Rat Ghrelin Stimulates GH Release and GH mRNA Expressionin the Pituitary of Orange-spotted Grouper, <Emphasis Type="BoldItalic">Epinephelus Coioides</Emphasis>

Ghrelin was recently demonstrated as an endogenous ligand of the growth hormone (GH) secretagogue receptor (GHS-R), which could promote the release of GH in mammal significantly. The present study conducted to determine whether ghrelin stimulate the release and synthesis of GH in orange-spotted grouper (Epinephelus coioides). Rat ghrelin was incubated with the pituitary fragments of grouper in static culture system. The culture medium was collected at 1, 6, 12, 18 and 24 h after incubation to detect the contents of GH by homologous radioimmunoassay. The level of GH mRNA in the pituitary fragments was measured by a sensitive chemiluminescent ribonuclease protection assay. The results showed that rat ghrelin not only stimulated the release of GH but also augmented the GH mRNA level in grouper. It suggested that the ghrelin-like peptide and the GHS-R involved in the regulation of GH synthesis and release in grouper. The present study would provide a better understanding of the regulatory mechanism of GH release in marine fish.     

Lack of growth hormone-releasing peptide-6 action on in vivo and in vitro growth hormone secretion in sexually immature grass carp (Ctenopharyngodon idellus)

Studies in mammals have shown that synthetic Met-enkephalin derivatives, called growth hormone-releasing peptides (GHRPs), stimulate growth hormone (GH) release. In the present study, GHRP-6 action on GH secretion was examined in vivo and in vitro in sexually immature grass carp. GHRP-6 injected intraperitoneally had no influences on serum GH levels in juvenile grass carp. Following intraperitonal injection of GHRP-6 and dopamine (DA) or cysteamine hydrochloride (CSH), alone and in combination into juvenile grass carp, DA and CSH were effective in elevating serum GH levels, but GHRP-6 was not effective in this respect; in addition, the synergistic action of GHRP-6 and DA or CSH on GH secretion was not seen. In this work, we had adapted and validated a perifusion system and a culture system for GH regulation studies. In a perifusion system, GHRP-6 (1000 to 0.1 nM), GHRP-6 (0.1 to 1000 nM), GHRP-6 (1 μM), and Hexarelin (an analog of GHRP, 1 μM) had no action on GH release from juvenile grass carp pituitary fragments or cells. Under static incubation conditions, GHRP-6 was inactive on GH release from juvenile grass carp pituitary fragments after 1 h and 6 h incubation, but human growth hormone-releasing hormone (hGHRH; 1 to 100 nM) as positive control could stimulate GH release in a dose-dependent manner. Furthermore, when GHRP-6 (100 nM) in combination static incubation with neuropeptides [e.g., hGHRH (100 nM), salmon gonadotropin-releasing hormone analogue (sGnRH-A) (100 nM), or D-Ala⁶,Pro⁹-NEt-luteinizing hormone-releasing hormone (D-Ala⁶,Pro⁹-NEt-LHRH, LHRH-A) (100nM)], GHRP-6 did not strengthen GH secretion actions of neuropeptides, and at the same time neuropeptides also did not modify the effects of GHRP-6 on GH secretion. The present results obtained using in vivo and in vitro techniques adapted for GH regulation studies show that GHRP-6 does not function as a GH-releasing factor in juvenile grass carp as it does in tilapia, amphibians, chickens, and mammals.     

Gene expression levels of growth hormone receptor and insulin-like growth factor-I in gonads of maturing coho salmon (Oncorhynchus kisutch)

The expression levels of growth hormone receptor (GHR) and insulin-like growth factor (IGF-I) in gonads of maturing coho salmon were measured by real-time PCR. The GHR and IGF-I mRNAs showed different gene expression pattern between male and female. These results suggest that a sexual dimorphism may exist in the GHR and IGF-I mRNA expressions of coho salmon during gonadal maturation     

Genetic and structural characterization of the growth hormone gene and protein from tench, Tinca tinca

The analysis of the tench growth hormone gene structure revealed a comparable organization of coding and non-coding regions than other from cyprinid species. Based on the performed mRNA and amino acid sequence alignments, gh tench is related to Asian than to European representatives of Cyprinidae family. Second aim of the work was to characterize and predict protein structure of the tench growth hormone. Tinca tinca GH share many common features with human GH molecule. The Tench GH protein binds to the growth hormone receptor (GHR) using two regions I and II that are situated at opposite sites of molecule. Binding site I is placed in the central part of T. tinca GH and H 189 amino acid in the middle region of the IV helix is crucial for GH–GHR interactions.     

Effect of dietary lipid level on growth performance and feed utilization of juvenile kelp grouper Epinephelus bruneus

A growth trial was conducted to evaluate the effect of dietary lipid level on juvenile kelp grouper Epinephelus bruneus. Juvenile kelp grouper were fed isonitrogenous diets (470 g/kg crude protein) with four levels of crude lipid at 60 g/kg (CL60), 130 g/kg (CL130), 210 g/kg (CL210), and 270 g/kg (CL270) for 56 days. The highest growth performance and feed utilization were found in the CL130 diet group. A high dietary lipid level (CL270 diet) significantly decreased growth performance and feed utilization. A significant difference in apparent digestibility was only observed in protein, which was highest in the CL130 diet groups. The highest retention for protein, energy, and lipid was found in the CL130 diet group. The dietary lipid levels significantly changed whole-body and liver compositions, the highest being the CL60 diet group for crude protein level and the CL210 diet group for crude lipid level. Based on a second-order polynomial regression analysis of crude lipid level against specific growth rate and protein efficiency ratio, the optimum dietary lipid level for kelp grouper was estimated to be 152 and 154 g/kg diet, respectively.     

Cichlasoma dimerus responds to refeeding with a partial compensatory growth associated with an increment of the feed conversion efficiency and a rapid recovery of GH/IGFs axis

Many fish species display compensatory growth (CG), a phenomenon by which fasted fish grow faster during refeeding. However, most studies use a group‐housed fish approach that could be problematic in social fish when interaction between individuals is not considered or eliminated. Additionally, the growth hormone (GH)/insulin‐like growth factors’ (IGF‐1 and IGF‐2) axis is implicated in postnatal growth in vertebrates, but its relevance in CG is not fully understood. Thus, the aim of this work was to determine whether CG occurs in a social fish, Cichlasoma dimerus, using an individually held fish approach and secondly, to evaluate the GH/IGFs expression profile during refeeding by 3 days and 3 weeks. C. dimerus showed partial CG. The feed conversion efficiency (FCE) was higher in three‐day‐refed fish, which presented higher GH plasma and mRNA levels than controls but shown no differences in liver and muscle GH receptors (GHR1 and GHR2) and IGFs mRNA levels. Surprisingly, three‐week‐refed fish exhibited GHR1 and IGF‐2 increments, but a reduction in GHR2 expression in muscle. These results show a strong association between GH levels, growth rate and FCE during refeeding, and a long‐lasting effect of refeeding on muscular expression of GHRs and IGF‐2.     

Molecular cloning and dimorphic expression of growth hormone (gh) in female and male spotted scat Scatophagus argus

Spotted scat Scatophagus argus exhibits a typical sexual growth dimorphism in which the females grow faster than the males. Growth hormone (GH) is best known as an anterior pituitary hormone fundamental in regulating growth. To clarify the roles in sexual growth dimorphism in the spotted scat, gh cDNA was isolated from the pituitary. A phylogenetic tree was constructed based on the GH amino acid sequences of spotted scat and other vertebrates, and the resulting topology clearly reflects the taxonomic relationship of the Perciformes species selected. Alignment of GH amino acid sequences displayed very high similarity between the spotted scat and the other Perciformes species. The qRT-PCR analysis demonstrated that females exhibited higher pituitary gh mRNA levels than males at 6, 18, and 30 months (P < 0.05), which was 1.84, 4.61 and 6.34 times greater, respectively. In addition, immature males (6 months) exhibited higher pituitary gh mRNA levels than mature males (18 and 30 months). These results imply that the sexual growth dimorphism may be ascribable to the gh levels in pituitary in spotted scat.     

Identification,characterization of selenoprotein W and its mRNA expression patterns in response to somatostatin 14, cysteamine hydrochloride, 17β-estradiol and a binary mixture of 17β-estradiol and cysteamine hydrochloride in topmouth culter (<Emphasis Type="Italic">Erythroculter ilishaeformis</Emphasis>)

In this study, a selenoprotein W cDNA was cloned from topmouth culter (Erythroculter ilishaeformis), and it was designated as EISelW. The EISelW open reading frame was composed of 261 base pairs (bp), encoding 86-amino-acid protein. The 5′ untranslated region (UTR) consisted of 104 bp, and the 3′-UTR was composed of 365 bp. A selenocysteine insertion sequence (SECIS) element was found in the 3′-UTR of EISelW mRNA. The SECIS element was classified as form II because of a small additional apical loop presented in SECIS element of EISelW mRNA. Bioinformatic approaches showed that the secondary structure of EISelW was a β1-α1-β2-β3-β4-α2 pattern from amino-terminal to carboxy-terminal. Real-time PCR analysis of EISelW mRNAs expression in 17 tissues showed that the EISelW mRNA was predominantly expressed in liver, ovary, pituitary, various regions of the brain, spinal cord and head kidney. Study of intraperitoneal injection showed that the levels of EISelW mRNA in brain, liver, ovary and spleen were regulated by somatostatin 14 (SS14), 17β-estradiol (E2), cysteamine hydrochloride (CSH) and a binary mixture of E2 and CSH, dependent on the dosage. These results suggest that E2, SS14 and CSH status may affect tissues of selenium metabolism by regulating the expression of SelW mRNA, as SelW plays a central role in selenium metabolism.     

Stimulation of insulin-like growth factor-I production by recombinant bovine growth hormone in Mozambique tilapia, Oreochromis mossambicus

We have previously reported growth-promoting effects of recombinant bovine growth hormone (rbGH) in Mozambique tilapia, Oreochromis mossambicus, after 4 weekly injections or a single injection of slow-releasing formulation (Posilac^®) (Leedom et al. 2002). In order to obtain further understanding of the role of the growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis in growth in the tilapia, the effects of rbGH on plasma and mRNA levels of IGF-I were examined. Plasma IGF-I levels were significantly increased after rbGH and Posilac^® injections, and a significant correlation was observed between plasma IGF-I levels, body length and mass in both treatments. IGF-I mRNA levels in the liver and in the skeletal muscle were also significantly increased after rbGH and Posilac^® injections, indicating that IGF-I gene expression in these tissues is under control of circulating GH. IGF-I mRNA levels in the gill were not affected by treatment. Liver IGF-I mRNA levels were significantly correlated with body length and with body mass after rbGH and Posilac^® injections. These results indicate that the growth-promoting effect of rbGH in this species is mediated to a significant extent via its stimulation of hepatic production of IGF-I and the resulting increase in plasma IGF-I, and also possibly through locally produced IGF-I in the skeletal muscle, acting in a paracrine or autocrine fashion.     

Effects of largely different feeding intensities on serum insulin‐like growth factor‐1 concentrations,quantified by enzyme immunoassay,leptin and growth hormone receptor 1 mRNA in rainbow trout (Oncorhynchus mykiss)

Insulin‐like growth factor‐1 (IGF‐1), somatolactin and leptin are involved in growth regulation and energy metabolism in fish. We herein focused on serum IGF‐1 concentration analysed by enzyme‐linked immunosorbent assay in restrictively fed rainbow trout (Oncorhynchus mykiss). The animals were fed a high‐fat/low‐protein diet at daily feed increases (DFI) ranging from 0.5% to 2% of initial body weight (IBW), starting either at 62 or 176 g IBW. In selected groups, growth hormone receptor 1 (GHR1) and leptin mRNA were quantified in liver, and GHR1 mRNA also in visceral adipose tissue. Serum IGF‐1 concentrations in both IBW groups were highest at 2% and 1% DFI and were nonlinearly decreasing with reduced DFI. The low‐IBW groups had mostly lower IGF‐1 concentrations than the high‐IBW groups. Leptin and GHR1 mRNA decreased with feeding intensity in liver, but GHR1 mRNA increased in adipose tissue. IGF‐1 is related to growth and may help to mitigate oxidative stress in consequence of lipid mobilization during restrictive feeding. IGF‐1 secretion associated with stress response in addition to its function in growth and energy metabolism seemed to reach a point of inflection at DFI 1%. Leptin and GHR1 might be linked to lipid metabolism and free fatty acid partitioning towards liver.     

Expression of insulin-like growth factor genes in olive flounder,Paralichthys olivaceus,fed a diet with partial replacement of dietary fish meal

This study analyzed the expression of growth-related genes of olive flounder, Paralichthys olivaceus, fed a low-fish meal (FM) diet to investigate the replacement of the FM diet in the field. The expression of growth hormone (GH)/insulin-like growth factor (IGF) axis genes in the brain/pituitary/liver and plasma hormone concentrations were measured. A basal experimental diet was formulated using 63% FM and 12% soybean meal as the primary protein sources, and then two other diets were made by replacing 20% and 30% of the FM with soy protein concentrate, tankage meal, and poultry by-products meal. Each diet was fed to duplicate groups of juvenile flounder (150 ± 3.0 g) twice a day. After 20 weeks, the plasma growth-related hormone concentrations were similar between the control and FM20 groups. Moreover, GH/IGF axis gene mRNA expression in the brain/pituitary/liver was similar between the control and FM20 groups. Immunoblotting of muscle and liver showed the same pattern. This study showed the utility of replacing 20% of the FM diet and supports the possibility of field application.     

Disrupting actions of bisphenol A and malachite green on growth hormone receptor gene expression and signal transduction in seabream

Environmental estrogen could mimic natural estrogens thereby disrupting the endocrine systems of human and animals. The actions of such endocrine disruptors have been studied mainly on reproduction and development. However, estrogen could also affect the somatotropic axis via multiple targets such as growth hormone (GH). In the present study, two endocrine disruptors were chosen to investigate their effects on the expression level and signal transduction of growth hormone receptor (GHR) in fish. Using real-time PCR, it was found that exposure to both the estrogenic (bisphenol A) and anti-estrogenic (malachite green) compounds could attenuate the expression levels of GHR1 and GHR2 in black seabream (Acanthopagrus schlegeli) hepatocytes. The expression level of IGF-I, the downstream effector of GHR activation in the liver, was decreased by bisphenol A but not by malachite green. Luciferase reporter assay of the β-casein promoter was used to monitor GHR signaling in transfected cells. In the fish liver cell line Hepa-T1, both GHR1 and GHR2 signaling were attenuated by bisphenol A and malachite green. This attenuation could only occur in the presence of estrogen receptor, indicating that these agents probably produce their actions via the estrogen receptor. Results of the present study demonstrated that estrogenic or anti-estrogenic compounds could down-regulate the somatotropic axis in fish by affecting both the gene expression and signaling of GHR. In view of the increasing prevalence of these compounds in the environment, the impact on fish growth and development both in the wild and in aquaculture would be considerable.     

Transitioning to a Pelleted Diet and Compensatory Growth of Pacific Goliath Grouper Epinephelus quinquefasciatus in Captivity

The objective of this study was to adapt juvenile Pacific goliath grouper to a diet of pelleted feed. Groupers were captured in nature, individually marked, and randomly assigned to two groups. One group was fed pieces of raw fish and the second group a pelleted feed. The experiment lasted 12 weeks, and adaptation to the pelleted feed was done gradually. Fish growth, weight gain, and specific growth rate (SGR) were estimated. The Student’s t-test, at the 0.01 level of significance, was used to compare SGR means. All the fish survived. The rate of growth for grouper fed pieces of raw fish was 1.6 g per day. Although the groupers fed the pellets lost weight at the beginning of the experiment, at the end their SGR was 5.5 grams per day. The average weight gain for juvenile grouper fed pellets was 14.6 ± 4.1 g per day.     

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.