Unique arylalkylamine N-acetyltransferase-2 polymorphism in salmonids and profound variations in thermal stability and catalytic efficiency conferred by two residues.

Melatonin contributes to synchronizing major biological and behavioral functions with cyclic changes in the environment. Arylalkylamine N-acetyltransferase (AANAT) is responsible for a daily rhythm in melatonin secretion. Teleost possess two enzyme forms, AANAT1 and AANAT2, preferentially expressed in the retina and the pineal gland, respectively. The concomitant action of light and temperature shapes the daily and seasonal changes in melatonin secretion: the former controls duration while the latter modulates amplitude. Investigating the respective roles of light and temperature is particularly relevant in the context of global warming, which is likely to affect the way fish decode and anticipate seasonal changes, with dramatic consequences on their physiology and behavior. Here we investigated the impact of temperature on pineal melatonin secretion of a migratory species, the Arctic charr (Salvelinus alpinus), the northernmost living and cold-adapted salmonid. We show that temperature directly impacts melatonin production in cultured pineal glands. We also show that one organ expresses two AANAT2 transcripts displaying high similarity between them and with trout Oncorhynchus mykiss AANAT2, differing by only two amino acid sites. We compared the kinetics and 3D models of these enzymes as well as of a chimeric construct, particularly with regard to their response to temperature. Our study brings interesting and new information on the evolutionary diversity of AANAT enzymes in teleosts and the role played by specific residues in the catalytic properties of the enzymes.

Functional diversity of Teleost arylalkylamine N-acetyltransferase-2: is the timezyme evolution driven by habitat temperature?

Arylalkylamine N-acetyltransferase-2 (AANAT2) is the enzyme responsible for the rhythmic production of the time-keeping hormone melatonin. It plays a crucial role in the synchronization of biological functions with changes in the environment. Annual and daily fluctuations in light are known to be key environmental factors involved in such synchronization. Previous studies have demonstrated that AANAT2 activity is also markedly influenced by temperature but the mechanisms through which it impacts the enzyme activity need to be further deciphered. We investigated AANAT2 primary to tertiary structures (3D models) and kinetics in relation to temperature for a variety of Teleost species from tropical to Arctic environments. The results extend our knowledge on the catalytic mechanisms of AANAT enzymes and bring strong support to the idea that AANAT2 diversification was limited by stabilizing selection conferring to the enzyme well conserved secondary and tertiary structures. Only a few changes in amino acids appeared sufficient to induce different enzyme activity patterns. It is concluded that AANAT2 evolution is mainly driven by phylogenetic relationships although catalytic properties (enzyme turnover and substrate affinity) are also under the influence of the respective species normal habitat temperature.

Melatonin modulates secretion of growth hormone and prolactin by trout pituitary glands and cells in culture.

In Teleost fish, development, growth, and reproduction are influenced by the daily and seasonal variations of photoperiod and temperature. Early in vivo studies indicated the pineal gland mediates the effects of these external factors, most probably through the rhythmic production of melatonin. The present investigation was aimed at determining whether melatonin acts directly on the pituitary to control GH and prolactin (PRL) secretion in rainbow trout. We show that 2-[125I]-iodomelatonin, a melatonin analog, binds selectively to membrane preparations and tissue sections from trout pituitaries. The affinity was within the range of that found for the binding to brain microsomal preparations, but the number of binding sites was 20-fold less than in the brain. In culture, melatonin inhibited pituitary cAMP accumulation induced by forskolin, the adenyl cyclase stimulator. Forskolin also induced an increase in GH release, which was reduced in the presence of picomolar concentrations of melatonin. At higher concentrations, the effects of melatonin became stimulatory. In the absence of forskolin, melatonin induced a dose-dependent increase in GH release, and a dose-dependent decrease in PRL release. Melatonin effects were abolished upon addition of luzindole, a melatonin antagonist. Our results provide the first evidence that melatonin modulates GH and PRL secretion in Teleost fish pituitary. Melatonin effects on GH have never been reported in any vertebrate before. The effects result from a direct action of melatonin on pituitary cells. The complexity of the observed responses suggests several types of melatonin receptors might be involved.

Fish growth hormone receptor: molecular characterization of two membrane-anchored forms.

A RT-PCR approach was used to clone and sequence the full-length growth hormone receptor (GHR) of a teleost fish, the turbot (Scophthalmus maximus). Total liver RNA was amplified by RT-PCR with degenerate primers designed in extracellular and cytoplasmic regions, and a single DNA fragment of 1100 bp was obtained. The entire coding region was obtained by 5' and 3' RACE assays, and comprises an open-reading frame of 633 amino acids. This sequence shows the characteristic motifs of the class I cytokine receptor superfamily, and its amino acid identity with mammalian, avian, reptilian and amphibian GHRs is 32-36%. The 3' RACE also revealed the occurrence of an alternate messenger encoding a membrane-anchored truncated receptor, which could facilitate the production of GH-binding protein in fish species. This report represents the first data on fish GHR sequence, and it provides evidence for the conservation of this receptor throughout vertebrate evolution.

Ultrastructural study of the saccular epithelium of the inner ear of two teleosts, oncorhynchus mykiss and psetta maxima

The secretory cells and ionocytes of the saccular epithelium of the inner ear of trout (Oncorhynchus mykiss) and turbot (Psetta maxima) have been studied by electron microscopy. In these species, the saccular epithelium may be subdivided into four zones: the "macula", the "meshwork area", the "patches area", and the "intermediate area". In addition to the sensory "hair cells" and their supporting cells, the macula contains, at its periphery, "granular cells" that have the ultrastructural characteristics of secretory cells. The "meshwork area" around the macula contains large ionocytes endowed with pseudopods, many mitochondria, and three intracytoplasmic membrane systems (endoplasmic reticulum, tubular, and vesicular systems). The patches area, located at some distance from the macula, consists of groups of small mitochondria-rich ionocytes characterized by infoldings of their lateral plasma membrane. In the intermediate area, the size and organelle-content of cells decrease from the meshwork area to the patches area. There is no significant difference in cell composition or structure of the saccular epithelium between the trout and the turbot. The secreting cells might be involved in secretion of endolymph and formation of the otolith, whereas the ionocytes probably regulate the ionic composition of the endolymph.

Characterization of the insulin-like growth factor type 1 receptor messenger in two teleost species.

The insulin-like growth factor type 1 receptor (IGF-1R) is a tyrosine kinase which plays essential role in the regulation of growth and development. In this study, we have cloned cDNAs encoding the tyrosine kinase domain of the IGF-1R from two species of fish. The turbot and trout nucleotide sequences share 82% identity. Moreover, the deduced polypeptides are also highly conserved (> 90% identity) compared with the IGF-1R sequences described in other vertebrates, particularly within domains involved in the catalytic activity and in the transduction pathway. Northern blot analyses have revealed a unique 13-kb mRNA transcript. Using an RT-PCR approach, we have also shown that the polyadenylation status seems to vary according to the developmental stage in turbot: polyadenylated in oocytes and in the first larval stages, the mRNA becomes undetectable in the polyadenylated fraction in later stages or in adult somatic tissues. These results suggest that IGF-1R mRNA undergoes complex post-transcriptional regulation.

Insulin and insulin-like growth factor-1 receptors in an evoluted fish, the turbot: cDNA cloning and mRNA expression.

The insulin receptor (IR) and the structurally related insulin-like growth factor type 1 receptor (IGF-1R) belong to the tyrosine kinase (TK) receptor family. In this study, we have carried out the molecular characterization of both receptors from turbot (Psetta maxima), an evoluted teleost flatfish. The cDNA encoding the precursors of IGF-1R and the nearly entire IR (only the first 16 amino acids of the alpha subunit are missing when compared to the published human sequence) were cloned from an embryonic cDNA library. The deduced polypeptides contain all the topological features characterizing the insulin/IGF-1 receptor family. They are highly conserved compared to their mammalian counterparts, particularly within domains involved in the catalytic activity and in the transduction pathways. Nevertheless, some differences in the primary sequences, especially in the carboxy-terminal domain of the precursors, may affect the functions fulfilled by these receptors. As in mammals, the long IGF-1R 5'-untranslated sequence contains open reading frames and potential Sp1 binding sites. Northern blot analyses have revealed a major IR transcript of 11 kb, which is approximately the size of IGF-1R transcript (Eliès, G., Groigno, L., Wolff, J., Boeuf, G., Boujard, D., 1996. Characterization of the insulin-like growth factor type 1 receptor messenger in two teleost species. Mol. Cell. Endocrinol. 124, 131-140.). If IR and IGF-1R transcripts are detected by RT-PCR at all developmental stages and in all tissues examined, in situ hybridization studies have shown that these mRNA can be visualized as ubiquitous signals only in young larvae, whereas IGF-1R and IR expression appears weaker during later development and in adult tissues.

Genetic, temporal and developmental differences between melatonin rhythm generating systems in the teleost fish pineal organ and retina.

Complete melatonin rhythm generating systems, including photodetector, circadian clock and melatonin synthesis machinery, are located within individual photoreceptor cells in two sites in Teleost fish: the pineal organ and retina. In both, light regulates daily variations in melatonin secretion by controlling the activity of arylalkylamine N-acetyltransferase (AANAT). However, in each species examined to date, marked differences exist between the two organs which may involve the genes encoding the photopigments, genes encoding AANAT, the times of day at which AANAT activity and melatonin production peak and the developmental schedule. We review the fish pineal and retinal melatonin rhythm generating systems and consider the evolutional pressures and other factors which led to these differences.