, MT1, in the golden rabbitfish, Siganus guttatus

The golden rabbitfish Siganus guttatus is a reef fish with a restricted lunar-synchronized spawning rhythmicity and releases gametes simultaneously around the first quarter moon period during the spawning season. In order to understand the molecular aspects of the "circa" rhythms in this species, the full-length melatonin receptor (MT1) cDNA was cloned, and its diurnal/circadian regulation was examined. The full-length MT1 cDNA (1257 bp) contained an open reading frame that encodes a protein of 350 amino acids; this protein is highly homologous to MT1 of nonmammalian species. A high expression of MT1 mRNA with a day-night difference was observed in the whole brain, retina, liver, and kidney. When diurnal variations in MT1 mRNA expression in the retina and whole brain were examined using real-time quantitative RT-PCR, an increase in the mRNA expression was observed during nighttime in both tissues under conditions of light/dark, constant darkness, and constant light. This suggests that MT1 mRNA expression is under circadian regulation. The expression of MT1 mRNA in the cultured pineal gland also showed diurnal variations with high expression levels during nighttime; this suggests that the increased expression level observed in the whole brain is partially of pineal origin. Alternation of light conditions in the pineal gland cultures resulted in the changes in melatonin release into the culture medium as well as MT1 mRNA expression in the pineal gland. The present results suggest that melatonin and its receptors play an important role in the exertion of daily and circadian variations in the neural tissues.     

Retinoid-related receptor (ROR) alpha mRNA expression is altered in the brain of male mice lacking all ligand-binding thyroid hormone receptor (TR) isoforms

In the vertebrate brain, the thalamus serves as a relay and integration station for diverse neuronal information en route from the periphery to the cortex. Deficiency of TH during development results in severe cerebral abnormalities similar to those seen in the mouse when the retinoic acid receptor (ROR)α gene is disrupted. To investigate the effect of the thyroid hormone receptors (TRs) on RORα gene expression, we used intact male mice, in which the genes encoding the α and β TRs have been deleted. In situ hybridization for RORα mRNA revealed that this gene is expressed in specific areas of the brain including the thalamus, pons, cerebellum, cortex, and hippocampus. Our quantitative data showed differences in RORα mRNA expression in different subthalamic nuclei between wild-type and knockout mice. For example, the centromedial nucleus of the thalamus, which plays a role in mediating nociceptive and visceral information from the brainstem to the basal ganglia and cortical regions, has less expression of RORα mRNA in the knockout mice (−37%) compared to the wild-type controls. Also, in the dorsal geniculate (+72%) and lateral posterior nuclei (+58%) we found more RORα mRNA in dKO as compared to dWT animals. Such differences in RORα mRNA expression may play a role in the behavioral alterations resulting from congenital hypothyroidism. The first three authors contributed equally to this work.     

Estrogen mimics bind with similar affinity and specificity to the hepatic estrogen receptor in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

A variety of organic chemicals have been documented to bind to and activate the estrogen receptor (ER) and consequently induce estrogenic effects in different animals. Although the function of the ER seems phylogenetically conserved in vertebrates, a considerable interspecies variation in the structure of the ER has been demonstrated. In this study, the hepatic ER from Atlantic salmon (Salmo salar) and rainbow trout (Oncorhunchus mykiss) were partly characterized, and the ligand-binding preference for a range of endogenous steroids and environmental estrogens (estrogen mimics) was determined by receptor-radio ligand studies. The results show that both Atlantic salmon and rainbow trout livers contain ERs that bind 1,2,4,6,7-[(3)H]estradiol ([(3)H]-E2) with high affinity and low capacity (K(d) = 2.5-4.4 nM and B(max) = 27-97 fmol/mg protein). The Atlantic salmon ER (asER) and rainbow trout ER (rtER) exhibit similar [(3)H]-E2 binding characteristics, although livers from female fish contained a two to three times higher amounts of ER than the males in the two species. In competition studies with [(3)H]-E2, the asER and rtER were found to bind both native steroids (E2 > estrone > 17 beta-estradiol 17-glucuronide > testosterone and 11-ketotestosterone) and putative estrogen mimics (diethylstilbestrol, 4-hydroxytamoxifen, ethynylestradiol > genistein, zearalenone > 4-t-octylphenol, 4-n-nonylphenol, and o,p'-DDT). The pesticides toxaphen and dieldrin, which are proposed to bind to and activate the human ER, did not display significant binding affinity for the fish ER, however. In general, the asER and rtER were found to bind both native steroids and estrogen mimics with similar affinity and specificity. The present results suggest that closely related species such as Atlantic salmon and rainbow trout display similar ER ligand-binding requirements, although interspecies differences in ER affinity and specificity between divergent species such as fish and humans may exist.     

Common genetic variation in the melatonin receptor 1B gene (MTNR1B) is associated with decreased early-phase insulin response

Aims/hypothesis  We investigated whether variation in MTNR1B, which was recently identified as a common genetic determinant of fasting glucose levels in healthy, diabetes-free individuals, is associated with measures of beta cell function and whole-body insulin sensitivity. Methods  We studied 1,276 healthy individuals of European ancestry at 19 centres of the Relationship between Insulin Sensitivity and Cardiovascular disease (RISC) study. Whole-body insulin sensitivity was assessed by euglycaemic–hyperinsulinaemic clamp and indices of beta cell function were derived from a 75 g oral glucose tolerance test (including 30 min insulin response and glucose sensitivity). We studied rs10830963 in MTNR1B using additive genetic models, adjusting for age, sex and recruitment centre. Results  The minor (G) allele of rs10830963 in MTNR1B (frequency 0.30 in HapMap Centre d’Etude du Polymorphisme [Utah residents with northern and western European ancestry] [CEU]; 0.29 in RISC participants) was associated with higher levels of fasting plasma glucose (standardised beta [95% CI] 0.17 [0.085, 0.25] per G allele, p = 5.8 × 10⁻⁵), consistent with recent observations. In addition, the G-allele was significantly associated with lower early insulin response (−0.19 [−0.28, −0.10], p = 1.7 × 10⁻⁵), as well as with decreased beta cell glucose sensitivity (−0.11 [−0.20, −0.027], p = 0.010). No associations were observed with clamp-assessed insulin sensitivity (p = 0.15) or different measures of body size (p > 0.7 for all). Conclusions/interpretation  Genetic variation in MTNR1B is associated with defective early insulin response and decreased beta cell glucose sensitivity, which may contribute to the higher glucose levels of non-diabetic individuals carrying the minor G allele of rs10830963 in MTNR1B. Electronic supplementary material  The online version of this article (doi:) contains a list of the members of the RISC Consortium, which is available to authorised users.     

Four gonadotropin releasing hormone receptor genes in Atlantic cod are differentially expressed in the brain and pituitary during puberty

Gonadotropin releasing hormones (GnRH) are an important part of the brain-pituitary-gonad axis in vertebrates. GnRH binding to its receptors (GnRH-R) stimulates synthesis and release of gonadotropins in the pituitary. GnRH-Rs also mediate other processes in the central nervous system such as reproductive behavior and neuromodulation. As many as five GnRH-R genes have been identified in two teleost fish species, but the function and phylogenetic relationship of these receptors is not fully understood. To gain a better understanding of the functional relationship between multiple GnRH-Rs in an important aquaculture species, the Atlantic cod (Gadus morhua), we identified four GnRH-Rs (gmGnRH-R) by RT-PCR, followed by full-length cloning and sequencing. The deduced amino acid sequences were used for phylogenetic analysis to identify conserved functional motifs and to clarify the relationship of gmGnRH-Rs with other vertebrate GnRH-Rs. The function of GnRH-R variants was investigated by quantitative PCR gene expression analysis in the brain and pituitary of female cod during a full reproductive cycle and in various peripheral tissues in sexually mature fish. Phylogenetic analysis revealed two types of teleost GnRH-Rs: Type I including gmGnRH-R1b and Type II including gmGnRH-R2a, gmGnRH-R2b and gmGnRH-R2c. All four gmGnRH-Rs are expressed in the brain, and gmGnRH-R1b, gmGnRH-R2a and gmGnRH-R2c are expressed in the pituitary. The only GnRH-R differentially expressed in the pituitary during the reproductive cycle is gmGnRH-R2a such that its expression is significantly increased during spawning. These data suggest that gmGnRH-R2a is the most likely candidate to mediate the hypophysiotropic function of GnRH in Atlantic cod.