A comprehensive analysis of teleost MHC class I sequences.

BACKGROUND: MHC class I (MHCI) molecules are the key presenters of peptides generated through the intracellular pathway to CD8-positive T-cells. In fish, MHCI genes were first identified in the early 1990's, but we still know little about their functional relevance. The expansion and presumed sub-functionalization of cod MHCI and access to many published fish genome sequences provide us with the incentive to undertake a comprehensive study of deduced teleost fish MHCI molecules. RESULTS: We expand the known MHCI lineages in teleosts to five with identification of a new lineage defined as P. The two lineages U and Z, which both include presumed peptide binding classical/typical molecules besides more derived molecules, are present in all teleosts analyzed. The U lineage displays two modes of evolution, most pronouncedly observed in classical-type alpha 1 domains; cod and stickleback have expanded on one of at least eight ancient alpha 1 domain lineages as opposed to many other teleosts that preserved a number of these ancient lineages. The Z lineage comes in a typical format present in all analyzed ray-finned fish species as well as lungfish. The typical Z format displays an unprecedented conservation of almost all 37 residues predicted to make up the peptide binding groove. However, also co-existing atypical Z sub-lineage molecules, which lost the presumed peptide binding motif, are found in some fish like carps and cavefish. The remaining three lineages, L, S and P, are not predicted to bind peptides and are lost in some species. CONCLUSIONS: Much like tetrapods, teleosts have polymorphic classical peptide binding MHCI molecules, a number of classical-similar non-classical MHCI molecules, and some members of more diverged MHCI lineages. Different from tetrapods, however, is that in some teleosts the classical MHCI polymorphism incorporates multiple ancient MHCI domain lineages. Also different from tetrapods is that teleosts have typical Z molecules, in which the residues that presumably form the peptide binding groove have been almost completely conserved for over 400 million years. The reasons for the uniquely teleost evolution modes of peptide binding MHCI molecules remain an enigma.

The saccus vasculosus of fish is a sensor of seasonal changes in day length.

The pars tuberalis of the pituitary gland is the regulatory hub for seasonal reproduction in birds and mammals. Although fish also exhibit robust seasonal responses, they do not possess an anatomically distinct pars tuberalis. Here we report that the saccus vasculosus of fish is a seasonal sensor. We observe expression of key genes regulating seasonal reproduction and rhodopsin family genes in the saccus vasculosus of masu salmon. Immunohistochemical studies demonstrate that all of these genes are expressed in the coronet cells of the saccus vasculosus, suggesting the existence of a photoperiodic signalling pathway from light input to neuroendocrine output. In addition, isolated saccus vasculosus has the capacity to respond to photoperiodic signals, and its removal abolishes photoperiodic response of the gonad. Although the physiological role of the saccus vasculosus has been a mystery for several centuries, our findings indicate that the saccus vasculosus acts as a sensor of seasonal changes in day length in fish.

Molecular cloning, molecular evolution and gene expression of cDNAs encoding thyrotropin-releasing hormone receptor subtypes in a teleost, the sockeye salmon (Oncorhynchus nerka).

Molecular cloning of thyrotropin-releasing hormone receptors (TRHR) was performed in a teleost, the sockeye salmon (Oncorhynchus nerka). Four different TRHR cDNAs were cloned and named TRHR1, TRHR2a, TRHR2b and TRHR3 based on their similarity to known TRHR subtypes in vertebrates. Important residues for TRH binding were conserved in deduced amino acid sequences of the three TRHR subtypes except for the TRHR2b. Seven transmembrane domains were predicted for TRHR1, TRHR2a and TRHR3 proteins but only five for TRHR2b which appears to be truncated. In silico database analysis identified putative TRHR sequences including invertebrate TRHR and reptilian, avian and mammalian TRHR3. Phylogenetic analyses predicted the molecular evolution of TRHR in vertebrates: from the common ancestral TRHR (i.e. invertebrate TRHR), the TRHR2 subtype diverged first and then TRHR1 and TRHR3 diverged. Reverse transcription-polymerase chain reaction analyses revealed TRHR1 transcripts in the brain (hypothalamus), retina, pituitary gland and large intestine; TRHR2a in the brain (telencephalon and hypothalamus); and TRHR3 in the brain (olfactory bulbs) and retina.

Lack of circadian regulation of in vitro melatonin release from the pineal organ of salmonid teleosts.

In many teleost species, the photoreceptive pineal organ harbors the circadian clock that regulates melatonin release in the pineal organ itself. However, the pineal organ of three salmonids (rainbow trout Oncorhynchus mykiss, masu salmon Oncorhynchus masou, and sockeye salmon Oncorhynchus nerka) did not exhibit circadian rhythms in melatonin release when maintained under constant darkness (DD) in vitro, suggesting that the pineal organs of all salmonids lack the circadian regulation of melatonin production. To test this hypothesis, the pineal organ of seven salmonids (common whitefish Coregonus lavaretus, grayling Thymallus thymallus, Japanese huchen Hucho perryi, Japanese charr Salvelius leucomaenis pluvius, brook trout Salvelius fontinalis, brown trout Salmo trutta and chum salmon Oncorhynchus keta) and closely related osmerids (ayu Plecoglossus altivelis altivelis and Japanese smelt Hypomesus nipponensis) were individually maintained in flow-through culture at 15 degrees C under several light conditions. Under light-dark cycles, the pineal organ of all species showed a rhythmic melatonin release with high rates during the dark phase. Under DD, the osmerid pineal organs exhibited circadian rhythms in melatonin release with high rates only during the subjective-night but the salmonid pineal organs constantly released melatonin at high rates. Under constant light, melatonin release was suppressed in all species. The pineal organ of rainbow trout maintained at different temperature (15, 20 or 25 degrees C) under DD released melatonin with high rates but the amount of melatonin released was temperature-sensitive (highest at 20 degrees C). Thus, melatonin release from the pineal organ of osmerids is regulated by both light and circadian clock but the circadian regulation is lacking in salmonids. These results indicate that ancestral salmonids lost the circadian regulation of melatonin production after the divergence from osmerid teleosts.

Lack of circadian regulation of melatonin rhythms in the sockeye salmon (Oncorhynchus nerka) in vivo and in vitro.

Melatonin profiles were determined in the plasma in vivo and in the pineal organ in vitro of the sockeye salmon (Oncorhynchus nerka) under various light conditions to test whether they are under circadian regulation. When serial blood samples were taken at 4-h intervals for 3 days via a cannula inserted into the dorsal aorta, plasma melatonin exhibited significant fluctuation under a light-dark cycle, with higher levels during the dark phase than during the light phase. No rhythmic fluctuations persisted under either constant dark or constant light, with constant low and high levels, respectively. Melatonin release from the pineal organ in flow-through culture exhibited a similar pattern in response to the change in light conditions, with high and low release associated with the dark and light phases, respectively. These results indicate that melatonin production in the sockeye salmon is driven by light and darkness but lacks circadian regulation.

Effects of acid water exposure on plasma cortisol, ion balance, and immune functions in the "cobalt" variant of rainbow trout.

This study was undertaken to examine physiological responses to acidification of environmental water in the "cobalt" variant of rainbow trout (Oncorhynchus mykiss), which exhibits malformation of the pituitary, by following changes in plasma levels of cortisol and electrolytes, blood pH, gill Na(+), K(+)-ATPase activity, and immune functions after exposure to acid water (pH 4.5). Resting levels of plasma cortisol and lysozyme were significantly lower in the cobalt variant than in the normal trout, whereas plasma ceruloplasmin was significantly higher in the cobalt variant, suggesting that some endocrine factors, lacking or deficient in the cobalt variant, are important for the regulation of its immune functions. Blood pH was slightly but significantly lower in the cobalt variant at rest. After exposure to acid water for 24 h, both the normal trout and cobalt variant showed a significant elevation in plasma cortisol, although the increased level in the cobalt variant was still lower than that in the normal trout transferred to neutral water. No differences were seen in blood pH, plasma electrolytes, and gill Na(+), K(+)-ATPase activity between the normal trout and the cobalt variant, indicating that the cobalt variant regulates ion balance when exposed to acid water, despite malformation of the pituitary. Although the normal trout showed a reduction in plasma lysozyme level after acid exposure, there was no significant change in the cobalt trout. Adverse effects of pituitary malformation on ion balance and immune functions may be compensated by extrapituitary factors in the cobalt variant when it is exposed to acid water.

Growth and behavioral traits in Donaldson rainbow trout (Oncorhynchus mykiss) cosegregate with classical major histocompatibility complex (MHC) class I genotype.

Although polymorphism in major histocompatibility complex (MHC) genes has been thought to confer populations with protection against widespread decimation by pathogens, this hypothesis cannot explain the type of large allelic diversity in classical MHC class I (Ia) in rainbow trout. Based on expression of Onmy-UBA (MHC class Ia) in trout neurons, we hypothesized that polymorphism in trout class Ia may contribute to polymorphism in behavioral traits. The present study examined whether polymorphism in Onmy-UBA was associated with behavioral variation in Donaldson rainbow trout (Oncorhynchus mykiss) using experiments on food competition, lure-catch, fright recovery, diel locomotor activity and activity characterized as dominance or aggression. These behavioral traits were investigated in fish having Onmy-UBA*401/*401 or *4901/*4901 homozygous, or Onmy-UBA*401/*4901 heterozygous genotypes (referred to as BB, FF and BF, respectively). The BB fish exhibited boldness, aggression, faster growth and crepuscular activity, while the FF fish showed little boldness, smaller body size, and diurnal activity with no aggressive behavior. The BF fish displayed traits intermediary to those of the BB and FF fish. These results are consistent with polymorphism in a single MHC class Ia locus driving variation in neural circuits, thereby creating behavioral variation in the trout. This is the first study in any animal to show a potential correlation between polymorphism in MHC class Ia genes with polymorphism of behavioral traits such as aggression.

Cortisol stimulates growth hormone gene expression in rainbow trout leucocytes in vitro.

Extrapituitary expression of the growth hormone (GH) gene has been reported for the immune system of various vertebrates. In the rainbow trout (Oncorhynchus mykiss), GH mRNA could be detected in several lymphoid organs and leucocytes by reverse transcriptase-polymerase chain reaction (RT-PCR). To understand the control of GH expression in the fish immune system, mRNA levels for two distinct GH genes (GH1 and GH2) in trout leucocytes isolated from peripheral blood were quantified using a real-time PCR method. Both GH mRNAs could be detected in trout leucocytes, although their levels were extremely low compared to those in pituitary cells. The levels of GH2 mRNA in leucocytes were several times higher than those of GH1, while no difference was observed between GH1 and GH2 mRNA levels in the pituitary. Administration of dibutyryl cyclic AMP and cortisol produced a significant elevation of GH mRNA levels in trout leucocytes, although the levels were unchanged by T3. GH1 and GH2 mRNA levels showed similarities in responses to those factors. The effect of cortisol on GH mRNA appears biphasic; a dose-depending elevation of GH gene expression was observed in leucocytes treated with cortisol at below 200 nM, however, cortisol had no effect at 2000 nM. Cortisol-treated leucocytes showed no significant change in the mRNA level of beta-actin or proliferative activity during the experiments. Our results thus show that, at the low levels, GH gene expression in trout leucocytes is regulated by cortisol, which has been known as a regulatory factor of GH gene expression in pituitary cells, and suggest a physiological significance of paracrine GH produced in the fish immune system.

Effects of prolactin and growth hormone on plasma levels of lysozyme and ceruloplasmin in rainbow trout.

In vivo and in vitro effects of prolactin (PRL) and growth hormone (GH) on plasma levels of lysozyme and ceruloplasmin were examined in the rainbow trout (Oncorhynchus mykiss). Hypophysectomy had no effect on the plasma lysozyme level. Implantation of PRL- or GH-containing cholesterol pellets increased the lysozyme level in a dose-related manner. After hypophysectomy and sham operation, plasma ceruloplasmin was elevated above the level in intact fish, suggesting inflammation caused by the surgery. PRL or GH treatment significantly attenuated the increased level of ceruloplasmin in the operated fish. Expression of lysozyme mRNA was detected in the leucocytes isolated from the peripheral blood by RT-PCR. In vitro administration of PRL or GH showed no effect on the proliferation of isolated leucocytes or on the total protein content; however, lysozyme activity in the medium increased in a dose-related manner. These results suggest that PRL and GH directly stimulate lysozyme production without affecting the proliferation of leucocytes, and the attenuated ceruloplasmin level increased in response to inflammation.

Effects of prolactin and growth hormone on proliferation and survival of cultured trout leucocytes.

The in vitro effects of prolactin (PRL) and growth hormone (GH) on the proliferation and survival of leucocytes isolated from the blood of the rainbow trout (Oncorhynchus mykiss) were examined, with special reference to the immunosuppression by cortisol. Both PRL and GH induced a mitogenic effect in trout leucocytes. Contrary, the frequencies of annexin V-positive apoptotic cells and propidium iodide-positive dead cells were decreased by the administration of PRL, but were uninfluenced by GH. Administration of cortisol suppressed the mitotic activity and induced cell death of leucocytes. PRL inhibited the decrease in mitosis and the increase in cell death due to cortisol. GH significantly assisted recovery of cortisol-suppressed mitosis but did not influence the survival of leucocytes. These results suggest that PRL and GH are involved in the maintenance of specific immune functions in fish through a prevention of immunosuppression by cortisol.

The MHC class I linkage group is a major determinant in the in vivo rejection of allogeneic erythrocytes in rainbow trout (Oncorhynchus mykiss).

Despite accumulating sequence data, information on the function of major histocompatibility complex (MHC) genes in fish is scarce. In contrast to the genome organization in higher vertebrates, the polymorphic MHC class I and II genes are not linked in the teleost genome. A previous study found an MHC class II linkage group to be a major determinant in the rejection of allogeneic scales by a teleost species (Cardwell et al. 2001). The present study investigated whether the teleost MHC class I linkage group can be involved in allograft rejection. Erythrocytes were chosen as grafts since they express MHC class I, but do not express class II. Rainbow trout erythrocytes expressing different MHC class I alleles were differentially stained, mixed and injected into recipients that were of the same sibling group as the donors. The MHC class I linkage group was the major determinant for in vivo graft rejection.

Relationships between obesity and metabolic hormones in the "cobalt" variant of rainbow trout.

The "cobalt" variant of rainbow trout (Oncorhynchus mykiss) lacks most of the pars intermedia of the pituitary, and shows significant obesity with an enlarged liver and a fat accumulation in the abdominal cavity. Plasma levels of growth hormone, prolactin, and somatolactin were significantly lower in the cobalt variant than those in the normal trout. In contrast, plasma insulin level was four times higher than that in the normal. Plasma levels of total protein, free cholesterol, and triacylglycerol were higher in the cobalt, while those of glucose and fatty acids were not different from the normal levels. In the white muscle, red muscle, liver, and mesenteric fat, the cobalt showed higher contents of triacylglycerol than the normal fish. There was no significant difference in tissue contents of phosphatidylcholine between the two groups of the trout, except for that in the mesenteric fat, exhibiting significantly lower content than in the normal fish. Activity of triacylglycerol lipase in the liver in vivo was lower in the cobalt than that in the normal trout, while there was no significant difference between the two in the cultured liver slices. Desacetyl-alpha-MSH stimulated lipolysis of triacylglycerol similarly in the cultured liver slices from the normal trout and from the cobalt variant. Results from this study suggest that the lack of pars intermedia and the increased plasma level of insulin are involved in a depression of lipid mobilization and obesity in this variant of rainbow trout.

Differences in MHC class I genes between strains of rainbow trout (Oncorhynchus mykiss).

In rainbow trout there is only one dominant classical MHC class I locus, Onmy-UBA, for which four very different allelic lineages have been described. The purpose of the present study was to determine if Onmy-UBA polymorphism could be used for strain characterisation. This was performed by lineage-specific PCR investigation of 30 fish, each of the Nikko and Donaldson strains, and by sequence analysis of 25 of the amplified DNA fragments. Two new MHC class I lineages were detected in addition to the four previously described lineages, thus six distinct lineages were observed within the fish examined (Sal-MHCIa*A-F). The distribution of lineages appeared to be strain-specific. For example, the lineage Sal-MHCIa*A was very common in the Nikko strain but could not be detected in the Donaldson strain. Analysis of MHC class I variation may help to elucidate relationships between strains and the roles of MHC alleles in disease resistance.

Hypophysectomy depresses immune functions in rainbow trout.

As the immune system is known to be influenced by the endocrine system, the effects of hypophysectomy on immune functions were examined in the rainbow trout (Oncorhynchus mykiss). Superoxide anion (O2-) production, accompanied by phagocytosis, was significantly decreased in leucocytes isolated from the head kidney 7 days after hypophysectomy. Significant reduction was also observed in plasma immunoglobulin (Ig) M levels, whereas no change was observed in plasma lysozyme activity. The number of Ig-secreting leucocytes in peripheral blood had decreased after hypophysectomy, although total leucocyte number was not affected. The percentage of Ig-producing leucocytes as assessed by flow cytometry using a monoclonal antibody to trout IgM showed significant reduction in the head kidney. However, hypophysectomy did not affect the number of Ig-producing leucocytes in spleen, thymus or peripheral blood. By RT-PCR, expression of two growth hormones (GH I and II) and prolactin (PRL) mRNA was detected in lymphoid tissues, such as head kidney, spleen, thymus and intestine, as well as in leucocytes from blood and head kidney, indicating the local production of these hormones. These results indicate important roles of hypophyseal hormones produced not only in the pituitary, but also in the lymphoid tissues, in the maintenance of the immune functions in trout.