Characterization of a novel growth hormone receptor-encoding cDNA in rainbow trout and regulation of its expression by nutritional state

To clarify the divergence of the growth hormone receptor (GHR) family, we characterized a novel GHR from a teleost fish (rainbow trout). A 2357-nt cDNA was isolated and found to contain a single initiation site 71 nt from the most 5′ end, an open reading frame of 1971 nt encoding a 657-amino acid protein, and a single polyadenylation site 229 nt from the poly-A tail. Based on structural analysis, the protein was identified as a type 1 GHR (GHR1). The new GHR1 shares 42% and 43% amino acid identity, respectively, with GHR2a and GHR2b, the two type 2 GHRs isolated from trout previously. GHR1 mRNA was found in a wide array of tissues with the highest expression in the liver, red muscle, and white muscle. Fasting animals for 4 weeks reduced steady state levels of GHR1 in the liver, adipose, and red muscle. These findings help clarify the divergence and nomenclature of GHRs and provide insight into the function of duplicated GHR types.     

Medaka receptors for somatolactin and growth hormone: phylogenetic paradox among fish growth hormone receptors

Somatolactin (SL) in fish belongs to the growth hormone/prolactin family. Its ortholog in tetrapods has not been identified and its function(s) remains largely unknown. The SL-deficient mutant of medaka (color interfere, ci) and an SL receptor (SLR) recently identified in salmon provide a fascinating field for investigating SL's function(s) in vivo. Here we isolated a medaka ortholog of the salmon SLR. The mRNA is transcribed in variable organs. Triglycerides and cholesterol contents in the ci are significantly higher than those in the wild type, providing the first evidence of SL's function in suppressing lipid accumulation to organs. Interestingly, phylogenetic comparisons between the medaka SLR and growth hormone receptor (GHR), which is also isolated in this study, in relation to GHRs of other fish, suggested that all GHRs reported from nonsalmonid species are, at least phylogenetically, SLRs. An extra intron inserted in medaka and pufferfish SLRs and flounder and sea bream GHRs also supports their orthologous relationship, but not with tetrapod GHRs. These results may indicate lineage-specific diversification of SLR and GHR functions among fish or just an inappropriate naming of these receptors. Further functional and comparative reassessments are necessary to address this question.     

Ichthyophonus infections in cultured marine fish from Spain

Ichthyophonus sp. is reported for the first time in Mugil capito (thinlip grey mullet) and Li a saliens (leaping grey mullet). The fungus was also found in L. aurata (golden grey mullet), Dicentrarchus labrax (sea bass), Sparus aurata (gilthead sea bream) and Scophthalmus maximus (turbot), whereas Mugil cephalus (grey mullet) was not parasitized. In fish sampled periodically, the highest prevalences were observed in sea bass and the lowest in turbot. Among the fish sampled occasionally, the fungus was found associated to an epizootic in thinlip grey mullet. Ichthyophonus was never found in fish weighing <0·5 g. An increase in the prevalence of infection with the age of turbot and gilthead sea bream was observed. Gilthead sea bream and sea bass showed higher prevalences in a closed system than in open and semi-intensive systems. Multinucleate spherical spores, hyphae and endospores of Ichthyophonus sp. parasitized different organs of thinlip and leaping grey mullets, though infection intensity was maximal in the spleen. In the remaining fish, the fungus was found mainly in the trunk kidney, where it appeared frequently in a necrotic form. Ichthyophonus sp. can be considered a potential threat for marine fish aquaculture, especially in culture conditions which may favour the introduction and transmission of the fungus.     

Nutritional assessment of somatolactin function in gilthead sea bream (Sparus aurata): concurrent changes in somatotropic axis and pancreatic hormones

The role of somatolactin (SL) in the regulation of energy homeostasis in gilthead sea bream (Sparus aurata) has been analysed. First, a down-regulation of plasma SL levels in response to gross shifts in dietary amino acid profile and the graded replacement of fish meal by plant protein sources (50%, 75% and 100%) has been observed. Thus, the impaired growth performance with changes in dietary amino acid profile and dietary protein source was accompanied by a decrease in plasma SL levels, which also decreased over the course of the post-prandial period irrespective of dietary nitrogen source. Secondly, we examined the effect of SL and growth hormone (GH) administration on voluntary feed intake. A single intraperitoneal injection of recombinant gilthead sea bream SL (0.1 microg/g fish) evoked a short-term inhibition of feed intake, whereas the same dose of GH exerted a marked enhancement of feed intake that still persisted 1 week later. Further, we addressed the effect of arginine (Arg) injection upon SL and related metabolic hormones (GH, insulin-like growth factor-I (IGF-I), insulin and glucagon) in fish fed diets with different nitrogen sources. A consistent effect of Arg injection (6.6 micromol/g fish) on plasma GH and IGF-I levels was not found regardless of dietary treatment. In contrast, the insulinotropic effect of Arg was found irrespective of dietary treatment, although the up-regulation of plasma glucagon and glucose levels was more persistent in fish fed a fish meal based diet (diet FM) than in those fed a plant protein diet with a 75% replacement (diet PP75). In the same way, a persistent and two-fold increase in plasma SL levels was observed in fish fed diet FM, whereas no effect was found in fish fed diet PP75. Taken together, these findings provide additional evidence for a role of SL as a marker of energy status, which may be perceived by fish as a daily and seasonal signal of abundant energy at a precise calendar time.     

Cloning and analysis of expression of a gilthead sea bream (Sparus aurata) Mx cDNA

In the current work, we have cloned and sequenced the full cDNA for a Mx protein in the gilthead sea bream (Sparus aurata) by RACE PCR. The Mx cDNA of 2182 bp contained an open reading frame of 1857 bp that codes for a protein of 618 aa. Within the coding sequence, characteristic features of Mx proteins were found, such as a tripartite guanosine-5'-triphosphate (GTP)-binding motif (GXXXSGKS/T, DXXG and T/NKXD), the signature of the dynamin family, LPRG(S/K)GIVTR, and a sequence that codes for a leucine zipper at the C-terminal region of the protein. An RT-PCR was optimised to estimate the level of expression of Mx protein in sea bream. Through this method we determined that Mx is constitutively expressed in head kidney, liver, spleen, heart, gills, muscle and brain of healthy sea bream. Intramuscular challenge of sea bream with polyinosinic:polycytidylic acid (Poly I:C) up-regulated Mx expression in liver, head kidney, spleen and muscle. Constitutive expression was also found in isolated head kidney macrophages and blood leukocytes. This expression was significantly up-regulated by addition of Poly I:C. Mx was not constitutively expressed in the sea bream established cell line, SAF-1, but Poly I:C and nodavirus were also capable of inducing Mx expression in this cell line.     

Amino acid composition and endocrine control of vitelline envelope proteins in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata)

The vitelline envelopes of European sea bass and gilthead sea bream are both composed of mainly four proteins with the molecular masses of 90, 52, 48, 45 kDa and 75, 50, 48, 44 kDa, respectively. Each protein has an amino acid composition that is characterized by a high content of proline and glutamic acid and a low content of cysteine, similar to the whole vitelline envelope of both species. The amino acid composition suggests that each protein is distinct but related to the other vitelline envelope proteins. The use of homologous antisera shows that both species have vitelline envelope proteins that are induced by estradiol-17β. As males of both species synthesize these proteins after treatment with estradiol-17β, the origin is not restricted to the ovaries. Vitellogenin of both Eurpoean sea bass and gilthead sea bream has the apparent molecular mass of 170 kDa. © 1995 Wiley-Liss, Inc.     

大熊猫生长激素受体(GHR) cDNA 的克隆与序列分析

根据已报道的若干物种GHR 基因cDNA 序列设计引物, 利用RT- PCR 技术首次从大熊猫肝脏组织总RNA中扩增出GHR 基因编码区全长cDNA 序列, 克隆于pGEM®-T 载体后进行测序和序列分析。结果表明,大熊猫GHR 的ORF为1 917 bp , 编码638 个氨基酸的前体蛋白, 由18 个氨基酸的信号肽和620 个氨基酸的成熟肽组成,与人、狗、猪GHR 结构相似, 大熊猫GHR 成熟肽由246 个氨基酸的胞外区、24 个氨基酸的跨膜区和350 个氨基酸的胞内区组成, 并具GHR 的特征性结构。序列相似性比较显示, 大熊猫GHR 与哺乳类GHR 具有69 %～93 %的高序列相似性, 与爬行类和鸟类的序列相似性也达到60 % , 而与鱼类的序列相似性较低, 仅为30 %左右。与其它哺乳动物GHR 相比, 大熊猫GHR 在氨基酸序列上也存在明显的特异性。     

Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.     

The cytokine IL-1β from the crocodile icefish Chionodraco hamatus (Perciformes: Channichthyidae)

The immune system of the so-called crocodile icefishes, members of the family Channichthyidae, has been recently investigated to study its morphological and functional organisation, to evidence the presence of specific immune humoral responses, and to analyse immunoglobulin genes. In the present study, primers designed to conserved regions of interleukin-1β were used for the homology cloning of the icefish Chionodraco hamatus IL-1β gene. The full length cDNA consists of 1,289 nucleotides that translate in a single reading frame to give a predicted 250-amino acid molecule. The sequence had the highest nucleotide identity (75.7%) and amino acid similarity (69.8%) and identity (63.2%) with turbot IL-1β, followed by European sea bass and gilthead sea bream. Studies of IL-1β expression indicated that this molecule is induced, both in vitro and in vivo, by bacterial lipopolysaccharide (LPS). Finally, similar to other Perciformes IL-1β genes, the C. hamatus gene is organised in five exons and four introns.     

北极狐GHR基因cDNA的克隆及序列分析

本文根据狗(AF133835)的GHR基因cDNA编码全序列设计了三对引物,利用RT-PCR方法克隆出北极狐GHR基因编码区全长cDNA序列(GenBank accession No.EU304325)。结果表明,北极狐GHR的ORF为1917bp,编码638个氨基酸的前体蛋白,由18个氨基酸的信号肽和620个氨基酸的成熟肽组成。通过同源性比较发现北极狐与狗的同源性最高,达到98%。另外,利用邻接法(NJ法)构建的分子系统进化树聚类结果表明,北极狐与狗先聚为一类,该聚类结果与传统的物种进化关系基本一致。另外,通过氨基酸对位序列比较发现,北极狐GHR在氨基酸序列上存在明显的特异性,如45和451位分别为A和E,而其它物种均分别为T(大鼠为K)和A(牛羊为V,鼠为T)。     

Homologous growth hormone (GH) binding in gilthead sea bream (Sparus aurata). Effect of fasting and refeeding on hepatic GH-binding and plasma somatomedin-like immunoreactivity

Specific binding of gilthead sea bream growth hormone (sbGH) to liver membrane preparations was a time and temperature dependent process, and was saturable by increasing amounts of membrane proteins. Scatchard analysis evidenced a single class of high-affinity and lowcapacity binding sites. Ovine prolactin, recombinant tilapia prolactin, carp gonadotropin and chinook salmon gonadotropin did not compete for the¹²⁵I-sbGH binding sites, while recombinant trout GH, bovine GH and human GH displaced iodinated sbGH in a dose dependent-manner. IGF-I-like immunoreactivity was detected after acidification of plasma and removal of IGF-I binding activity. A parallel displacement to the rhIGF-1 standard was observed with extracted plasma samples. Free and total hepatic GH-binding decreased during long-term starvation (3–9 weeks), returning to control values during the refeeding period. Plasma IGF-I-like immunoreactivity showed a similar trend. To our knowledge, this is the first report that indicates a coordinated regulation of GH-binding and plasma somatomedin-like activity in a typical marine fish.     

Lysine and Leucine Deficiencies Affect Myocytes Development and IGF Signaling in Gilthead Sea Bream (Sparus aurata)

Optimizing aquaculture production requires better knowledge of growth regulation and improvement in diet formulation. A great effort has been made to replace fish meal for plant protein sources in aquafeeds, making necessary the supplementation of such diets with crystalline amino acids (AA) to cover the nutritional requirements of each species. Lysine and Leucine are limiting essential AA in fish, and it has been demonstrated that supplementation with them improves growth in different species. However, the specific effects of AA deficiencies in myogenesis are completely unknown and have only been studied at the level of hepatic metabolism. It is well-known that the TOR pathway integrates the nutritional and hormonal signals to regulate protein synthesis and cell proliferation, to finally control muscle growth, a process also coordinated by the expression of myogenic regulatory factors (MRFs). This study aimed to provide new information on the impact of Lysine and Leucine deficiencies in gilthead sea bream cultured myocytes examining their development and the response of insulin-like growth factors (IGFs), MRFs, as well as key molecules involved in muscle growth regulation like TOR. Leucine deficiency did not cause significant differences in most of the molecules analyzed, whereas Lysine deficiency appeared crucial in IGFs regulation, decreasing significantly IGF-I, IGF-II and IGF-IRb mRNA levels. This treatment also down-regulated the gene expression of different MRFs, including Myf5, Myogenin and MyoD2. These changes were also corroborated by a significant decrease in proliferation and differentiation markers in the Lysine-deficient treatment. Moreover, both Lysine and Leucine limitation induced a significant down-regulation in FOXO3 gene expression, which deserves further investigation. We believe that these results will be relevant for the production of a species as appreciated for human consumption as it is gilthead sea bream and demonstrates the importance of an adequate level of Lysine in fishmeal diet formulation for optimum growth.