Low oxygen tolerance of different life stages of temperate freshwater fish species.

Data on low dissolved oxygen (DO2) tolerance of freshwater fish species of north-western Europe were used to create species sensitivity distributions (SSD). Lowest observed effect concentrations (LOEC) and 100% lethal concentrations (LC100) data were collected from the scientific literature. Comparisons were made among life stages as well as between native and exotic species. In addition, lethal DO2 concentrations were compared to oxygen concentrations corresponding to maximum tolerable water temperatures of the same species. Fish eggs and embryos were the least tolerant. Juveniles had a significantly lower mean LOEC than adults, but there was no difference in mean LC100 between the two groups. The difference in lethal oxygen concentrations between adults and juveniles was largest for three salmonids, although it remains uncertain if this was a result of smoltification. There were no significant differences between native and exotic species; however, data on exotics are limited. DO2 concentrations converted from maximum tolerable water temperatures were 3·9 times higher than the measured lethal DO2 concentrations, which may reflect changes in respiration rates (Q10) and may also relate to the simplicity of the model used.

The effects of arachidonic acid on the endocrine and osmoregulatory response of tilapia (Oreochromis mossambicus) acclimated to seawater and subjected to confinement stress.

In previous studies in freshwater tilapia (Oreochromis mossambicus), dietary supplementation with arachidonic acid (ArA; 20:4n - 6) had considerable, opposing effects on the main ion-transporting enzyme Na(+)/K(+)-ATPase in gills and kidneys and changed the release of osmoregulatory hormones, such as cortisol. The present study was performed to assess the influence of dietary ArA on (1) the osmoregulatory capacity of tilapia acclimated to seawater (SW) (34‰) and (2) the osmoregulatory imbalance associated with acute stress. The increased ambient salinity was associated with significant alterations in the tissue fatty acid composition, particularly the n - 6 polyunsaturated fatty acids (PUFAs). Tissue levels of ArA were further increased as a result of dietary supplementation, whereas docosahexaenoic acid (DHA, 22:6n - 3) and eicosapentaenoic acid (EPA, 20:5n - 3) decreased in gills and kidneys. Basal plasma cortisol as well as lactate levels were elevated in the ArA-supplemented SW-acclimated tilapia compared with the control group. The 5 min of confinement (transient stress) increased plasma cortisol, glucose, and lactate levels with significantly higher levels in ArA-supplemented tilapia. Confinement was also associated with significantly elevated plasma osmolality, sodium, chloride, and potassium levels. ArA-supplemented tilapia showed markedly lower ionic disturbances after confinement, suggesting that dietary ArA can attenuate the hydromineral imbalance associated with acute stress. These results emphasize the involvement of ArA and/or its metabolites in the endocrine and osmoregulatory processes and the response to confinement stress.

Cellular responses in the skin of the gilthead sea bream Sparus aurata L. and the sea bass Dicentrarchus labrax (L.) exposed to high ammonia.

Adult gilthead sea bream Sparus aurata and sea bass Dicentrarchus labrax were exposed for 24 and 48 h, respectively, to two concentrations of ammonia each (mean values of 3·34 and 13·10 mg l(-1) TA-N in S. aurata; 2·99 and 11·90 mg l(-1) TA-N in D. labrax). Light microscopy and computerized morphometry were used to evaluate ammonia-induced alterations in skin structure during exposure and following recovery in normal water. In S. aurata, ammonia exposure induced a concentration-dependent increase in the number (hyperplasia) of neutral mucous cells (mc), with peak values at 24 h recovery after exposure. An increase in the dispersion of melanosomes in skin melanocytes was also observed in the dermis and occasionally in the epidermis of S. aurata, with peak values at 24 h of ammonia exposure. Exposure of D. labrax to ammonia had, likewise, concentration-dependent effects on mucous secretion. Of the two types of mc in this species, there was an increase in the number of the neutral mc and a reduction in the much more numerous acid mc, with peak values at 24 and 48 h, respectively, of ammonia exposure. The more intense mucous secretion in D. labrax compared to S. aurata could be related to the lower tolerance to ammonia in D. labrax, as reported elsewhere. Finally, the increase in melanosome dispersion was less evident in D. labrax, due to highly variable control values. These morphological alterations to the skin could be useful indicators of non-specific stress in cultured fishes.

Calcium homeostasis in low and high calcium water acclimatized Oreochromis mossambicus exposed to ambient and dietary cadmium.

The effects of cadmium administered via ambient water (10 microg/l) or food (10 microgCd/fish/day) on plasma calcium, corpuscles of Stannius and bony tissues of Oreochromis mossambicus acclimated to low calcium (0.2 mM) and high calcium (0.8 mM) water were studied for 2, 4, 14 and 35 days. In low calcium water acclimated fish, ambient cadmium induced significant hypocalcemia, while the structure and morphometry of type-1 and type-2 cells of corpuscles of Stannius were not affected on day 2 and 4. Subsequently on day 14 and 35, recovery of plasma calcium to normal levels was observed followed by a decrease in corpuscles of Stannius index (CSI), cell size, volume of granular endoplasmic reticulum (p < 0.05) of type-1 cells in both, fish exposed to ambient or dietary cadmium. The type-2 cells were not affected. In high calcium water acclimated fish both, ambient and dietary cadmium caused a significant reduction of plasma calcium levels on day 2 and 4. In these fish, there was a significant transient increase in the size of corpuscles of Stannius on day 4, followed by recovery on day 14 and 35. Ultrastructural observations of corpuscles of Stannius revealed that cadmium did not cause any cellular damage on type- and type-2 cells during 35 days exposure. In low or high calcium water acclimatized tilapia exposed to ambient or dietary cadmium had no effect on the calcium and phosphate composition of the scales, operculum and vertebrae. Thus, it is unlikely that recovery of hypocalcemia was due to the dissolution of calcium from bony tissues. This study also revealed that cadmium does not mediate stimulation of the corpuscles of Stannius gland, and that high Ca2+ water had a protective effect against ambient and dietary cadmium.

Cloning and sequence analysis of a hypothalamic cDNA encoding a D1c dopamine receptor in tilapia.

Physiological and pharmacological studies have indicated that during acid stress a D1-like dopamine receptor becomes functional on intermediate pituitary melanocyte-stimulating hormone cells of tilapia (Oreochromis mossambicus). As a first step towards physiological expression studies we isolated a D1-like dopamine receptor from a tilapia hypothalamus cDNA library. Construction of a phylogenetic tree of most of the D1-like receptors known in human, rat, Xenopus, goldfish and Drosophila revealed that the here presented clone is most likely the tilapia equivalent of the Xenopus D1c dopamine receptor.

Parathyroid hormone-like effects of rainbow trout Stannius products on bone resorption of embryonic mouse calvaria in vitro.

Products of the Stannius corpuscles (SC) of rainbow trout were tested in an established PTH bioassay involving bone resorption in embryonic mouse calvaria. Aqueous extracts from Stannius corpuscles (SC-homogenate) showed a bone-resorbing activity comparable to PTH in 24-h cultures of calvaria, indicated by a dose-dependent stimulation of lactate production and of calcium, phosphate as well as beta-glucuronidase release. Moreover, SC-homogenates induced an increase in osteoclastic activity. The PTH-like SC-principle is released during in vitro incubations of the glands. These results and the lack of an additive effect of SC-products and PTH on bone resorption suggest that both products activate the same receptor. We hypothesize that the hypocalcemic hormone of the SC of fish shares structural resemblance with PTH, the major hypercalcemic hormone of terrestrial vertebrates.

Prolactin cells and plasma prolactin levels in the aging male Wistar rat: an immunocytochemical and biochemical study.

Non-neoplastic pituitary glands of 12 age-groups of 10 male Wistar rats (subpopulation 1) and of 228 rats with age-related disorders (population 2) were studied. For subpopulation 1 a positive correlation was found between body weight, pars distalis volume and total prolactin (PRL)-cell volume. These values increased during the first half of the life span and decreased to values similar to that found in young rats, after the 50% survival age was reached. Age-related changes were found with respect to the distribution of the PRL cells (from homogeneous into non-homogeneous) and to their ultrastructural features (PRL cells with polymorphic granules were substituted by cells with round granules). Pituitary glands of disordered aged rats (subpopulation 2) showed a similar non-homogeneous distribution of PRL cells. Plasma PRL levels of both subpopulations were similar. No age-related increase of plasma PRL level was found in rats with non-neoplastic pituitary glands.

Expression of tilapia prepro-melanin-concentrating hormone mRNA in hypothalamic and neurohypophysial cells.

Melanin-concentrating hormone (MCH) is a neuropeptide involved in background adaptation in teleost fish, and in multiple regulatory functions in mammals and fish. To study the expression of the MCH preprohormone (ppMCH) in teleosts, we first cloned a hypothalamic cDNA encoding the complete ppMCH of tilapia (Oreochromis mossambicus), and a cRNA probe derived from a 270 bp ppMCH cDNA fragment was used for the expression studies. The level of ppMCH mRNA expression in tilapia hypothalamus, measured by dot blot analysis, was significantly higher in fish adapted to a white background than in black-adapted animals, which is in accordance with the reported MCH plasma and tissue concentrations in fish. Northern blot analysis not only revealed a strong ppMCH mRNA signal in the hypothalamus, but also the presence of ppMCH mRNA in the neurointermediate lobe (NIL) of the pituitary. In situ hybridization and immunocytochemistry showed that ppMCH mRNA as well as MCH immunoreactivity are located in perikarya of two hypothalamic regions, namely in the nucleus lateralis tuberis (NLT) and the nucleus recessus lateralis (NRL). Quantitative analysis by dot blot hybridization revealed about eight times more ppMCH mRNA in the NLT than in the NRL and NIL of mature tilapias. ppMCH mRNA in the NIL could be localized to cell bodies of the neurohypophysis, which were also MCH immunoreactive.

Melanin-concentrating hormone gene-related peptide stimulates ACTH, but not alpha-MSH, release from the tilapia pituitary.

Tilapia (Oreochromis mossambicus; teleostei) melanin-concentrating hormone gene-related peptide (tMgrp) was tested for tropic actions on adenocorticotropin hormone (ACTH) and alpha-melanocyte stimulating hormone (alpha-MSH) producing cells in the tilapia pituitary gland in vitro. Up to 100 microM synthetic tilapia Mgrp (tMgrp) had no effect on alpha-MSH release from tilapia neuro-intermediate lobes in a superfusion set up. However, at concentrations above 1 microM, tMgrp concentration dependently stimulated ACTH release from tilapia anterior lobes. This is the first evidence that Mgrp modulates ACTH release from teleost corticotropes, and this might implicate the peptide in the regulation of the pituitary-interrenal axis of fish.

A role for di-acetyl alpha-melanocyte-stimulating hormone in the control of cortisol release in the teleost Oreochromis mossambicus.

In stressed tilapia, Oreochromis mossambicus, total alpha-melanocyte-stimulating hormone (alpha-MSH) levels and di-acetyl alpha-MSH/mono-acetyl alpha-MSH (di:mono) ratios are elevated. We therefore investigated the role of alpha-MSH in the regulation of the pituitary-interrenal axis. The corticotrophic activities of des-acetyl alpha-MSH, mono-acetyl alpha-MSH and di-acetyl alpha-MSH were compared. These forms of alpha-MSH were isolated from neurointermediate lobes and tested in a superfusion experiment with homologous interrenal tissue. The corticotrophic activity of di-acetyl alpha-MSH was the highest, followed by that of des-acetyl alpha-MSH and mono-acetyl alpha-MSH. Apparently, acetylation of alpha-MSH is of functional significance for corticotrophic action. Di-acetyl alpha-MSH proved to be about 100 times less potent than ACTH(1-39): the half-maximal stimulating concentrations for ACTH and di-acetyl alpha-MSH were 0.89 nmol/l and 110 nmol/l respectively. Surprisingly, a superfusate from neurointermediate lobes proved to be only about three times less active than a superfusate from the pituitary pars distalis, in which the corticotrophic activity is attributable to its ACTH content. When selectively stripped of all forms of alpha-MSH by passage through a Sepharose column coated with an antiserum against alpha-MSH, the neuro-intermediate lobe superfusate was devoid of corticotrophic activity. Thus alpha-MSH appears to be the corticotrophic factor in the superfusate of the neurointermediate lobe. After the same treatment, the corticotrophic activity of the pars distalis superfusate was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning of a proopiomelanocortin cDNA from the pituitary gland of the sea bass (Dicentrarchus labrax) and assessment of mRNA expression in different tissues by means of real-time PCR.

Proopiomelanocortin (POMC) cDNA was cloned from sea bass (Dicentrarchus labrax) pituitary gland. A 743 nucleotide sequence was obtained coding for the following sequences flanked by sets of proteolytic cleavage sites: ACTH (Ser(88)-Met(127)), alpha-MSH (Ser(88)-Gly(102)), CLIP (Pro(106)-Met(127)), beta-LPH (Glu(131)-Gln(208)), gamma-LPH (Glu(131)-Ser(175)), beta-MSH (Asp(159)-Ser(175)), and beta-endorphin (Tyr(178)-Gln(208)). No region homologous to gamma-MSH/joining peptide (a tetrapod POMC feature) was found. Amino acid sequence identity was high with other teleostean species considered (tilapia: 73%) and lower with elasmobranchs (dogfish: 42%). However, the presumed biologically active peptides were highly conserved within all species considered: alpha-MSH (93-100%), ACTH (80-95%) and beta-endorphin (54-90%). Real-time PCR allowed us to quantify the expression of the POMC in different tIssues of the sea bass: pituitary gland, liver, gonad and head kidney. No significant POMC expression was found in the integument. In pituitary gland, gonads, head kidney and liver, POMC expression was respectively, 1.26x10(10), 2.67x10(5), 2.06x10(4) and 1.67x10(4) copies/ micro g mRNA.

Corticotropin-releasing hormone in the teleost stress response: rapid appearance of the peptide in plasma of tilapia (Oreochromis mossambicus).

High concentrations (up to 600 pg/ml) of corticotropin-releasing hormone (CRH) were detected in plasma of the teleost fish Oreochromis mossambicus (tilapia) when screening peripheral tissues of tilapia exposed to stress. Notably, the plasma CRH response to stressors in tilapia is much more pronounced than that in higher vertebrates, such as rats. After characterisation by RIA, by spiking plasma with synthetic tilapia CRH and by methanol-acid extraction, it is concluded that the immunoreactive (ir) material in plasma represents tilapia CRH(1-41). Results indicate that a CRH-binding protein is absent in tilapia plasma. Unstressed fish had plasma CRH levels under the limit of detection (<2 pg/ml), but following capture stress plasma CRH levels (170-300 pg/ml) as well as plasma cortisol levels (120 ng/ml) increased rapidly to plateau levels, which were reached after approximately 5 min. Tilapia CRH(1-41) tested at concentrations between 10(-11) and 10(-7) M in vitro did not stimulate the cortisol release from interrenal tissue. Also pretreatment of interrenal tissue with 10(-9) M CRH did not sensitise the cortisol-producing cells to a subsequent ACTH challenge. Forty-eight hours of net confinement or 48 h of cortisol treatment abolished the plasma CRH response and cortisol response to capture stress. The rapidity of the plasma CRH response and its inhibition after 48 h of stress or cortisol treatment point to release by central nervous tissue. Therefore the distribution of glucocorticoid receptors (GRs) in the brain and pituitary of tilapia was investigated. Main GR-ir cell clusters were found in the medial part (Dm) and posterior part of the dorsal telencephalon, in the preoptic region, in the inferior lobe of the hypothalamus and in the cerebellum. We conclude from comparison of CRH brain contents of unstressed and stressed fish that plasma CRH was released by CRH-ir cells located in the lateral part of the ventral telencephalon (Vl), and suggest that the cortisol feedback on CRH release by Vl is mainly exerted via the forebrain Dm region. We propose that CRH is mobilised during stress to fulfil peripheral functions, such as the regulation of circulating leukocytes or of cardiac output, as CRH receptors have been reported in these organs for fish species.

The stress response of the gilthead sea bream (Sparus aurata L.) to air exposure and confinement.

We investigated short-term effects (up to 24 h) of air exposure and confinement, and long-term effects (up to 11 days) of confinement, to elucidate signalling pathways in the stress response of gilthead sea bream Sparus aurata L. Plasma glucose and lactate were taken as indicators of sympathetic activation, and alpha-melanocyte stimulating hormone (alpha-MSH), adrenocorticotrophic hormone (ACTH) and cortisol as indicators of activation of the brain-pituitary-interrenal (BPI) axis. Air exposure for 3 min resulted, within 30 min, in an increase in plasma concentrations of cortisol, alpha-MSH, glucose, lactate, osmolality and plasma Na, Cl and Mg. Plasma ACTH and beta-endorphin and plasma K, Ca and P did not change. We conclude that air exposure mainly activates the brain-sympathetic-chromaffin cell (BSC) axis. In fish confined at a density of 70 kg/m(3) (compared with 4 kg/m(3) in controls), cortisol, ACTH and alpha-MSH increased within 1 h, indicating activation of the BPI axis. Plasma glucose, Na, Cl and Mg increased with an 8 h delay compared with the response to air exposure. No changes in plasma lactate, osmolality, K, Ca and P were observed. Long-term confinement induced a biphasic cortisol response with peaks at 1 h and at 2 and 3 days. A gradual increase in plasma beta-endorphin concentrations peaked at 7 days; the concentration of alpha-MSH increased rapidly within 1 h and then declined to control values 4 days after the onset of confinement. No changes in ACTH were detected. Our data provide evidence that a stressor-specific activation of the BSC and BPI axes may occur in Sparus aurata.

Differential expression of two pro-opiomelanocortin mRNAs during temperature stress in common carp (Cyprinus carpio L.).

Pro-opiomelanocortin (POMC) is the precursor of a number of biologically active peptides, including adrenocorticotropic hormone, alpha-melanocyte-stimulating hormone and beta-endorphin, which are released by the pituitary glands of fish as well as mammals. To quantify the levels of expression of the two POMC mRNAs relative to one another during the response of the common carp to temperature-induced stress, we used reverse transcriptase PCR combined with capillary electrophoresis and laser-induced fluorescence detection. The ratio of POMC-I mRNA to POMC-II mRNA determined in wild-type and four isogenic carp strains was found to be strain-dependent and influenced by temperature. In strain E20xR8, the ratio had altered in favour of POMC-I from 1:3.2 (POMC-I:POMC-II) in fish adapted to 24 degreesC to 1:1.2 in fish adapted to a decrease of 9 degreesC in ambient temperature. A rapid drop in temperature from 24 to 15 degreesC decreased the POMC mRNA ratio at the expense of POMC-I from 1:1.9 in the control fish (strain E4xR3R8) to 1:4.2 3 h after the temperature drop of 9 degreesC. We conclude that both POMC genes are expressed in the common carp and that their expression ratio is strain-dependent and changes in response to ambient temperature.

Regulation of differential release of alpha-melanocyte-stimulating hormone forms from the pituitary of a teleost fish, Oreochromis mossambicus.

Using high-performance liquid chromatography (HPLC) in combination with radioimmunoassay, three forms of alpha-MSH (des-acetyl, mono-acetyl and di-acetyl alpha-MSH) were separated and identified in tilapia neurointermediate lobes and plasma, and in medium from lobes superfused in vitro. The presence of acetylated forms in lobe extracts indicated that the peptides are acetylated intracellularly. Di-acetyl alpha-MSH was, especially in comparison with monoacetyl alpha-MSH, relatively more abundant in lobe extracts than in plasma. This suggests that the three forms of alpha-MSH are not released according to their relative intracellular abundances. The possibility of regulation of this differential release by dopamine and TRH was investigated, using a microsuperfusion system. Dopamine was a potent inhibitor of alpha-MSH release, but did not modulate the relative abundance of the different forms of alpha-MSH released from the MSH cells. TRH was a potent stimulator of alpha-MSH release. It enhanced in vitro the release of di-acetyl alpha-MSH more than the release of mono-acetyl alpha-MSH. Thus tilapia may be able to modulate not only the quantitative but also the qualitative signal from the MSH cells. This might enhance the flexibility of the animals to respond to environmental challenges.

Stress responsiveness of the pituitary-interrenal axis during early life stages of common carp (Cyprinus carpio).

Whole-body levels of ACTH, alpha-MSH and cortisol in eggs and larvae of the common carp (Cyprinus carpio) were determined periodically up until 168 h after fertilisation. ACTH, alpha-MSH and cortisol immunoreactivity was detected in unfertilised eggs, and endogenous production of ACTH and alpha-MSH was observed 24 h after fertilisation and that of cortisol 36 h after fertilisation. ACTH immunoreactivity reached peak levels before hatching (56-72 h after fertilisation) and remained relatively stable thereafter, while alpha-MSH immunoreactivity started to increase after hatching. At 36 h after fertilisation, whole-body cortisol levels increased rapidly reaching peak levels at the end of hatching (72 h after fertilisation), remaining stable until the end of the experiment. From 50 h after fertilisation onwards, embryos and larvae increased their whole-body cortisol levels when subjected to handling (mechanical pressure during egg stage or netting during the larval stage). It is concluded that the pituitary-interrenal axis in carp is fully functional at the time of hatching. No indications of a stress non-responsive period after hatching were observed. To characterise ACTH and alpha-MSH immunoreactivities in carp larvae, whole-body homogenates were analysed by HPLC, with pituitary homogenates of adult carp serving as a reference. ACTH and alpha-MSH immunoreactivity in carp larvae homogenates consisted of three and two products respectively. HPLC of adult carp pituitaries revealed the presence of two ACTH immunoreactive products, which may represent a phosphorylated and a non-phosphorylated ACTH variant, while the three alpha-MSH peaks most likely represent des-acetylated, mono-acetylated and di-acetylated alpha-MSH, the latter being the predominant form. In carp larvae, however, one of the ACTH immunoreactive products co-eluted with the non-phosphorylated ACTH, while the two alpha-MSH products identified co-eluted with des-acetylated and mono-acetylated alpha-MSH, indicating that POMC processing at this stage of development is different from prohormone processing in adult fish.

Production and purification of biologically active recombinant tilapia (Oreochromis niloticus) prolactins.

Recombinant expression vectors carrying tilapia prolactin-I or -II (tiPRL-I or tiPRL-II) cDNA were constructed and the tiPRL-I and II proteins were produced in E. coli as inclusion bodies. These inclusion bodies were dissolved in 6 mol urea/l. Refolding of the proteins was followed by SDS-PAGE under non-reducing conditions so as to visualize the oxidized state of the molecules. Proteins tiPRL-I and tiPRL-II were purified by gel filtration and ion-exchange chromatography. The N-terminal sequence and bioactivities of both purified proteins were then analysed. Recombinant tiPRL-I and tiPRL-II induced a significant rise in plasma calcium levels as well as in mucocyte density in the abdominal skin epithelium. When tested on kidney membrane, both proteins exhibited potency in competing with 125I-labelled tiPRL-I for binding sites, but tiPRL-I seemed to be more potent than tiPRL-II in competing for these sites. The results obtained for the biological activities tested suggest that both recombinant prolactins were correctly refolded and had retained the full biological activity previously observed with the natural hormone preparations extracted from the animals.

Identification of beta-endorphins in the pituitary gland and blood plasma of the common carp (Cyprinus carpio).

Carp beta-endorphin is posttranslationally modified by N-terminal acetylation and C-terminal cleavage. These processes determine the biological activity of the beta-endorphins. Forms of beta-endorphin were identified in the pars intermedia and the pars distalis of the pituitary gland of the common carp (Cyprinus carpio), as well as the forms released in vitro and into the blood. After separation and quantitation by high performance liquid chromatography (HPLC) coupled with radioimmunoassay, the beta-endorphin immunoreactive products were identified by electrospray ionisation mass spectrometry and peptide sequencing. The release of beta-endorphins by the pituitary gland was studied after stimulation with corticotrophin-releasing factor (CRF) in vitro. In the pars intermedia, eight N-acetylated truncated forms were identified. Full length N-acetyl beta-endorphin(1-33) coeluted with N-acetyl beta-endorphin(1-29) and these forms together amounted to over 50% of total immunoreactivity. These products were partially processed to N-acetyl betaendorphin(1-15) (30.8% of total immunoreactivity) and N-acetyl beta-endorphin(1-10) (3.1%) via two different cleavage pathways. The acetylated carp homologues of mammalian alpha- and gamma-endorphin were also found. N-acetyl beta-endorphin(1-15) and (1-29) and/or (1-33) were the major products to be released in vitro, and were the only acetylated beta-endorphins found in blood plasma, although never together. CRF stimulated the release of opioid beta-endorphin from the pars distalis. This non-acetylated beta-endorphin represents the full length peptide and is the most abundant form in plasma.

Cloning and expression of two proopiomelanocortin mRNAs in the common carp (Cyprinus carpio L.).

Proopiomelanocortin (POMC) is the precursor for a number of biologically active peptides such as adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin. It is well known that these peptides are involved in the stress response in fish as well as in mammals. We have cloned two different carp POMC cDNAs called, POMC-I and POMC-II. The nucleotide sequences of 955 bp for POMC-I and 959 bp for POMC-II share 93.5% identity in their cDNAs, and the deduced amino acid sequences (both 222 amino acids) are 91.4% identical. In the ACTH and beta-MSH domain, two amino acid substitutions are found, whereas alpha-MSH and beta-endorphin are identical. For beta-MSH, the serine replacement (in POMC-I) by a glycine (in POMC-II) results in a putative amidation site Pro-X-Gly for POMC-II. We used RT-PCR to show that both POMC mRNAs are expressed in the hypophysis, hypothalamus and other parts of the brain of a single fish. Furthermore, in a phylogenetic tree based on POMC sequences the divergence of carp POMC-I and -II from tetraploid animals (salmon, trout and xenopus) is demonstrated.

Differential melanin-concentrating hormone gene expression in two hypothalamic nuclei of the teleost tilapia in response to environmental changes.

For some teleosts, a role has been established for melanin-concentrating hormone (MCH) background adaptation and stress response. In teleost fishes, prepro-MCH (ppMCH) mRNA is expressed in the hypothalamus, predominantly in neurons of the nucleus lateralis tuberis (NLT) and in scattered cells of the nucleus recessus lateralis (NRL). The response of mature tilapia to different environmental challenges was studied by assessing ppMCH mRNA levels in these two hypothalamic nuclei by quantitative dot blot analysis. Changes in background colour induced pronounced differences in ppMCH mRNA expression in the NLT, but not in the NRL. The NLT of tilapia adapted to a white background contained 2.5 to 3 times more ppMCH mRNA than the NLT of black-adapted fish. The NLT of fish kept on neutral background contained intermediate levels of ppMCH mRNA, which were significantly lower than the levels in white-adapted fish. Oral administration of dexamethasone lowered plasma cortisol concentrations, but had no effect on ppMCH mRNA levels in white- and black-adapted fish. In tilapia exposed to strongly acidified water (pH 3.5), plasma cortisol and ACThH concentrations were highly elevated, and plasma chloride concentrations considerably lower than in controls. These fish responded with a 70% rise in ppMCH mRNA levels in the NLT, which is most probably associated with a stress response evoked by inadequate osmoregulation. After exposure to a milder acidification (pH 4.0) or to seawater no significant changes in ppMCH mRNA levels occurred in either the NLT or the NRL, nor in plasma chloride, cortisol and ACTH levels. A specific increase of ppMCH mRNA levels in the NRL was observed in repeatedly disturbed tilapia.(ABSTRACT TRUNCATED AT 250 WORDS)