Molecular characterization and expression pattern of insulin-like growth factor binding protein-3 (IGFBP-3) in common carp, Cyprinus carpio

A full-length cDNA encoding the insulin-like growth factor binding protein-3 (IGFBP-3) was cloned from the liver of common carp (Cyprinus carpio) by RT-PCR. The IGFBP-3 cDNA sequence is 1,680 bp long and has an open reading frame of 882 bp encoding a predicted polypeptide of 293 amino acid residues. The deduced amino acid sequence contains a putative signal peptide of 25 amino acid residues resulting in a mature protein of 268 amino acids. A single band of approximate 1.9 kb was found in liver by Northern blot analysis. IGFBP-3 mRNA was observed in all regions of brain with high levels. In peripheral tissues, high levels of IGFBP-3 mRNA were found in retina, red muscle, liver, heart, posterior intestine, spleen, and testis. Relatively lower levels were found in white muscle, kidney, thymus gland, and ovary, while in head kidney, blood, skin, gill, middle intestine, and anterior intestine, the IGFBP-3 mRNA levels were much lower. IGFBP-3 mRNA was first detected in the blastula stage with significantly high level. The level sharply decreased in gastrula stage, and it became to increase in the following stages. During the reproductive cycle, the abundance of IGFBP-3 mRNA significantly decreased between the recrudescing stage and the matured stage in ovary, although in testis, IGFBP-3 mRNA expression level did not exhibit a significant change. The mRNA expression profiles in the present study imply that the IGFBP-3 may play important physiological functions in common carp development and reproduction.     

牙鲆胰岛素样生长因子结合蛋白IGFBP-1 cDNA全长的克隆及表达分析

利用RACE-PCR技术,从牙鲆(Paralichthys olivaceus)的肝脏组织总RNA中克隆得到胰岛素样生长因子结合蛋白-1(insulin-like growth factor binding protein 1, IGFBP-1)基因的全长cDNA序列,该cDNA全长为1070 bp,开放阅读框为729 bp,编码242个氨基酸。通过系统进化树分析,牙鲆IGFBP-1与鱼类IGFBP-1基因聚为一支;通过同源性比对,牙鲆IGFBP-1基因的核苷酸序列与大菱鲆同源性最高,为95%,而其推导的氨基酸序列与其它鱼类如大菱鲆、五条鰤、黄金鲈、红点鲑、鲤鱼和斑马鱼的同源性分别为89%、89%、84%、79%、67%和67%。半定量RT-PCR分析表明,牙鲆IGFBP-1基因存在母源转录本,合子基因在孵化前的胚胎阶段及早期仔鱼中仅有较低水平的表达,在后期仔鱼中表达逐渐增高;牙鲆IGFBP-1基因在肝脏中表达量最高,在胃、脾、肠、性腺、肾、鳃、脑、心脏和肌肉中也有不同程度的表达。     

Insulin-like growth factor binding protein-2 (IGFBP-2) in Japanese flounder, Paralichthys olivaceus: molecular cloning, expression patterns and hormonal regulation during metamorphosis

In this study, we cloned and characterized cDNA sequences of two insulin-like growth factor binding protein-2 (IGFBP-2a and IGFBP-2b) from Japanese flounder, Paralichthys olivaceus. The full-length cDNA of IGFBP-2a is 1,046 bp long and consists an open frame (ORF) of 876 bp, a 5′-untranslated region (UTR) of 125 bp and a 3′-UTR of 45 bp. IGFBP-2b is 1,067 bp, including a 5′-UTR of 53 bp, a 3′-UTR of 198 bp and an ORF of 816 bp. Real-time quantitative PCR results revealed that IGFBP-2a -2b mRNA was expressed in all detected tissues. Interestingly, the levels of IGFBP-2a mRNA in all detected tissues were higher in female than male, but IGFBP-2b was precisely the opposite. At different embryonic stages, the levels of IGFBP-2a mRNA were typically higher than IGFBP-2b. After hatching, IGFBP-2a mRNA was gradually decreased to a relatively lower level. However, the expression of IGFBP-2b mRNA was increased after hatching, including 3, 7, 10, 14, 17, 20 and 23 days post-hatching (dph), and it presents a higher level until 29 (metamorphic climax), 36 (post-climax) and 41 dph (the end of metamorphosis). In levothyroxine sodium salt (T4, the main form of thyroid hormone in animals)-treated and thiourea (TU)-treated larvae, the expressions of IGFBP-2a had not visibly changed, except in T4-treated 17 dph larvae. The expressions of IGFBP-2b mRNA were distinctly increased from 17 to 23 dph, but suddenly dropped to a lower level in and after 29 dph. However, the levels of IGFBP-2b mRNA during metamorphosis were greatly down-regulated after TU treatment. These results provided basic information for further studies on the role of IGF system in flatfish development and metamorphosis.     

Dopamine functions as a growth hormone-releasing factor in the goldfish,Carassius auratus

In vivo and in vitro approaches have been used to examine the role of dopamine (DA) as a growth hormone (GH)-releasing factor in the goldfish. DA stimulated GH release from perifused pituitary fragments of goldfish in a dose-dependent manner. The GH-releasing effect of DA was seasonal, being the highest in sexually regressed fish, intermediate in recrudescent fish, and the lowest in sexually mature (prespawning) fish. The GH response to DA was blocked by the D1 antagonist (+)SCH23390, confirming the involvement of D1 receptors in DA-stimulated GH release. In studies using static incubation of pituitary cells, somatostatin, a known physiological GH-release inhibitor in the goldfish, abolished the GH response to DA. Intraperitoneal injection of apomorphine, a non-selective DA agonist, also increased the plasma GH levels and enhanced the linear body growth of goldfish. These results strongly suggest that DA, by acting through DA D1 receptors, functions as a GH-releasing factor in the goldfish.     

鲫血清转铁蛋白cDNA片段的克隆与序列分析

取体重约300g鲜活鲫(Carassius auratus)的肝组织进行总RNA提取。从GenBank数据库查询已发表的8种鱼转铁蛋白cDNA或基因序列,根据铁离子结合转运功能位点,设计并合成了2对引物P1、P4以及P2、P3,克隆出鲫血清转铁蛋白cDNA中的从大约550bp至1450bp的核心片段,长度为0．9kb。同时,比较了几种鱼血清转铁蛋白cDNA序列的同源性,并推导出鲫血清转铁蛋白cDNA核心片段的氨基酸序列,并分析了该氨基酸序列的结构和特性。     

鲫血清转铁蛋白cDNA克隆及系统发育进化序列分析

鱼类血清转铁蛋白是鱼类血清中一种非血红素结合铁的β—球蛋白。从GenBank数据库查询发表的鱼类转铁蛋白cDNA或基因序列,根据铁离子结合及转运功能位点,设计并合成了两对引物P1、P4以及P2、P3,克隆出鲫血清转铁蛋白cDNA中的核心片段,长度为866bp。再根据克隆出的核心片段分别设计上游及下游两对引物P5、P6以及P7、P8,随后用RACE方法分别克隆出鲫血清转铁蛋白cDNA的5’端(787bp)和3’端(1081bp)以及全长cDNA,最后在计算机上排列出鲫血清转铁蛋白全长cDNA,长度为2444bp。比较了14种鱼血清转铁蛋白cDNA序列的同源性,其同源性在30％～80％之间,结果显示鲤科鱼类(鲫、银鲫、鲤及斑马鱼等)具有很近的亲缘关系;同时进行了系统发育进化分析,证实了鱼类血清转铁蛋白进化的保守性和氨基酸序列的高度同源性。     

Hypothalamic peptides influencing growth hormone secretion in the goldfish,Carassius auratus

In vivo andin vitro techniques were used to examine the influence of various vertebrate peptides on growth hormone (GH) secretion in the goldfish. Tetradecapeptide somatostatin (SRIF-14) was found to inhibit GH secretionin vitro from perifused pituitary fragments, whereas similar concentrations of a salmonid SRIF peptide (sSRIF-25) did not affect GH secretion from the goldfish pituitary fragments. This indicates that SRIF receptors on the goldfish pituitary are very specific for SRIF-14-like peptides. Salmon gonadotropin (GTH)-releasing hormone (sGnRH) was found to elevate serum GH levels in male goldfish. The dopamine antagonist pimozide alone or injected in combination with sGnRH did not influence serum GH levels, although injection of pimozide alone significantly elevated serum GTH levels, in addition to potentiating the effects of sGnRH on GTH secretion. sGnRH stimulated GH secretion from goldfish pituitary fragmentsin vitro, indicating that sGnRH acts directly at the level of the pituitary to stimulate GH secretion in the goldfish. These results suggest that GnRH may also function as a GH-releasing factor in the goldfish, although the release-inhibitory factors for GH and GTH secretion do appear to be separate and distinct. Two human GH-releasing hormone (hGHRH) peptides were found to be ineffective in altering GH secretionin vitro from the perifused pituitary fragments. Consequently, a role for a mammalian GHRH-like peptide in the hypothalamic regulation of GH secretion in the goldfish remains questionable.     

Characterization of growth hormone binding sites in the goldfish,Carassius auratus: effects of hypophysectomy and hormone injection

A recombinant carp growth hormone (rcGH) was used to develop for a GH radioreceptor binding assay in the goldfish (Carassius auratus). Specific binding of¹²⁵I-rcGH to goldfish liver membranes was a pH, time, temperature, and membrane protein dependent process. Scatchard and LIGAND analysis indicated a single class of high affinity and low capacity binding site, with an association constant (Ka) of 1.9×10¹⁰ M^–1 and a maximum binding capacity (B_max) of 9 fmol mg^–1 protein. Liver tissue displayed the highest¹²⁵I-rcGH binding of all the tissues examined. Displacement of¹²⁵I-rcGH with various unlabeled teleost and mammalian GHs and prolactins revealed that the goldfish hepatic binding site was highly specific for teleost GH. Intraperitoneal administration of 0.1, 1.0, and 10 g rcGH g^–1 body weight to hypophysectomized goldfish resulted in a 27, 52, and 68% decrease in total binding sites, respectively. Injection of a high dose of rat prolactin (rPRL) (5 g rPRL g^–1 body weight) also resulted in a 32% decrease in total binding sites. These results suggest that endogenous GH may have a role in the regulation of its own receptors in the goldfish.     

银鲫crh基因的克隆、组织表达谱及其对摄食的影响

为研究crh (Corticotropin-releasing hormone)基因对鱼类摄食的调控作用,首次克隆了银鲫(Carassius auratus gibelio) crh基因c DNA全长序列。运用实时荧光定量PCR(RT-qPCR)技术检测crh基因m RNA在不同组织的表达及餐前、餐后和禁食对其表达的影响。结果显示,银鲫crh基因的c DNA序列全长为920 bp,其中,5’-UTR为53 bp,3’-UTR为378 bp,开放阅读框(Open reading frame,ORF)为489 bp。推导银鲫crh基因的ORF区编码蛋白由162个氨基酸组成,其中,含有11个氨基酸的保守区,24个氨基酸的信号肽,41个氨基酸的成熟肽。氨基酸序列的多重比较分析显示,银鲫crh基因与金鱼(Carassiusauratus)的同源性为99%,与鲤(Cyprinuscarpio)的同源性为96%。荧光定量PCR表达分析显示,银鲫crh基因在下丘脑中的表达量最高,其次是端脑和心脏。crh基因的表达量在餐前与餐后没有显著变化(P0.05),在禁食第5天、第7天出现极显著降低(P0.01),而在复投喂后第9天、第11天出现极显著升高(P0.01)。以上结果显示,crh基因在银鲫摄食调控方面可能扮演着重要角色。     

Intracellular mechanisms mediating gonadotropin and growth hormone release in the goldfish,Carassius auratus

Evidence for the involvement of Ca²⁺, protein kinase C, cAMP, and arachidonic acid metabolism in mediating gonadotropin (GTH) and growth hormone (GH) release in the goldfish is reviewed. Models for the signal transduction pathways mediating GTH-releasing hormone (GnRH) and dopamine actions on GTH and GH secretion are postulated. A novel hypothesis that two GnRHs which bind to the same receptor type activate different transduction cascade in two different cell types (GTH vs. GH) as well as within the same cell type (GTH) is presented.

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Résumé Cette revue présente les données expérimentales démontrant l'implication de Ca⁺⁺, de la protéine kinase C et du métabolismes de l'acide arachidonique dans les mécanismes régulant la sécrétion des hormones gonadotrope (GTH) et de croissance (GH). Des modèles de signaux de transduction de l'action de la gonadolibérine (GnRH) et de la dopamine sur la sécrétion de GTH et de GH sont proposés. Les deux GnRHs existant chez le poisson rouge pourraient se lier au même type de récepteur et activer différentes voies de transduction dans deux différents types cellulaires (GTH vs. GH) ou dans un seul type (GTH).

     

哲罗鲑胰岛素样生长因子－I（IGF－I）cDNA 分子克隆、序列分析及组织表达

采用逆转录聚合酶链式反应（RT－PCR）方法,从哲罗鲑（Hucho taimen）肝脏的总 RNA 中扩增出胰岛素样生长因子－I（IGF－I）的 cDNA 开放阅读框（Open reading frame, ORF）序列,运用软件对其进行生物信息学分析,并利用荧光实时定量 PCR 技术检测了哲罗鲑成鱼不同组织中 IGF－I mRNA 的表达情况。结果显示, IGF－I 基因的 cD－NA 开放阅读框为573 bp,编码190个氨基酸,蛋白质等电点为9．21,氨基酸结构由信号肽、 B、 C、 A、 D 结构域及 E 肽组成;氨基酸序列与其他鲑科鱼类具有较高的同源性,其中与北极红点鲑的 IGF－I 同源性最高（99．2％）;组织表达分析显示,哲罗鲑 IGF－I mRNA 在肝脏中表达量最高,在鳃、前肠中次之,在脑、头肾、脾、心、胃和肌肉等组织中的表达量较低。     

Herpesviral haematopoietic necrosis of goldfish, Carassius auratus (L.)

Abstract. In the spring of 1992 and 1993, an epizootic with severe mortality occurred among cultured goldfish. Carassius auratus (L.), in Aichi and Nara Prefectures in Japan. A herpes-virus was isolated from moribund goldfish that induced cytopathic effects in FHM and EPC cells. The isolate was sensitive to IUdR (5-iodo-2-deoxyuridine), acid (pH 3) and ether. Transmission studies confirmed the pathogenicity of the viral isolate for goldfish while it was not pathogenic for fancy carp, Cyprinus carpio L. Diseased fish had no visible external signs except for listlessness, but internally there was softening and discolouration of the spleen and kidney, and necrotic foci in the haematopoietic tissue, splenic pulp, pancreas, and lamina propria and submucosa of the intestine. Electron microscopy revealed enveloped visions ranging from 170 to 220nm in diameter with hexagonal nucleocapsids (115–117nm edge-to-edge diameter). The disease has been designated as herpesviral haematopoietic necrosis (HVHN), and to date, is the only herpesvirus infection to be described in goldfish.     

First detection of Cyprinid Herpesvirus 2 (CyHV‐2) in goldfish (Carassius auratus) in France

Massive mortalities of Carassius auratus (L.) occurred in a farm in France during summer 2014. Fish presented anorexia, loss of scales and large amounts of mucus on the gills. Necrosis of the distal tip of the filament and the lamellae, combined with fusion of the lamellae, was observed, as well as necrosis in the hematopoietic organs and in the digestive tract. The histological examination led to hypothesize the implication of a virus in the mortality. The presence of cyprinid herpesvirus 2 (CyHV‐2) in dead fish was demonstrated by amplification and sequencing of portions of the DNA polymerase and helicase genes, both sequences exhibiting 100% identity with CyHV‐2 from Japan. In an attempt to find genetic markers of variation, two regions containing tandem repeats in the Japanese genome were amplified from a virus‐positive sample from the present outbreak. A first region (mB) was fully identical to the Japanese isolate. However, the second region (mA) exhibited a range of deletions and substitutions compared to CyHV‐2 from Japan. This is the first report of CyHV‐2 in France in association with mortality of goldfish and the first identification of a molecular marker for its tracing.