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.

How should salinity influence fish growth?

Development and growth (continuous in fish) are controlled by 'internal factors' including CNS, endocrinological and neuroendocrinological systems. Among vertebrates, they also are highly dependent on environmental conditions. Among other factors, many studies have reported an influence of water salinity on fish development and growth. In most species, egg fertilization and incubation, yolk sac resorption, early embryogenesis, swimbladder inflation, larval growth are dependent on salinity. In larger fish, salinity is also a key factor in controlling growth. Do the changes in growth rate, that depend on salinity, result from an action on: (1) standard metabolic rate; (2) food intake; (3) food conversion; and/or (4) hormonal stimulation? Better growth at intermediate salinities (8-20 psu) is very often, but not systematically, correlated to a lower standard metabolic rate. Numerous studies have shown that 20 to >50% of the total fish energy budget are dedicated to osmoregulation. However, recent ones indicate that the osmotic cost is not as high (roughly 10%) as this. Data are also available in terms of food intake and stimulation of food conversion, which are both dependent on the environmental salinity. Temperature and salinity have complex interactions. Many hormones are known to be active in both osmoregulation and growth regulation, e.g. in the control of food intake. All of these factors are reviewed. As often, multiple causality is likely to be at work and the interactive effects of salinity on physiology and behaviour must also be taken into account.

Smoltification and seawater adaptation in Atlantic salmon (Salmo salar): plasma prolactin, growth hormone, and thyroid hormones.

To obtain more information on the role of prolactin and growth hormone during the parr-smolt transformation of Atlantic salmon, a population of fish in fresh water was sampled from January to June during two consecutive years. Gill Na+,K+-ATPase activity increased steadily during smoltification and a plasma thyroxine peak was observed 2-3 weeks before the gill Na+,K+-ATPase peak. On the basis of these two parameters, smoltification was considered complete in our populations in April 1985 and May 1986. Two peaks in plasma growth hormone levels occurred in 1986, one in mid-April and the second in mid-May. In both cases, these peaks coincided with a peak in plasma triiodothyronine and preceded the thyroxine peak by 1-2 weeks. Moreover, the second peak which lasted for 1 month coincided with maximal gill Na+,K+-ATPase activity. A decrease in plasma prolactin levels was observed during smoltification of Atlantic salmon in 2 consecutive years. During this period of decreasing and low plasma prolactin levels, gill Na+,K+-ATPase activity increased to its highest values. Atlantic salmon smolts were also directly transferred into seawater. After 2 days or more in seawater, plasma prolactin levels were not significantly different from those on Day 0, whereas in fresh water they showed large fluctuations. All these data indicate that growth hormone may play an important role in the development of hypoosmoregulatory activity. Increased hypoosmoregulatory ability also appears to be associated with low prolactin levels.

Ultrastructural features of chloride cells in the gill epithelium of the Atlantic salmon, Salmo salar, and their modifications during smoltification.

To elucidate the ultrastructural modifications of the gill epithelium during smoltification, gills of the Atlantic salmon (Salmo salar) were examined by electron microscopy at three stages of this process, which were defined as follows: "parrs" were freshwater fish that had not yet started their transformation; "freshwater smolts" were freshwater fish that were ready to enter seawater; and "seawater smolts" were smolts that had been transferred from fresh water and maintained for 4 days in seawater (35%). In the gill epithelium of parrs, there were two types of chloride cells. The large chloride cells contained deeply stained mitochondria and numerous apical, irregular, dense, membrane-bound bodies that formed 77% of the chloride cell population and were distinguished easily from small chloride cells that have distinctly paler mitochondria and no dense bodies in their apical cytoplasm. In freshwater smolts, the large chloride cells formed 95% of the chloride-cell population. In contrast to the small chloride cells that were not modified, they almost doubled in size. Their tubular system developed extensively to form a tight network with regular meshes significantly smaller than those observed in parr chloride cells. Forty percent of the large chloride cells were associated with a new type of cell, the accessory cell, to which they were bound by shallow apical junctions. Half of these accessory cells were not seen to be in contact with the external medium. In seawater smolts, 80% of the large chloride cells were associated with accessory cells. Most accessory cells reached the external medium and sent numerous cytoplasmic interdigitations within the apical portion of the adjacent chloride cells. As a result, a section through the apical portion of the chloride cells and their associated accessory cells revealed a mosaic of interlocked cell processes bound together by an extended, shallow apical junction. It was concluded that the Atlantic salmon develops in fresh water most of the ultrastructural modifications of the gill epithelium which in most euryhaline fish are triggered by exposure to seawater. The effective transfer into seawater would act only as a final stimulus to achieve some adequacy between the freshwater smolt and its new environment.

Plasma and pituitary prolactin levels in rainbow trout during adaptation to different salinities.

The development of a highly specific radioimmunoassay for salmonid prolactin (PRL) using chinook salmon PRL allowed us to study plasma and pituitary PRL profiles in large sedentary rainbow trout (Salmo gairdneri) transferred from fresh water to seawater and vice versa. Plasma osmotic pressure and chloride levels were also measured for 3 weeks following change of salinity. Within 1 day after transfer to full seawater we observed a plasma PRL decrease, which stayed significantly lower (3-5 ng/ml) than the fresh water control group (10-15 ng/ml) during the entire experiment. Pituitary PRL content showed an initial abrupt increase, but after 3 weeks in seawater pituitary PRL content had decreased to the same level as in the fresh water control group. On the contrary, transfer from seawater to fresh water was followed within 1 day by a rise in plasma PRL levels, which stayed high (10-15 ng/ml) after 3 weeks in fresh water. Simultaneously, pituitary PRL content decreased significantly. These results may indicate an important role of PRL in fresh water adaptation of sedentary rainbow trout.

Ultrastructural features of mitochondria-rich cells in stenohaline freshwater and seawater fishes.

In order to elucidate the functional significance of accessory cells in freshwater fishes, such as the rainbow trout, which displays a poor adaptability to seawater life, a search for such cells was performed in two stenohaline freshwater fishes: the loach and the gudgeon. Accessory cells were never encountered in these species; but, in contrast, two types of chloride cells were observed consistently that strikingly resembled the alpha- and beta-cells previously described in the guppy, a freshwater-adapted euryhaline fish. The alpha-cell, a pale and elongated chloride cell, was located at the base of the secondary lamellae in close contact with the arterioarterial pillar capillary. Darker, ovoid chloride cells resembling the beta-cell were found exclusively in the interlamellar region of the primary epithelium facing the central venous sinous. The latter cells frequently formed multicellular complexes linked together by deep, narrow, apical junctions. In another experiment, a stenohaline seawater fish, the turbot, was adapted to diluted 5% saltwater and to fresh water. In seawater, the gill epithelium contained only one type of chloride cell, always associated with accessory cells. Due to numerous cytoplasmic interdigitations between the accessory cells and the apical portion of the chloride cell, there was a noticeable increase in the length of the shallow apical junction, sealing off the intercellular space between the two cell types. In 5% saltwater, there was a decrease in the number of these interdigitations and a concomitant decrease in the length of the shallow apical junction. In fresh water, chloride cells were partially or completely separated from the outside medium by modified accessory cells. It is thus concluded that accessory cells are found exclusively in fish living in seawater or preadapted to seawater and that they probably are involved in the formation and modulation of paracellular pathways for ionic excretion. In contrast, the respective roles of the two types of chloride cells observed in freshwater fishes are still to be determined.

Effects of growth hormone on plasma ionic regulation, respiration and extracellular acid-base status in trout (Oncorhynchus mykiss) transferred to seawater.

The effects of trout recombinant growth hormone (rtGH) treatment (0.25 µg g(-1) by intraperitoneal implant) on plasma ionic regulation, extracellular acid-base status and respiration were investigated in freshwater rainbow trout and during a 4-day period after direct transfer into seawater (35 g 1(-1)).In freshwater, rtGH treatment resulted in a significant increase in gill (Na(+), K(+)) ATPase activity and in standard metabolism (MO2). The latter would mainly result from a higher rate of protein synthesis. Direct transfer from freshwater to seawater induced a decrease in arterial blood pH, far more pronounced in controls than in treated fish. This effect could be regarded in both groups mainly as a metabolic acidosis resulting from extracellular ion composition changes (i.e., an increase higher in chloride than in sodium, more marked in controls than in treated fish). As the rise in PaCO2, in spite of an increase in ventilatory activity, is more significant in controls than in treated fish, it can be assumed that rtGH treatment lightened the decrease in the gas diffusing capacity of gills induced by transfer to seawater. The initial increase in MO2 in both controls and treated fish could be the consequence of an increase in energetic cost of ventilation and osmoregulation. Then, in treated fish, the persistent high level of M may indicate a stimulation of intermediary metabolism by rtGH. In addition, the absence in treated fish of an increase in plasma lactate concentration, as observed in controls, would indicate that rtGH attenuated the decrease in O2 affinity of haemoglobin foreseeable from the metabolic acidosis.

Biochemical relationships between endolymph and otolith matrix in the trout (Oncorhynchus mykiss) and turbot (Psetta maxima).

This paper compares the organic compositions of the otolith and endolymph of trout and turbot. Irrespective of the method of demineralization (0.5 M EDTA or acetic acid), trout otoliths were found to be largely composed of proteins (48%), collagens (23%), and proteoglycans (29%). Collagen was only detectable in the EDTA-insoluble (0.30 microg/mg) and in the acetic acid-soluble fractions (0.53 microg/mg). The same compounds were found in the endolymph but in different proportions (proteins 85%, collagens 12%, and proteoglycans 3%). It was shown that the distribution of these compounds was not uniform within the endolymph. Proteins, collagens, and amino acids were 4, 10, and 3 times, respectively, more concentrated in the proximal (facing the macula) than the distal side whereas proteoglycans were 10 times more concentrated at the distal side. SDS PAGE analyses of proximal and distal samples of endolymph showed similar patterns suggesting that the spatial gradient of protein is quantitative and not qualitative. SDS PAGE comparison of endolymph and otolith samples showed only two proteins with similar molecular weights. We propose that collagen and protein gradients are involved in the organic matrix formation and otolith calcification process. Endolymphs from both trout and turbot display inhibitions of in vitro calcification although these inhibitions were 50 and 80 times, respectively, less than that of the otoliths. The inhibitory factor probably plays a significant role in the regulation of otolith calcification.

Individual diurnal plasma profiles of thyroid hormones in rainbow trout (Oncorhynchus mykiss) in relation to cortisol, growth hormone, and growth rate.

In order to characterize the individual diurnal plasma profiles of triiodothyronine (T3) and thyroxine (T4) in rainbow trout (Oncorhynchus mykiss), blood samples from 41 fish were taken every hour during a 24-hr period, through a catheter inserted into the dorsal aorta. The possible influences of day-night alternation, sex, and diet (feed intake, time of meals) on thyroid hormone (TH) profiles were analyzed. The existence of relations between diurnal plasma profiles of T3, T4, T3/T4 ratio, and those of the growth hormone (GH), cortisol (previously described in Gomez et al., J. Exp. Zool. 274, 171-180, 1996), and the growth rate was monitored. Average daily T3 and T4 concentrations were, respectively, 2.6 +/- 0.2 and 5.5 +/- 0.3 ng/ml (n = 41). Our study showed little or no variation in plasma T3 concentrations during one 24-hr period, while those of T4 fluctuated markedly. T4 peaks occurred from a baseline of 4.0 +/- 0.2 ng/ml at a frequency of 2.5 +/- 0.2 peaks/24 hr, with an amplitude of 3.0 +/- 0.4 ng/ml, and a duration of 4.3 +/- 0.4 hr. There was a significant difference between the average circulating T3 level during the day and that at night (2.4 +/- 0.2 vs 2.7 +/- 0.2 ng/ml). No influence of sex or food factors was observed on daily TH concentrations. TH peaks occurred irregularly and asynchronously without apparent influence of day-night alternation, sex, and diet. The growth rate was significantly correlated with the daily T3 concentration (r = 0.77), but not with T4. No significant relationships were found between daily concentrations of T3, T4, GH, and cortisol. The absence of a relationship between TH and GH concentrations suggests that, in salmonids, GH may have no observable short-term action on the conversion of T4 to T3.

Chemical composition of saccular endolymph and otolith in fish inner ear: lack of spatial uniformity.

Fish otoliths provide a record of age, growth, and environmental influences. In both trout and turbot, spatial chemical investigation of the endolymph surrounding the otolith (sagitta) showed a lack of uniformity. Proteins, PO(3-)(4), and Mg(2+) were significantly more concentrated in the proximal (facing the macula) than distal zone, whereas the opposite was observed for K(+) and total CO(2) (totCO(2)). Na(+) concentration ([Na(+)]) was 20% higher in the proximal zone in trout but not in turbot. Total Ca and Cl(-) contents were uniformly distributed in both species. We propose that the endolymphatic gradients of protein and totCO(2) concentration within the endolymph are involved in the otolithic biocalcification process. Microchemical analyses of otolith sections by wavelength dispersive spectrometry showed a lack of spatial uniformity in the K/Ca and Na/Ca ratios, whereas the Sr/Ca ratio was uniform. There is a clear relationship between endolymph and otolith [K(+)], but the interpretation of the results for [Na(+)] needs further investigation. Thus the lack of uniformity in the otolith composition must be taken into account when investigating otolith microchemistry.