Response of rainbow trout transcriptome to model chemical contaminants

We used high-density cDNA microarray in studies of responses of rainbow trout fry at sublethal ranges of beta-naphthoflavone, cadmium, carbon tetrachloride, and pyrene. The differentially expressed genes were grouped by the functional categories of Gene Ontology. Significantly different response to the studied compounds was shown by a number of classes, such as cell cycle, apoptosis, signal transduction, oxidative stress, subcellular and extracellular structures, protein biosynthesis, and modification. Cluster analysis separated responses to the contaminants at low and medium doses, whereas at high levels the adaptive reactions were masked with general unspecific response to toxicity. We found enhanced expression of many mitochondrial proteins as well as genes involved in metabolism of metal ions and protein biosynthesis. In parallel, genes related to stress and immune response, signal transduction, and nucleotide metabolism were down-regulated. We performed computer-assisted analyses of Medline abstracts retrieved for each compound, which helped us to indicate the expected and novel findings.     

Characterization of p53 expression in rainbow trout

The tumour suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. Given the high incidence of p53 mutations in human cancers, it has been extensively studied, though only a small fraction of these investigations have been in non-mammalian systems. For the present study, an anti-rainbow trout p53 polyclonal antibody was generated. A variety of rainbow trout (Oncorhynchus mykiss) tissues and cell lines were examined through western blot analysis of cellular protein extracts, which revealed relatively high p53 levels in brain and gills. To evaluate the checkpoint response of rainbow trout p53, RTbrain-W1 and RTgill-W1 cell lines were exposed to varying concentrations of the DNA damaging agent bleomycin and ribonucleotide reductase inhibitor hydroxyurea. In contrast to mammals, these checkpoint-inducing agents provoked no apparent increase in rainbow trout p53 levels. These results infer the presence of alternate DNA damage checkpoint mechanisms in rainbow trout cells.     

Suppression of first egg mitosis induced by heat shocks in the rainbow trout

Chromosome doubling by mitotic interference was achieved by heat-shocking rainbow trout ( Oncorhynchus mykiss ) eggs fertilized with either intact or genetically inactivated sperm. Tetraploid and mitotic gynogenetic individuals resulted from these treatments respectively. The temperature (27–33° C), duration (2–30min) and application time (2–4 h 40min after egg activation, at 10° C) of the thermal shock were investigated. The best yields of gynogenetics usually resulted from shocks of medium intensity (30° C for 9 min, 31° C for 5 min, 32° C for 4 min). A range of optimal application times was determined between 3 and 4 h after egg activation. A strong maternal effect on gynogenetic yield, irrespective of the application time, was observed. The treatments found to produce the highest yields of gynogenetics (up to 23% survival at hatching, relative to that of the diploid control) should not be used for tetraploid induction because of their rather low efficiency in chromosome doubling (around 70%). Tetraploid populations where viable residual diploids are undesirable should be produced by more intense shocks.     

Mass spectrometric evidence that proteolytic processing of rainbow trout egg vitelline envelope proteins takes place on the egg

The rainbow trout egg vitelline envelope (VE) is constructed of three proteins, called VEalpha,VEbeta, and VEgamma, that are synthesized and secreted by the liver and transported in the bloodstream to the ovary, the site of VE assembly around eggs. All three proteins possess an N-terminal signal peptide, a zona pellucida domain, a consensus furin-like cleavage site (CFLCS) close to the C terminus, and a short propeptide downstream of the CFLCS. Proteolytic processing at the CFLCS results in loss of the short C-terminal propeptide from precursor proteins and enables incorporation of mature proteins into the VE. Here mass spectrometry (matrix-assisted laser desorption ionization time-of-flight-mass spectrometry and liquid chromatography-mass spectrometry with a micromass-quadrupole TOF hybrid mass and a QSTAR Pulsar i mass spectrometer) was employed with VE proteins isolated from rainbow trout eggs in a peptidomics-based approach to determine the following: 1) the C-terminal amino acid of mature, proteolytically processed VE proteins; 2) the cellular site of proteolytic processing at the CFLCS of VE precursor proteins; and 3) the relationship between proteolytic processing and limited covalent cross-linking of VE proteins. Peptides derived from the C-terminal region were found for all three VE proteins isolated from eggs, indicating that processing at the CFLCS occurs after the arrival of VE precursor proteins at the egg. Consistent with this conclusion, peptides containing an intact CFLCS were also found for all three VE proteins isolated from eggs. Furthermore, peptides derived from the C-terminal propeptides of VE protein heterodimers VEalpha-VEgamma and VEbeta-VEgamma were found, suggesting that a small amount of VE protein can be covalently cross-linked on eggs prior to proteolytic processing at the CFLCS. Collectively, these results provide important evidence about the process of VE formation in rainbow trout and other non-cyprinoid fish and allow comparisons to be made with the process of zona pellucida formation in mammals.     

Characterization of rainbow trout cell lines using microsatellite DNA profiling

Ten microsatellite loci (Omy27DU,Omy325(A3)UoG, OmyFGT5TUF,OmyFGT14TUF, OmyFGT15TUF,OmyFGT23TUF, Omy77DU,Ssa20.19NUIG, Ots1BML, andOne18ASC) were amplified using the polymerase chain reaction to create genetic profiles for nine cell lines (RTG-2, RTH-149,RTL-W1,RTgill-W1, RTS-11, RTS-34st, RTP-2, RTP-91E and RTP-91F) from rainbow trout(Oncorhynchus mykiss) and one cell line (CHSE-214) from Chinook salmon (O. tschawytscha). A cell line (PHL) from anon-salmonid, the Pacific herring (Clupea harengus pallasi), was included as a control. The ten loci clearly revealed the uniqueness of each cell line, except for two cell lines (RTP-91E andRTP-91F) from the same fish. RTP-91E and RTP-91F were identical at all loci except Ssa20.19NUIG. The most useful locus for demonstrating uniqueness was Ots1BML. The information was used to demonstrate that an uncharacterized rainbow trout cell line (Clone 1A)was in fact CHSE-214, illustrating the usefulness of multiplexed microsatellites for the creation of genetic profiles for salmonid cell lines and for the testing of cell line cross-contamination. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of a monoclonal antibody specific to rainbow trout thrombocytes.

A monoclonal antibody (MAb) specific for rainbow trout thrombocytes was produced and its reactivity was demonstrated by flow cytometry and immuno-electron microscopy. Flow cytometry analysis showed that this MAb (TTL-7D11) reacted positively with about 30% of the peripheral blood leucocytes (PBL) and about 1%, 2%, and 11% of the pronephros, mesonephros, and spleen cells, respectively. Electron microscopy using immunogold labeling demonstrated that this MAb reacted strongly with thrombocytes, where gold beads could be seen attached only to the membrane and canalicular system of these cells. Positive and negative leucocytes for this MAb were obtained by magnetic cell separation. In the positive fraction, 96% of the cells were thrombocytes, while in the negative fraction no more than 3% were, which clearly showed a high purity of the positive fraction. Aggregation studies showed that about 75% of the positive fraction cells aggregated after being mixed with U-46619 thromboxane-mimetic, whereas in the negative fraction only 10% of the cells did so. Thus, utilizing the TTL-7D11 we have succeeded in isolating a pure thrombocyte population, and this would facilitate further studies, particularly on their characteristics and function(s).     

Characterization of morphology and physiological actions of scale osteoclasts in the rainbow trout

The morphology of scale osteoclasts in rainbow trout Oncorhynchus mykiss was characterized by light and scanning electron microscopy, and the effects of oestradiol-17β-treatment and sexual maturation on scale osteoclast morphology were investigated. The cells associated with resorption cavities could be distinguished morphologically as two types: symmetrical, compact cells lacking or having only a few cell processes, termed type 1 cells, and asymmetrical cells covered with folds and having several cell processes, termed type 2 cells. In adult sexually maturing fish, where scale resorption was high, type 1 cells were predominant. In juveniles and spawned adults where scale resorption was assumed to be relatively low, mostly type 2 cells were present. Oestradiol 17-β-treatment of juvenile rainbow trout increased the osteoclast activity, but did not affect the osteoclast morphology. Using light microscopy, the majority of the cells observed in, and closely associated with, the resorption cavities were mononucleated in both maturing and spawned fish. Occasionally, bi- and multinucleated osteoclasts were observed in the maturing, but not in the spawned fish. Light microscopic enzyme-histochemistry showed that the majority of the mononucleated cells, as well as the bi- and multinucleated ones, were tartrate resistant acid phosphatase positive in both groups of fish, thus implying that both type 1 and type 2 cells were osteoclasts. It is thus apparent that scale resorption in rainbow trout is carried out by two morphologically distinct osteoclast populations, representing different stages of osteoclast activity and/or different stages of osteoclast differentiation.     

Post-ovulatory ageing and egg quality: A proteomic analysis of rainbow trout coelomic fluid

Background  

In fish, oocyte post-ovulatory ageing is associated with egg quality decrease. During this period, eggs are held in the body cavity where they bath in a semi-viscous liquid known as coelomic fluid (CF). CF components are suspected to play a role in maintaining oocyte fertility and developmental competence (egg quality). However, CF proteic composition remains poorly studied. Thus rainbow trout CF proteome was studied during the egg quality decrease associated with oocyte post-ovulatory ageing.     

Characterization of Saccharomyces cerevisiae CBS 7764 isolated from rainbow trout intestine

A wild-type Saccharomyces cerevisiae, strain CBS 7764, isolated from the intestine of rainbow trout, was analyzed with respect to general growth parameters and global protein expression. Characterization of this strain was of interest since previous data show non-typical S. cerevisiae cell surface properties and because data suggest a probiotic potential of CBS 7764. The heat production rate (dQ/dt), monitored by microcalorimetry, showed that the typical growth phases resulting from diauxic growth on glucose were present in the fish isolate. However, CBS 7764 differentiated from a reference strain by becoming limited in the respiratory phase as demonstrated by a plateau in the dQ/dt signal. The global protein expression, as studied by two-dimensional gel electrophoresis (2D-PAGE), revealed a large degree of resemblance of the fish isolate to the reference strain, however, also clear qualitative and quantitative expression differences were detected; e.g. 14% of the proteins differed in expression level by a factor of at least 2. In addition, the fish isolate expressed 12 unique proteins. The heat shock proteins, which for other organisms have been identified as important in mucosal colonization, were generally expressed to a higher level in CBS 7764.     

Characterization of rainbow trout egg quality: a case study using four different breeding protocols, with emphasis on the incidence of embryonic malformations

The aim of this study was to set up a methodology to accurately evaluate the effects of various husbandry practices on a fish broodstock based on the developmental potential of the egg. For that purpose, long-short photoperiod manipulations (tested twice, PM1 and PM2 groups), spawning induction by injection of a GnRH analog (SI group), and a 16-day post-ovulatory ageing of eggs (POA group) were used in rainbow trout (Oncorhynchus mykiss). Females without any treatment were used as a control group. Survival at eying (E) and yolk-sac resorption (YSR) were recorded and malformations at YSR were monitored according to a detailed typology that included cyclopia, torsion, incomplete YSR, prognathia, and others. Egg weight was also monitored. A deleterious effect of photoperiod manipulation was observed on egg quality in both PM1 and PM2 groups. Incomplete YSR appeared as the predominant malformation while cyclopia type was nearly absent. In the SI group, a limited effect on egg quality was observed in comparison to the other experimental groups, although the percentage of normal alevins at YSR was significantly lower than in the control group. Finally, the most important effects on egg quality were observed in the POA group. The percentage of normal alevins was only 14+/-6% (mean+/-95% confidence interval) while the percentage of malformed embryos reached 49+/-11%. The proportion of cyclopia was significantly higher than in the control group. In conclusion, the type of egg quality alteration is extremely dependent on the applied breeding protocols, and the proposed methodology is able to discriminate those experimental conditions even when the impact on egg quality is limited.     

Characterization of adrenergic receptors and related transduction pathways in the liver of the rainbow trout

Epinephrine (EPI) is thought to act by stimulating adenylyl cyclase (ACase) and cAMP production through β-adrenoceptors in the liver of more primitive vertebrates. Recent observations, however, point to an involvement of α₁-adrenoceptors in EPI action, at least in some fish species. The role of the α₁- and β-adrenergic transduction pathways has been investigated in rainbow trout (Oncorhynchus mykiss) hepatic tissue. Radioligand-binding assays with the β-adrenergic antagonist ³H-CGP-12177 using hepatic membranes purified on a discontinuous sucrose gradient confirmed the presence of β-adrenoceptors (K_d0.36 nM, B_max 8.61 fmol · mg⁻¹ protein). We provide the first demonstration of α₁-adrenoceptors in these same membranes; analysis of binding data with the α₁-adrenergic antagonist ³H-prazosin demonstrated a single class of binding sites with a K_dof 15.4 nM and a B_max of 75.2 fmol · mg⁻¹ protein. There is a straight correlation between β-adrenoceptor occupancy, ACase activation and cAMP production. On the contrary, the role of inositol 1,4,5-trisphosphate (IP₃) has to be elucidated; in fact, despite the presence of specific microsomal binding sites for IP₃ (K_d 6.03 nM, B_max 90.2 fmol · mg⁻¹ protein), its cytosolic concentration was not modulated by EPI. On the other hand, we have previously shown in American eel and bullhead hepatocytes that α₁-adrenergic agonists are able to increase intracellular concentrations of IP₃ and Ca²⁺ and to activate glycogenolysis. These data suggest a marked variation in the liver of different fish both in terms of α₁-binding sites affinity and of α₁-adrenoceptor/IP₃/Ca²⁺ transduction systems.     

Cloning of rainbow trout egg envelope proteins: members of a unique group of structural proteins

All vertebrate eggs are surrounded by an extracellular envelope that protects the egg and is vital for a successful fertilization. The terminology and functions of the egg envelope vary in different vertebrate groups, but the envelope itself is consistently composed of a few major proteins that are deposited around the oocyte during oocyte growth. Here, we describe the deduced amino acid sequences and tissue expression patterns of the three major egg envelope proteins for rainbow trout (Oncorhynchus mykiss). All three vitelline envelope proteins (VEPs) are expressed in the livers of both male and female fish, with higher expression in females. In addition, VEPgamma mRNA is also detected in the female gonads. To our knowledge, this is the first time that expression of a VEP protein gene has been demonstrated to occur in more than one organ. Sequence comparison reveals that all three VEP proteins share distinct homology with their amphibian, avian, and mammalian counterparts. Whereas mammalian zona pellucida protein 3 isoforms contain two conserved serines needed for sperm binding, these are not conserved in teleost species, in which sperm entry is restricted to the micropyle. Besides the difference in VEPgamma sperm-binding function, the high sequence homology suggests that the egg envelope proteins from these distinct vertebrate groups share a common ancestry and form a unique group of structural proteins.     

Activation of rainbow trout macrophages

Rainbow trout peritoneal macrophages were stimulated in vitro using Concanavalin A (Con A) and in vivo using formalin-killed Aeromonas salmonicida in Freund's incomplete adjuvant (FIA). Whether these cells had been activated was determined by the measurements of oxygen anions (NBT reduction), H₂O₂ production (oxidation of phenol red), RNA synthesis (³H-uridine incorporation), acid phosphatase activity and bactericidal activity.
In vitro -stimulated macrophages showed an increased NBT reduction and ³H-uridine incorporation over a range of Con A concentrations, compared with untreated control macrophages, but no detectable increases in H₂O₂ production or bactericidal activity were observed. On the other hand, in vivo -stimulated peritoneal cells showed increases in all the assays compared with FIA-elicited control cells, and were considered to have been activated.     

Characterization of extracellular substance of Vibrio anguillarum toxic for rainbow trout and mice

An extracellular toxic substance was separated from the cell-free culture filtrate of Vibrio anguillarum (strain NCMB571). Two fractions (GI and GII + III) obtained by Sephadex G-200 chromatography following DEAE-cellulose chromatography were lethal to rainbow trout and mice. Material separated from the GI fraction by Sepharose 4B affinity chromatography (GI-A fraction) was lethal to these animals. By sodium dodecylsulfate polyacrylamide gel electrophoresis, the GI and GI-A fractions were found to be composed of components with molecular weights of 44K and 34K, and 44K, respectively. The 44K protein band was associated with carbohydrate. Peripheral vascular disorder was observed in fish and mice that died after inoculation with GI or GI-A fraction. The toxic substance was sensitive to potassium periodate but was resistant to trypsin and acetone. Heat inactivation of the toxic substance was almost complete at 100 C for 20 min and complete at 121 C for 20 min. The toxic activity was not associated with hemolytic or proteolytic activity. Homologous antitoxin completely neutralized the toxic activity.