Gene Characterization of Nocturnin Paralogues in Goldfish: Full Coding Sequences, Structure, Phylogeny and Tissue Expression.

The aim of this work is the full characterization of all the nocturnin (noc) paralogues expressed in a teleost, the goldfish. An in silico analysis of the evolutive origin of noc in Osteichthyes is performed, including the splicing variants and new paralogues appearing after teleostean 3R genomic duplication and the cyprinine 4Rc. After sequencing the full-length mRNA of goldfish, we obtained two isoforms for noc-a (noc-aa and noc-ab) with two splice variants (I and II), and only one for noc-b (noc-bb) with two transcripts (II and III). Using the splicing variant II, the prediction of the secondary and tertiary structures renders a well-conserved 3D distribution of four α-helices and nine ß-sheets in the three noc isoforms. A synteny analysis based on the localization of noc genes in the patrilineal or matrilineal subgenomes and a phylogenetic tree of protein sequences were accomplished to stablish a classification and a long-lasting nomenclature of noc in goldfish, and valid to be extrapolated to allotetraploid Cyprininae. Finally, both goldfish and zebrafish showed a broad tissue expression of all the noc paralogues. Moreover, the enriched expression of specific paralogues in some tissues argues in favour of neo- or subfunctionalization.

Daily Rhythms in the IGF-1 System in the Liver of Goldfish and Their Synchronization to Light/Dark Cycle and Feeding Time.

The relevance of the insulin-like growth factor-1 (IGF-1) system in several physiological processes is well-known in vertebrates, although little information about their temporal organization is available. This work aims to investigate the possible rhythmicity of the different components of the IGF-1 system (igf-1, the igf1ra and igf1rb receptors and the paralogs of its binding proteins IGFBP1 and IGFBP2) in the liver of goldfish. In addition, we also study the influence of two environmental cues, the light/dark cycle and feeding time, as zeitgebers. The hepatic igf-1 expression showed a significant daily rhythm with the acrophase prior to feeding time, which seems to be strongly dependent on both zeitgebers. Only igfbp1a-b and igfbp1b-b paralogs exhibited a robust daily rhythm of expression in the liver that persists in fish held under constant darkness or randomly fed. The hepatic expression of the two receptor subtypes did not show daily rhythms in any of the experimental conditions. Altogether these results point to the igf-1, igfbp1a-b, and igfbp1b-b as clock-controlled genes, supporting their role as putative rhythmic outputs of the hepatic oscillator, and highlight the relevance of mealtime as an external cue for the 24-h rhythmic expression of the IGF-1 system in fish.

Characterization of Ghrelin O-Acyltransferase (GOAT) in goldfish (Carassius auratus).

Ghrelin is the only known hormone posttranslationally modified with an acylation. This modification is crucial for most of ghrelin's physiological effects and is catalyzed by the polytopic enzyme ghrelin O-acyltransferase (GOAT). The aim of this study was to characterize GOAT in a teleost model, goldfish (Carassius auratus). First, the full-length cDNA sequence was obtained by RT-PCR and rapid amplification of cDNA ends methods. Two highly homologous cDNAs of 1491 and 1413 bp, respectively, named goat-V1 and goat-V2 were identified. Deduced protein sequences (393 and 367 amino acids, respectively) are predicted to present 11 and 9 transmembrane regions, respectively, and both contain two conserved key residues proposed to be involved in catalysis: asparagine 273 and histidine 304. RT-qPCR revealed that both forms of goat mRNAs show a similar widespread tissue distribution, with the highest expression in the gastrointestinal tract and gonads and less but considerable expression in brain, pituitary, liver and adipose tissue. Immunostaining of intestinal sections showed the presence of GOAT immunoreactive cells in the intestinal mucosa, some of which colocalize with ghrelin. Using an in vitro approach, we observed that acylated ghrelin downregulates GOAT gene and protein levels in cultured intestine in a time-dependent manner. Finally, we found a rhythmic oscillation of goat mRNA expression in the hypothalamus, pituitary and intestinal bulb of goldfish fed at midday, but not at midnight. Together, these findings report novel data characterizing GOAT, and offer new information about the ghrelinergic system in fish.

Melatonin effects on gut motility are independent of the relaxation mediated by the nitrergic system in the goldfish.

Melatonin is a key neuroendocrine transducer in the circadian organization of vertebrates. However, its role in gastrointestinal physiology has not been explored in depth. In goldfish, a role for melatonin as a modulator of intestinal motility has been reported, whereby it attenuates the cholinergic contraction. The aim of the present work was to investigate this relaxation induced by melatonin in the gut smooth muscle of the goldfish, studying the possible involvement of nitric oxide. An in vitro model of isolated goldfish intestine was used to test the effects on intestinal motility. The addition of melatonin (10 pM-100 µM) to the organ bath relaxed acetylcholine- and serotonin-stimulated gut strips, but no effect was observed on KCl-contracted preparations. The addition of L-NAME (nitric oxide synthase inhibitor) increased the amplitude of the spontaneous slow waves, while sodium nitroprusside (SNP, nitric oxide donor) abolished them. All these results support a role for the nitrergic system in goldfish gut motility. However, neither L-NAME, nor SNP nor the nitric oxide precursor, l-arginine, modified the melatonin relaxing effect. These results highlight the existence of a basal nitrergic tone in the gut of goldfish, where melatonin would exert a calcium-dependent, nitric oxide-independent relaxing effect on serotonergic and cholinergic contraction.

Melatonin-synthesizing enzymes in pineal, retina, liver, and gut of the goldfish (Carassius): mRNA expression pattern and regulation of daily rhythms by lighting conditions.

It has been suggested that melatonin is synthesized in nonphotosensitive organs of vertebrates in addition to the well-known sites of the pineal gland and retina. However, very few studies have demonstrated the gene expression of melatonin-synthesizing enzymes in extrapineal and extraretinal locations. This study focuses on the circadian expression of the two key enzymes of the melatoninergic pathway, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), in central and peripheral locations of a teleost fish, the goldfish (Carassius auratus). First, the full-length cDNA sequences corresponding to the goldfish AANAT-2 (gAanat-2) and HIOMT-2 (gHiomt-2) were cloned, showing high similarity with other teleost sequences. Two forms of AANAT exist in teleosts. Here, for the first time, two isoforms of HIOMT are deduced from phylogenetic analysis. Moreover, both HIOMT and AANAT were detected in several peripheral locations, including liver and gut, the present results being the first to find HIOMT in nonphotosensitive structures of a fish species. Second, quantitative real-time polymerase chain reaction (PCR) studies were performed to investigate regulation of gAanat-2 in pineal and peripheral locations of goldfish maintained under different lighting conditions. The current results show circadian rhythms in Aanat-2 and Hiomt-2 transcripts in liver and hindgut, suggesting a local melatonin synthesis in goldfish. Moreover, the analysis of daily expression of gAanat-2 under different lighting conditions, including continuous light (24L) and darkness (24D) revealed light-dependent rhythms in the pineal and retina, as expected, but also in liver and hindgut. The persistence in hindgut of these gAanat-2 rhythms under both constant conditions, 24L and 24D, suggests expression of this transcript is governed by a circadian clock and entrained by nonphotic cues. Finally, the current results support the existence of melatonin synthesis in gut and liver of the goldfish.

Melatonin attenuates the acetylcholine-induced contraction in isolated intestine of a teleost fish.

The present study investigates the possible direct actions of melatonin (N-acetyl-5-methoxytryptamine) on intestinal motility in goldfish (Carassius auratus) using an in vitro system of isolated intestine in an organ bath engaged to an isometric transducer. The longitudinal strips from goldfish intestine in the organ bath showed a resting spontaneous myogenic rhythmic activity which is not altered by melatonin. The addition of acetylcholine (1 nmol l(-1)-10 mmol l(-1)) to the organ bath induces a significant contraction of the intestinal strips in a concentration-dependent manner. The addition of melatonin and its agonist, 2-iodomelatonin, induced a concentration-dependent attenuation of acetylcholine-induced contractile response. The specificity of this effect is tested by the preincubation of the intestine strips in the presence of two melatoninergic antagonists, luzindole (a non-selective MT(1)/MT(2) melatonin receptor antagonist) and 4-P-PDOT (preferred antagonist of MT2 receptor subtype), which counteracted the melatonin-induced relaxation in a concentration-dependent manner. Finally, present results demonstrate that this melatoninergic effect on intestinal strips is a process highly dependent on extracellular calcium. In conclusion, this is the first study demonstrating the role of melatonin in the control of gut motility in a non-mammalian vertebrate. The melatonin effects on isolated intestine from goldfish are mediated by melatoninergic membrane receptors, and could suggest a delay in food transit time, supporting its anorectic effect reported on in vivo studies.

Melatonin binding sites in senegal sole: day/night changes in density and location in different regions of the brain.

We localized melatonin binding sites in different brain regions (optic tectum, telencephalon, cerebellum, hypothalamus, olfactory bulbs, and medulla oblongata) of Senegal sole, a species of aquaculture interest, and checked day/night changes in density (B(max)) at mid-light (ZT06) and mid-dark (ZT18). Plasma melatonin was measured using a radioimmunoassay, while binding assays were performed using 2-[(125)I]iodomelatonin as a radioligand. Plasma melatonin concentrations were significantly lower at mid-light (189.5+/-46 pg/ml) than mid-dark (455.5+/-163 pg/ml). Values of B(max) were statistically significantly higher in the optic tectum (5.6+/-0.6 and 12.3+/-1 fmol/mg prot, at mid-light and mid-dark, respectively) and in the cerebellum (7.7+/-1.1 and 10.6+/-1.3 fmol/mg prot, at mid-light and mid-dark, respectively). Significant day/night differences were only observed in these two tissues. These results show for the first time the distribution of melatonin binding sites within the brain of a flatfish species and their lack of down-regulation.