Responses to ROS inducer agents in zebrafish cell line: differences between copper and UV-B radiation

Fish are commonly exposed to environmental pollutants, which in turns could induce an oxidative stress. So, it is important to understand the effects and the responses elicited by these toxicants in fish species, being fish cell lines important tools for this purpose. Thus, the aim of the present study was to compare the effects of copper and UV-B radiation exposure on zebrafish hepatocytes (ZFL lineage) in terms of reactive oxygen species (ROS) levels, sulfhydril groups content and mRNA levels of important genes related to cellular response to toxic agents. Exposure of ZFL cells to UV-B radiation (23.3 mJ/cm²) significantly increased levels of intracellular ROS and mRNA of both superoxide dismutase isoforms (sod1 and sod2), three glutathione S-transferase isoforms (gstα, gstµ and gstπ) and a heat shock protein (hsp70). However, no changes in nonprotein sulfhydryl groups (NP-SH) content, as well as in the mRNA levels of genes related to glutathione (GSH) synthesis and recycling, were observed. Contrary to this, copper exposure (20 mg/L) diminished NP-SH content and increased the levels of mRNA of genes related to GSH synthesis (gclc and gs). Moreover, copper exposure increases the mRNA levels of some genes related to antioxidant defenses (gpx and gstπ), biotransformation reactions (cyp1a1) and protein repair (hsp70). In conclusion, these results demonstrated that both toxicants could increase ROS levels in ZFL cell line, but the responses are different, which could be related to activation of different signaling pathways.     

Molecular cloning,characterization, and expression analysis of luteinizing hormone receptor gene in turbot (Scophthalmus maximus)

The luteinizing hormone receptor (LHR) plays a crucial role in female reproduction. In the present study, full-length sequence coding for the LHR was obtained from female turbot (Scophthalmus maximus) by homology cloning and a strategy based on rapid amplification of cDNA end-polymerase chain reaction. The full-length LHR cDNA was 3,184 bp long and contained a 2,058-bp open reading frame which encoded a protein of 685 amino acids. Multiple sequence alignments of the turbot LHR manifested high homologies with the corresponding sequences of available teleosts and representative vertebrates, and significant homology with that of Hippoglossus hippoglossus. In addition, the turbot LHR showed typical characteristics of glycoprotein receptors, including a long N-terminal extracellular domain, seven transmembrane domains, and a short C-terminal intracellular domain. LHR mRNA was abundant in the ovary, but was deficient in extra-ovarian tissues. Furthermore, LHR mRNA gradually developed from previtellogenesis to migratory nucleus stage, with the highest values observed in migratory nucleus stage during reproductive cycle. However, LHR mRNA sharply decreased in atresia stage. These results suggested that LHR is a typical G protein-coupled receptor that is involved in the promotion of turbot ovarian development and may be related to the final maturation and ovulation of oocyte. These findings contribute to the understanding of the potential roles of LHR in controlling the fish reproductive cycle.     

Cloning,molecular characterization and expression analysis of insulin-like growth factor binding protein-2 (IGFBP-2) cDNA in goldfish,Carassius auratus

In the present study, a full-length cDNA encoding the insulin-like growth factor binding protein-2 (IGFBP-2) was cloned from the liver of goldfish (Carassius auratus) by rapid amplification of cDNA ends technique. The goldfish IGFBP-2 cDNA sequence was 1,513 bp long and had an open reading frame of 825 bp encoding a predicted polypeptide of 274 amino acid residues. Semi-quantitative RT-PCR results revealed that goldfish IGFBP-2 mRNA was expressed in all detected tissues. In liver, central nervous system and pituitary gland, goldfish IGFBP-2 expressed at high levels, followed by anterior intestine, middle intestine and kidney. In posterior intestine, ovary, skin, fat, spleen, muscle and gill, the goldfish IGFBP-2 expression levels were very low. Fasting and refeeding experiment showed that the mRNA expression of goldfish IGFBP-2 was up-regulated significantly in liver compared to the fed group and restored rapidly to normal level after refed. However, the mRNA expressions of IGFBP-2 in hypothalamus and pituitary of goldfish were insensitive to fasting. Furthermore, the mRNA expressions of IGFBP-2 in hypothalamus, pituitary and liver were varied in periprandial changes and significantly down-regulated at 2 and 4 h after meal. These results imply that the IGFBP-2 mRNA expression may be associated with anabolic and catabolic metabolism and regulated by metabolic factors in goldfish.     

Schizothorax prenanti corticotropin-releasing hormone (CRH): molecular cloning,tissue expression,and the function of feeding regulation

Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioral, and immune responses to stress. For a better understanding of the structure and function of the CRH gene and to study its effect on feeding regulation in cyprinid fish, the cDNA of the CRH gene from the brain of Schizothorax prenanti was cloned and sequenced. The full-length CRH cDNA consisted of 1,046 bp with an open reading frame of 489 bp encoding a protein of 162 amino acids. Real-time quantitative PCR analyses revealed that CRH was widely expressed in central and peripheral tissues. In particular, high expression level of CRH was detected in brain. Furthermore, CRH mRNA expression was examined in different brain regions, especially high in hypothalamus. In addition, there was no significant change in CRH mRNA expression in fed group compared with the fasted group in the S. prenanti hypothalamus during short-term fasting. However, CRH gene expression presented significant decrease in the hypothalamus in fasted group compared with the fed group (P < 0.05) on day 7; thereafter, re-feeding could lead to a significant increase in CRH mRNA expression in fasted group on day 9. The results suggest that the CRH may play a critical role in feeding regulation in S. prenanti.     

Molecular cloning and biochemical characterization of medaka (Oryzias latipes) lysosomal neu4 sialidase

Glycoconjugates are known to be involved in many physiological events in vertebrates. Sialidase is one of the glycosidases, which removes sialic acid from glycoconjugates. In mammals, the properties and physiological functions of sialidases have been investigated, while there is little understanding of fish sialidase. Here, to investigate the significance of fish neu4 sialidase, neu4 gene was cloned from medaka brain mRNA and identified. Sialidase-specific motifs (GPG, YRVP and Asp-Box) were well conserved in the medaka neu4 polypeptide. Optimal pH of medaka neu4 sialidase was 4.6, but its activity was sustained even at neutral and weak alkaline pH. The neu4 considerably cleaved sialic acid from 4-methylumbelliferyl-N-acetyl-α-d-neuraminic acid and sialyllactose, but not from ganglioside and fetuin, which are good substrates for human NEU4. neu4 activity was mostly detected in mitochondria/lysosome fraction after biochemical fractionation, and indirect immunofluorescence assays revealed neu4 localization in lysosome in neu4 overexpressed cells. Next, developmental change in medaka neu4 and other sialidase mRNA levels were estimated by real-time PCR. Each sialidases showed different expression patterns in embryonic development: neu4 was up-regulated at late developmental stage in embryo, and neu3a mRNA level was quite high in 0.5 dpf. On the other hand, neu3b expression was drastically increased after hatching, suggesting that each sialidase may play a different role in embryonic development.     

Dynamic expression pattern of corticotropin-releasing hormone,urotensin I and II genes under acute salinity and temperature challenge during early development of zebrafish

Corticotropin-releasing hormone (CRH), urotensin I (UI) and urotensin II (UII) are found throughout vertebrate species from fish to human. To further understand the role of crh, uI and uII in teleosts during development, we investigated the expression pattern of crh, uI, uIIα and uIIβ genes, and their response to acute salinity and temperature challenge during early development of zebrafish, Danio rerio. The results reveal that crh, uI, uIIα and uIIβ mRNA are detected from 0hpf, and the expression levels increase to a maximum at 6 days post fertilization (dpf), with the exception of uIIα that peak at 5dpf. Exposure of zebrafish embryos and larvae to acute osmotic (30ppt) stress for 15 min failed to modify expression levels of crh, uI, uIIα and uIIβ mRNA from levels in control fish except at 6dpf when uIIα and uIIβ were significantly (P < 0.05) modified. Exposure of embryos and larvae to a cold (18 °C) or hot stress (38 °C) generally down-regulated mRNA levels of crh, uI, uIIα and uIIβ apart from at 3dpf. The results indicate that the contribution of crh, uI, uIIα and uIIβ genes to the stress response in zebrafish may be stressor-specific during early development. Overall, the results from this study provide a basis for further research into the developmental and stressor-specific function of crh, uI, uIIα and uIIβ in zebrafish.     

Profiles of thyrotropin,thyroid hormones,follicular cells and type I deiodinase gene expression during ontogenetic development of tilapia larvae and juveniles

The aims of the present study are to determine whether triiodothyronine (T3) and/or thyroxine (T4) in tilapia larvae is gifted through the mother, and to investigate the change profiles of thyrotropin (TSH), thyroid follicular cells and type I deiodinase (D1) gene expression following larval development. T3 and T4 contents were measured using radioimmunoassay, thyrotropin was observed using immunocytochemistry, and the D1 gene was cloned and measured using real-time PCR. Results indicated that the β-TSH-immunoreactive cells (thyrotropin ICC) signals were detected at 9 dph (i.e., 9 days of post-hatching). Thyroid follicular cells were observed first at 3 dph, while the T3 contents of the whole body gradually decreased before 11 dph. T4 contents were detected until 13 dph, with higher secretion during 19–21 dph. In addition, the T3 synthesis was not inhibited by thiourea (TU) before 13 dph, but the TU response in the larvae appeared after 13 dph. Type I deiodinase (D1: GenBank accession number KC591724) was found to contain 2444 bases and encoded 248 amino acids. The D1 mRNA expression began to increase at 13 dph, with a higher expression during 15–19 dph. These results suggested that the T3 contents were maternally derived before 13 dph. Both thyroid hormonal changes and some parameters related to thyroid hormone synthesis in ontogenetic tilapia are discussed.     

The bioactivity of gonadotropin releasing hormones and its regulation in the gilthead seabream,Sparus aurata: in vivo andin vitro studies

Thein vivo andin vitro potency of native and modified forms of gonadotropin releasing hormone (GnRH) to release gonadotropin (GtH) was studied inSparus aurata and correlated with their relative susceptibility to degradation by cytosolic-bound enzymes of the pituitary, kidney, and liver. Salmon (s) GnRH and luteinizing hormone-releasing hormone (LHRH) are equipotent whereas analogs of these peptides ((D-Arg⁶-Pro⁹NET)-sGnRH, (D-Ala⁶-Pro⁹NET)-LHRH, (D-Trp⁶)-LHRH) are superactive in inducingin vivo GtH release (at 10 μg/kg body weight). In anin vitro superfusion system of pituitary fragments all analogs are equipotent to the native peptides (at 10⁻¹⁰ to 2.5 × 10⁻⁷M). sGnRH and LHRH are rapidly degraded by cytosolic peptidases of the pituitary, liver, and kidney. The preferred site of cleavage is the Tyr⁵-Gly⁶ bond. Substitution of the position 6 glycine by D-amino acids renders the 5–6 bond resistant to degradation and shifts the main site of cleavage to the Pro⁹-Gly¹⁰NH₂ bond. Substitution at position 6 (as above) and at position 10 with Pro⁹NET results in analogs that are resistant to degradation. We propose that enzymatic cleavage terminates GnRH bioactivityin vivo and thus increased resistance to degradation is a major determinant of GnRH analog superactivity.     

Role of NPY receptor 8 in regulating of food intake in Chinese perch (Siniperca chuatsi)

Neuropeptide Y receptor Y8 (NPY8R) is a fish-specific receptor, whose role in feeding regulation remains poorly understood. In the present study, two subtypes of NPY8R (NPY8Ra and NPY8Rb) were identified in Chinese perch, and their amino acid sequences were highly conserved in fish. The predicted tertiary protein structures of human NPY1R, Chinese perch NPY8Ra, and NPY8Rb tended to be conserved as a whole. However, the predicted tertiary protein structures of sc-NPY8Ra and sc-NPY8Rb were different at several sites. Chinese perch NPY8Ra and NPY8Rb were mainly expressed in brain. Intracerebroventricular administration of siRNA-NPY8Rb significantly decreased the mRNA expression levels of NPY8Rb and POMC (appetite-suppressive gene) and significantly increased the food intake of Chinese perch at 2 h and 8 h after injection. Similar result has not been observed in the siRNA-NPY8Ra administration group. The above results suggested that NPY8Ra might not play a role in appetite regulation and NPY8Rb might be an effector in the negative feedback regulation of feeding in Chinese perch.

     

Cortisol treatment improves the development of hypoosmoregulatory mechanisms in the euryhaline rainbow trout,Salmo gairdneri

The effect of cortisol on osmoregulatory parameters was studied in rainbow trout, (Salmo gairdneri), kept in freshwater (FW) and/or transferred to seawater (SW). Repeated injections of 20 μg cortisol/g fish stimulated gill and gut Na⁺/K⁺-ATPase activity and reduced plasma Na⁺ and Cl⁻ levels after 2 weeks of treatment in FW-adapted fish. Cortisol doses of 0.05 and 1.0 μg/g were without effect. Repeated injections of 10 μg cortisol/g stimulated gill Na⁺/K⁺-ATPase activity and reduced plasma Na+ and Cl⁻ levels in fish in FW, and significantly improved ion regulation after their transfer to 28SW. Higher doses of cortisol (10 and 20 μg/g) induced hyperglycemia, whereas low doses (0.05 and 1.0 μg/g were without effect or induced hypoglycemia. Plasma glucose levels decreased in cortisol-treated fish transferred to SW, whereas transient hyperglycemia was seen in the control fish.     

The role of Ca2+ and Na+ membrane transport in brook trout (Salvelinus fontinalis) spermatozoa motility

The role of environmental ion composition and osmolality in Ca²⁺ signaled activation was assessed in spermatozoa of brook trout Salvelinus fontinalis. Milt from ten mature males was obtained by abdominal massage. Spermatozoa motility was evaluated in 0, 100, and 300 mOsm/kg NaCl or sucrose solutions, buffered by 10 mM Tris–HCl pH 8.5. For investigation of spermatozoa reaction to external Ca²⁺ concentration, 2 mM ethylene glycol tetraacetic acid (EGTA) was added to the activation media as a calcium ions chelator. For investigation of the effect of external Na⁺ concentration in conditions of low external Ca²⁺, 100 µM amiloride was added to the EGTA-containing solutions as a Na⁺ transport blocker. Low motility was observed in sucrose (Na⁺ free) solutions containing 2 mM EGTA but not in Na⁺ solutions containing 2 mM EGTA. Addition of amiloride led to significantly increased motility (P < 0.05) compared with sucrose (Na⁺ free) solutions containing 2 mM EGTA. We conclude that Na⁺ transport in Ca²⁺-free solutions plays a regulatory role in brook trout spermatozoa activation. The influence of competitive Na⁺ and Ca²⁺ transport on the control of spermatozoa activation requires further study with respect to its application for improvement of artificial activation and storage media.     

Molecular cytogenetic study of genome ploidy in the German mirror carp Cyprinus carpio

We examined the polyploidy of Cyprinus carpio, the German mirror carp. Specimens were collected in the field in Hulan, China, and treated with phytohemagglutinin and colchicine before the chromosome spreads and the karyotype of kidney cells were examined using silver staining, chromomycin A₃ (CMA₃)/distamycin (DA)/4,6-diamidino-2-phenylindole (DAPI) staining, and fluorescence in situ hybridization (FISH) with a 5.8S + 28S rDNA probe. One to twosilver stained (Ag) nucleolus organizing regions (NORs) were detected during metaphase and interphase events, with 80 % of the metaphase spreads and 69 % of the interphase nuclei showing two Ag-NORs signals. One CMA₃ signal was detected on the terminus of the short arm of each submetacentric chromosome 8 (SMC8) homolog (n = 2). The 5.8S + 28S rDNA probe hybridized at the terminus of the short arm of each SMC8 homolog (n = 2). The results of the Ag-NORs, CMA₃/DA/DAPI, and 5.8S + 28S rDNA FISH analyses were consistent with regard to the total number and location of the SMC8 NORs in the chromosome spreads and karyotype, indicating that, at the molecular cytogenetic level, the German mirror carp is an evolutionary tetraploid with two sets of chromosomes after diploidization.     

Modulation of GR activity does not affect the in vitro metabolism of cortisol by rainbow trout ovarian follicles

The goal of the study was to determine whether the metabolic clearance of cortisol from rainbow trout (Oncorhynchus mykiss) ovarian follicles is affected by the level of ovarian steroidogenesis, and whether it involves the activation of glucocorticoid receptors (GRs). Ovarian follicles were incubated in vitro; the adenylate cyclase activator, forskolin, was used to stimulate ovarian steroidogenesis, and the modulation of GR activity was brought about using GR agonists (cortisol and dexamethasone) or the GR antagonist, mifepristone (RU486). The follicles were co-incubated with [2, 4, 6, 7 ³H] cortisol, and the tritium-labelled steroid products were separated by HPLC. In addition, the rates of expression of genes encoding for the two forms of GR (gr1 and gr2) were measured. Cortisone, cortisol sulphate, and cortisone sulphate were the major glucocorticoid products of cortisol metabolism, indicative of the action of 11β-hydroxysteroid dehydrogenase and glucocorticoid sulphotransferase in the follicular cells. There were no effects of RU486 or forskolin on the rates of [³H]cortisol metabolism suggesting that cortisol metabolism by ovarian follicles was independent of GR activation, and not influenced by increased activation of gonadal reproductive steroidogenesis.     

Modulation of appetite,lipid and glucose metabolism of juvenile grass carp (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>) by different dietary protein levels

This study was undertaken to explore the systemic metabolic strategies of juvenile grass carp (Ctenopharyngodon idellus) to maintain growth when fed with different dietary protein levels. The optimal growth group and two growing discomfort groups were selected through the basic data, to explain the growth difference from appetite regulation and lipid and glucose metabolism perspective. Three experimental diets were formulated with three dietary protein levels at 200.3, 296.1 and 442.9 g kg^?1, named P1, P2 and P3, respectively. Juvenile grass carp (initial body weight 12.28 ± 0.14 g) were fed with three diets with 3 replications per dietary treatment in an indoor recirculation system for an 8-week feeding trial. Fish fed with diet P2 dietary group showed significantly higher WG, SGR, FI and PER than other groups. Compared with other groups, mRNA expressions of NPY, Y8a and Y8b in fish fed with P2 significantly down-regulated, while the expressions of CCK and CART in fish fed with P3 significantly down-regulated (P < 0.05). With increasing dietary protein levels, G6Pase, GK, PK and PEPCK were all significantly inhibited (P < 0.05). For lipid metabolism, the mRNA expression of ACC in P1 dietary group was significantly higher than P3 dietary group; besides, LPL expression in P3 group was significantly higher than other two groups (P < 0.05). PPARα expression in P2 was significantly lower than other groups (P < 0.05). These results suggested that grass carp fed with P2 (296.1 g kg^?1 protein level) showed highest weight gain, contributed to more balanced nutrient metabolism and appetite regulation. Too high dietary protein (442.9 g kg^?1) should be avoided because it induced lowest PER, body lipid and liver lipid, and inhibited glucose and lipid metabolism in juvenile grass carp.     

Exploration of the mechanisms of protein quality control and osmoregulation in gills of Chromis viridis in response to reduced salinity

Fish gills are the vital multifunctional organ in direct contact with external environment. Therefore, activation of the cytoprotective mechanisms to maintain branchial cell viability is important for fish upon stresses. Salinity is one of the major factors strongly affecting cellular and organismal functions. Reduction of ambient salinity may occur in coral reef and leads to osmotic stress for reef-associated stenohaline fish. However, the physiological responses to salinity stress in reef-associated fish were not examined substantially. With this regard, the physiological parameters and the responses of protein quality control (PQC) and osmoregulatory mechanisms in gills of seawater (SW; 33–35 ‰)- and brackish water (BW; 20 ‰)-acclimated blue-green damselfish (Chromis viridis) were explored. The results showed that the examined physiological parameters were maintained within certain physiological ranges in C. viridis acclimated to different salinities. In PQC mechanism, expression of heat-shock protein (HSP) 90, 70, and 60 elevated in response to BW acclimation while the levels of ubiquitin-conjugated proteins were similar between the two groups. Thus, it was presumed that upregulation of HSPs was sufficient to prevent the accumulation of aggregated proteins for maintaining the protein quality and viability of gill cells when C. viridis were acclimated to BW. Moreover, gill Na⁺/K⁺-ATPase expression and protein amounts of basolaterally located Na⁺/K⁺/2Cl^? cotransporter were higher in SW fish than in BW fish. Taken together, this study showed that the cytoprotective and osmoregulatory mechanisms of blue-green damselfish were functionally activated and modulated to withstand the challenge of reduction in salinity for maintaining physiological homeostasis.