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PubMed2000

Induction of gonadal intersex in genotypic male rainbow trout (Oncorhynchus mykiss) embryos following immersion in estradiol-17beta.

This investigation was conducted to determine the initial period of gonadal sensitivity to estrogen in genetically male rainbow trout (Oncorhynchus mykiss). Fish were immersed in approximately 250 microg estradiol-17beta, (E2)/l for two 2 hr periods during different stages of embryonic development beginning 30 days postfertilization (DPF) and continuing until 68 DPF. Histological analysis of gonad samples indicated a significant proportion of E2-treated fish had intersex gonads; these gonads were primarily comprised of testicular tissue with one or more oocytes scattered throughout. The most sensitive period for altering normal testicular development was found to occur between 44 and 51 DPF (63% intersex), while the labile period in general was determined to span 24 days (from 30 to 54 DPF). Additionally, quantitative RT-PCR was used to measure estrogen receptor (ER) mRNA expression in whole individual untreated embryos at six weekly time points throughout the period of E2-exposure. Although the intersex condition was not observed throughout the entire E2-exposure period, ER mRNA was detected at each time point assayed.

Animals↗
PubMed2003

Ontogeny of ultraviolet-sensitive cones in the retina of rainbow trout (Oncorhynchus mykiss).

In order to facilitate emerging models of retinal development, we developed electroretinogram and in situ hybridization protocols to examine the ontogeny of photoreceptors in the retina of a land-locked salmonid, the rainbow trout (Oncorhynchus mykiss). We cloned cDNA fragments corresponding to the rod opsin and each of the four cone opsin gene families, which we utilized to produce riboprobes. We established the specificity of the in situ hybridization protocol by examining subcellular signal localization and through double-labeling experiments. We confirm the assumption that the accessory corner cones in the square mosaic are the ultraviolet wavelength-sensitive (UVS) cone photoreceptor (i.e., they express an SWS1 opsin) and observed UVS cones throughout the retina of small trout. Larger fish have a decrease in sensitivity to short wavelength light stimuli and the distribution of UVS cones in the mature retina is limited to the dorsal-temporal quadrant. These larger fish also possess differentiated UVS cones in the peripheral germinal zone (PGZ), including within areas peripheral to mature retina lacking UVS cones. These data are consistent with the loss of putative UVS cones from the PGZ of a migratory salmonid of another genus, and thus the disappearance of UVS cones appears to be general to the Family Salmonidae, regardless of life history strategy. The generation, differentiation, and subsequent loss of UVS cones in the smolt PGZ is a dramatic example of the supposition that the mechanisms of PGZ development recapitulate the retinal embryogenesis of that species.

Aging↗
PubMed2003

Experimental study of the connections of the gustatory system in the rainbow trout, Oncorhynchus mykiss.

Salmonids are a group of teleosts with a nonspecialized gustatory system. With the aim of describing the gustatory connections in a member of this group, we carried out tract-tracing experiments using the lipophilic carbocyanine dye 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in fixed brains of the rainbow trout (Oncorhynchus mykiss). The neural tracer was applied to the primary viscerosensory column, secondary gustatory visceral nucleus (SGN), torus lateralis (TL), and tertiary gustatory nucleus (TGN), the dorsal part of the ventral area of the telencephalon (dorsal-Vv), and the medial area of the dorsal telencephalon (Dm). The primary viscerosensory column projects mainly to the SGN. DiI application to the SGN showed a bilateral and reciprocal connection with the TGN and a rostral portion of the nucleus of the lateral hypothalamic recess. The application of DiI in the dorsal-Vv and Dm at levels rostral to the anterior commissure led to labeling of a restricted group of diencephalic neurons in the TGN and sending dendrites to the TL. DiI application to the TL/TGN anterogradely labeled fibers that coursed in the medial forebrain bundle innervating the precommissural portion of the dorsal-Vv and Dm. Caudally, this type of application led to labeling of fibers in the viscerosensory column and perikarya in the SGN. Tract-tracing results showed direct projections from the diencephalic and rhombencephalic gustatory nuclei to the telencephalon. There was a direct and reciprocal connection between the SGN and the ventral telencephalon. The results showed that the gustatory connections of the trout are similar to those of teleosts, with highly specialized gustatory centers as in cyprinids and ictalurids, and to that observed in the percomorph tilapia, thus demonstrating a basic organization that is shared by most teleosts.

Animals↗
PubMed2001

Assessment of thyroid function in adult medaka (Oryzias latipes) and juvenile rainbow trout (Oncorhynchus mykiss) using immunostaining methods.

The effect of injected bovine TSH on the pattern of anti-T(4) and anti-T(3) immunostaining of the thyroid tissue was examined in rainbow trout (Oncorhynchus mykiss) and medaka (Oryzias latipes) to determine if the previously reported immunostaining of the cytoplasm of thyrocytes is due to the presence of colloid pinocytotic vesicles and is thus indicative of thyroid hormone release. We hypothesized that the number of immunostained thyrocytes should increase following a TSH challenge, and this should parallel other indicators of increased thyroid activity. In medaka, immunostained thyrocytes were only found following the TSH challenge, and were most marked after 24 to 72 hours; the immunostaining was associated with large colloid-filled cytoplasmic vesicles. In trout, the number and staining intensity of immunostained thyrocytes were increased after the TSH challenge; the immunostaining was present throughout the cytoplasm of the thyrocytes. These findings support the working hypothesis that the immunostaining of the thyrocytes is associated with the pinocytosis of thyroglobulin by the thyrocytes in parallel with an increase in release of thyroid hormone, and that this investigational approach provides a reliable indicator of thyroid hormone release activity.

Animals↗
PubMed2005

An in vitro system for the long-term tissue culture of juvenile rainbow trout (Oncorhynchus mykiss) testis.

In vitro cultivation of fish gonad fragments continues to be an important experimental approach to answer both fundamental and applied scientific questions. The aim of the present investigation was to test whether juvenile rainbow trout (Oncorhynchus mykiss) testes cultured for a week or more were physiologically competent. Trout testis fragments (approximately 1 mm2) were placed on pieces of flat, culture plate insert filter, in a drop of liquid medium (modified Leibovitz's L-15), and floated on the medium surface in a multi-well culture plate. Culture plates were covered and incubated in air at 15 degrees C. Three different endpoints were used to test whether cultured testis fragments remained healthy and functional. First, a comparison of the histological appearance of testis fragments cultured in vitro for different periods up to 8 days with intact testes indicated that no differences were evident. Secondly, testis fragments incubated in medium supplemented with bovine calf serum (BCS) at concentrations of 2.5% and 25% BCS had significantly greater proliferation of interstitial and spermatocyst cells as measured by nuclear 5-bromo-2'-deoxyuridine incorporation. Finally, testis fragments cultured for 5 days and transplanted back into the donor fish underwent precocious sexual maturity in response to a regime of salmon pituitary extract injections and produced fertile sperm as determined by their ability to successfully fertilize eggs. These experiments demonstrate the utility of this method for in vitro culture of juvenile rainbow trout testis fragments.

Animals↗
PubMed1993

Effect of lipoxins and other eicosanoids on phagocytosis and intracellular calcium mobilisation in rainbow trout (Oncorhynchus mykiss) leukocytes.

Rainbow trout (Oncorhynchus mykiss) macrophages generated lipoxin (LX) A4, LXA5, leukotriene (LT) B4, LTB5 and 12-hydroxyeicosatetraenoic acid (12-HETE) during the phagocytosis of zymosan and Escherichia coli, but not of the yeast Saccharomyces cerevisiae. Prostaglandin (PG) E2 was also detected in supernatants from macrophages incubated with either zymosan or calcium ionophore A23187. LXA4 (10(-8)-10(-6) M) and LTB4 (10(-9)-10(-7) M) provoked rapid and transient dose-dependent increases in intracellular calcium ([Ca]i) concentrations in leukocyte suspensions containing 40-60% macrophages. EC50 values were 14.9 and 1.2 nM, respectively. PGE2 and 12-HETE had no effect on [Ca]i at concentrations up to 30 microM. PGE2 and 12-HETE (10(-5)-10(-10) M) enhanced the in vitro phagocytosis of yeast test particles by trout macrophages, whereas LXA4 and LTB4 had no demonstrable effect on the responses of these cells at concentrations up to 10(-5) M. In conclusion, the processes involved in trout macrophage stimulation are complex but involve generation of both cyclooxygenase and lipoxygenase products. The increase in [Ca]i caused by LXA4 and LTB4 may form part of the chemotactic transduction mechanism that recruits granulocytes and macrophages to sites of inflammation. The effects of eicosanoids on phagocytosis appear to be independent of changes in [Ca]i.

Animals↗
PubMed1995

Elevation of rainbow trout Oncorhynchus mykiss macrophage respiratory burst activity with macrophage-derived supernatants.

A variety of supernatants were prepared by stimulating rainbow trout Oncorhynchus mykiss head kidney macrophages with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), or a leucocyte-derived macrophage-activating factor (I-MAF), individually and in combination. If generated using a 12-h stimulation period, such supernatants were found to elevate significantly the respiratory burst activity of target macrophages; that is, they contained a macrophage-derived MAF (m-MAF), but supernatants generated using a shorter incubation period showed no significant activity. Combinations of these treatments were particularly effective in generating m-MAF-containing supernatants. The elevation of respiratory burst activity by supernatants generated using combined treatments could be partially inhibited by prior treatment of the target macrophages with anti-TNF-alpha receptor 1 (TNFR1) monoclonal antibodies (mAbs). Similarly, treatment of macrophages with combinations of 1-MAF and m-MAF generated supernatants with potent m-MAF activity and this activity was partially inhibited by prior treatment of the target cells with anti-TNFR1 mAb. In addition, the presence of anti-transforming growth factor beta 1 (TGF-beta 1) serum while generating these latter supernatants resulted in significantly increased m-MAF activity. Such data suggest that fish leukocytes secrete a variety of potent macrophage-activating (TNF-alpha) and -deactivating (TGF-beta) factors.

Animals↗
PubMed2002

Molecular cloning and expression during spermatogenesis of a cDNA encoding testicular 11beta-hydroxylase (P45011beta) in rainbow trout (Oncorhynchus mykiss).

Cytochrome P450 1beta-hydroxylase (P450(11beta)) is an important steroidogenic enzyme for glucocorticoid and mineralocorticoid production in vertebrates. In teleosts, P450(11beta) also plays a role in the production of 11-ketotestosterone (11-KT), the predominant androgen in male fish. In this study, we cloned a cDNA encoding rainbow trout (Oncorhynchus mykiss) testicular P450(11beta). The cDNA contains 1,740 nucleotides that encode a protein of 551 amino acids which shares 65.2% homology with testicular P450(11beta) from Japanese eel, and 33-45% homology with adrenal P450(11beta) from rat, human, and frog. HEK293 cells transfected with an expression vector containing the rainbow trout P450(11beta) cDNA open reading frame showed 11beta-hydroxylating activity in the presence of exogenous testosterone. Analysis of tissue distribution by RT-PCR showed great abundance of P450(11beta) mRNA in testis and head kidney. In order to clarify the sites of P450(11beta) gene expression, cRNA in situ hybridization analysis was performed. Hybridization signals were detected in Leydig cells and head kidney inter-renal cells. The results of Northern blot analysis indicated a single 1.8-kb transcript encoding P450(11beta) in testis and in head kidney, suggesting that the testicular form of P450(11beta) may also be involved in cortisol production by inter-renal cells. Seasonal changes in total P450(11beta) mRNA levels in testes during spermatogenesis showed a pattern similar to that of plasma androgens. These results suggest that androgen production in male rainbow trout is partially regulated by changes in abundance of P450(11beta) mRNA.

Amino Acid Sequence↗
PubMed2005

Androgen and estrogen treatments alter steady state messengers RNA (mRNA) levels of testicular steroidogenic enzymes in the rainbow trout, Oncorhynchus mykiss.

Recent investigations have shown that estrogens have profound inhibitory effects on steroidogenic enzyme gene expressions before and after testicular differentiation in the rainbow trout, Oncorhynchus mykiss. This present study bring new data on juvenile rainbow trout treated with estrogens and androgens. Following a 8 days oral treatment of juvenile male with 17alpha-ethynyl-estradiol (EE2, 20 mg/kg diet) or 11beta-hydroxyandrostenedione (11betaOHDelta4, 10 mg/kg diet), we observed a fast and marked decrease of steady-state mRNA levels for 3betaHSD, P450scc, P450c17, and P450c11 enzymes in the testis. After completion of these treatments, mRNA levels of these enzymes remained low in EE2 treated males whereas in 11betaOHDelta4 treated males they recovered their initial levels in 8 days. This demonstrate that both androgen and estrogen treatments have profound effects on testicular steroidogenesis by decreasing steroid enzymes steady-state mRNA. After in vitro incubation of testicular explants with 17beta-estradiol (E2, 600 ng/ml of medium), we also observed a decrease of mRNA levels for 3betaHSD and P450c11. This suggest that estrogens effects could be triggered, at least to some extend, directly on the testis. We also investigated the hypothesis of a negative feedback of steroids on follicle stimulating hormone (FSH) secretion, but FSH plasmatic levels in treated fish did not showed any significant decrease. This demonstrate that FSH is not implied in this steroids inhibition of steroidogenic enzymes gene expression.

Androgens↗
PubMed1997

Characterization of rainbow trout (Oncorhynchus mykiss) growth hormone 1 gene and the promoter region of growth hormone 2 gene.

Studies by Agellon et al. (Mol. Reprod. Dev. 1, 11-17) showed the presence of two growth hormone (rtGH1 and rtGH2) mRNA species in pituitary glands of adult rainbow trout (Oncorhynchus mykiss). In this study, we have detected rtGH1 and rtGH2 mRNAs in pituitary glands of rainbow trout from fry to 2 years of age. The level of rtGH1 mRNA is notably higher than that of rtGH2 mRNA in 10-day-old fry and 2-year-old females. These results suggest differential expression of rtGH1 and rtGH2 genes in different sexes and developmental stages. As a step toward elucidating the mechanism of differential expression of both GH genes, DNA fragments encoding rtGH1 gene and the promoter/regulatory region of rtGH2 gene were isolated and characterized. Rainbow trout GH genes span approximately 4.5 kb and are composed of six exons and five introns. The 5'-flanking region of both genes contain consensus sequences for TATA boxes and several Pit-1 binding sequences. Consensus sequences related to the cAMP response element, thyroid hormone response element, retinoic acid response element, estrogen response element (ERE), and glucocorticoid response element are present not only in the 5'-flanking region, but also in introns and exons in rtGH1 gene. These hormone response elements, except ERE, are also present in rtGH2 gene.

Animals↗
PubMed1997

CYP4T1--a cytochrome P450 expressed in rainbow trout (Oncorhynchus mykiss) liver.

Total RNA isolated from a rainbow trout (Oncorhynchus mykiss) liver was subjected to RT/PCR using degenerate primers designed from homologous regions amongst cytochrome P450 CYP4 proteins. PCR amplification resulted in a single electrophoretic band which was excised, purified and sequenced directly, using cycle sequencing. The deduced protein sequence demonstrated the closest amino acid identity to rabbit CYP4B1 (54.6%) and rat CYP4B2 (55.4%). Phylogenic analysis of this sequence was found to be significantly different to any other CYP4 sequence and has been named CYP4T1. This represents the first CYP4 family member to be identified in an aquatic vertebrate.

Animals↗
PubMed1999

Cloning and expression of two carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase cDNAs in ovarian follicles of rainbow trout (Oncorhynchus mykiss).

In salmonid fish, 20beta-hydroxysteroid dehydrogenase (20beta-HSD) is a key enzyme involved in the production of oocyte maturation-inducing hormone (MIH), 17alpha, 20beta-dihydroxy-4-pregnen-3-one. Here we report the isolation of two cDNAs which encode proteins with high homology to carbonyl reductase-like 20beta-HSD (CR/20beta-HSD) from rainbow trout (Oncorhynchus mykiss) ovarian follicles. Genomic DNA analysis showed that the two CR/20beta-HSD cDNAs are derived from two different genes. Northern blot and RT PCR analysis demonstrated that trout CR/20beta-HSDs are broadly expressed in various tissues. Enzymatic characterization using recombinant CR/20beta-HSD proteins produced in E. coli showed that the product of one of the two cDNAs had both 20beta-HSD and CR activity, but the other had neither activity. Although the functional significance of the two genes remains unresolved, these results clearly demonstrate the presence of two distinct CR/20beta-HSD transcripts in the trout ovary.

Alcohol Oxidoreductases↗
PubMed1999

Cloning, functional characterization, and expression of a gonadotropin receptor cDNA in the ovary and testis of amago salmon (Oncorhynchus rhodurus).

A gonadotropin receptor was cloned from amago salmon (Oncorhynchus rhodurus) ovarian follicles. This receptor (sGTH-R) belongs to the glycoprotein hormone receptor family with a large extracellular and seven-transmembrane domains. Its sequence homology is highest with mammalian LH receptors. Phylogenetic analysis reveals that sGTH-R is grouped with mammalian and chicken FSH and LH receptors, but not with mammalian TSH receptors. sGTH-R is expressed dominantly in the ovary and testis. Functional characterization examined with transiently transfected mammalian cells revealed increased intracellular cAMP level when exposed to mammalian and fish gonadotropins; the most potent hormone was salmon GTH II. These results indicate that the cloned cDNA encodes a functional amago salmon GTH receptor protein.

Amino Acid Sequence↗
PubMed1999

The duality of fish gonadotropin receptors: cloning and functional characterization of a second gonadotropin receptor cDNA expressed in the ovary and testis of amago salmon (Oncorhynchus rhodurus).

We previously isolated a cDNA encoding a gonadotropin receptor (sGTH-R) from amago salmon (Oncorhynchus rhodurus) ovarian follicles. In the present study, we cloned a second gonadotropin receptor (sGTH-RI) from the same RNA preparations. Overall sequence homology between sGTH-RI and sGTH-R is 44%. The highest homology occurs with mammalian FSH receptors (49%). Functional characterization examined in COS-7 cells transiently transfected with sGTH-RI showed the largest increase in cAMP production when exposed to salmon GTH I. These results provide the first evidence in any ectothermic vertebrate, the amago salmon, of the duality of gonadotropin receptors.

Amino Acid Sequence↗
PubMed2000

Cloning, functional characterization, and expression of thyrotropin receptors in the thyroid of amago salmon (Oncorhynchus rhodurus).

Two thyrotropin receptor cDNAs (sTSH-Ra and sTSH-Rb) were cloned from thyroid tissue of the amago salmon, Oncorhynchus rhodurus. sTSH-Ra and sTSH-Rb showed the highest degrees of sequence homology to mammalian TSH receptors. Functional characterization in COS-7 cells transiently transfected with sTSH-Ra or sTSH-Rb showed the largest increase in cAMP when exposed to bovine TSH. RT-PCR analysis demonstrated that sTSH-Ra and sTSH-Rb were expressed in the basibranchial region, but not in the ovary, testis, liver, kidney or brain. In situ hybridization revealed that sTSH-Ra and sTSH-Rb were exclusively expressed in thyroid follicular epithelial cells of amago salmon undergoing smoltification. These results indicated that the cloned cDNAs encode functional TSH receptor proteins. This is the first report of isolation of TSH receptor molecules from nonmammalian vertebrates.

Amino Acid Sequence↗
PubMed2002

Cloning of a novel rainbow trout (Oncorhynchus mykiss) CC chemokine with a fractalkine-like stalk and a TNF decoy receptor using cDNA fragments containing AU-rich elements.

An activation-specific cDNA library was made from phytohaemagglutinin (PHA)-activated haematopoietic cells of the rainbow trout (Oncorhynchus mykiss) using the technique of suppression subtractive hybridization. Several immune system genes were identified, including an interleukin (IL)1 receptor related protein and two invariant chain-like proteins. Many clones showed no similarity by BLAST search, but had AU-rich elements. These fragments were labelled and used for hybridization with a PHA-activated head kidney cDNA library. Several immune system genes were isolated by this technique, including a tumour necrosis factor (TNF) decoy receptor and a novel chemokine, designated trout chemokine 2. The TNF receptor is 285 amino acids in length and is 32-36% identical to a brook trout and human homologue. The CC chemokine is 44% identical at the amino acid level to a carp CC chemokine and approximately 20% identical to several mammalian CC chemokines. However, it has a 91 amino acid stalk-like structure at its COOH end, which is similar to the glycosylated stalk of fractalkine, a mammalian CX(3)C chemokine. In summary, AU-rich fragments obtained from an activation-specific library proved useful as hybridization probes for isolating trout immune system genes.

Adenine↗
PubMed1994

Effects of lindane exposure on rainbow trout (Oncorhynchus mykiss) immunity. III. Effect on nonspecific immunity and B lymphocyte functions.

The effect of the organochlorine insecticide lindane (gamma-hexachlorocyclohexane) was examined on some major immune functions of rainbow trout (Oncorhynchus mykiss) on the chemiluminescent response of pronephric cells (PMA-induced) in phagocytosis, on the proliferation of lymphocytes with B and T mitogens, and on the number of B lymphocytes analyzed by cytofluorometry. Two different methods of exposure were tried via food (first protocol) and via a single intraperitoneal injection (second protocol). After the oral contamination at a daily body dose of 1 mg/kg for 30 days, a decreased chemiluminescent response was observed with persisted for two more weeks and disappeared over 1.5 months. No effect was observed on lymphocyte proliferation and on the number of circulating B lymphocytes. In the second protocol lindane was administered intraperitoneally at 10, 50, or 100 mg/kg body wt. After 45 days the lymphocyte proliferation of B cells was depressed but not the T cell one. The B cells number in head kidney as measured by cytofluorometry was not significantly modified. Some nonspecific immunity parameters in sera were significantly modified.

Administration, Oral↗
PubMed1995

Effect of lindane exposure on rainbow trout (Oncorhynchus mykiss) immunity. IV. Prevention of nonspecific and specific immunosuppression by dietary vitamin C (ascorbate-2-polyphosphate).

The effect of the organochlorine insecticide lindane (gamma-hexachlorocyclohexane) administered intraperitoneally at 10 or 50 mg/kg body wt on some major immune functions of rainbow trout (Oncorhynchus mykiss) was examined. Fish were fed vitamin C as ascorbate-2-polyphosphate at a basal level (60 mg ascorbic acid-(AA)-equivalent/kg of feed) or a high level (2000 mg AA-equivalent/kg) 1 month before lindane exposure and during the whole experiment. The aim of the experiment was to determine whether dietary vitamin C is able to prevent immunosuppression due to lindane. The concentration of ascorbic acid in organs and the circulation was controlled, and the number of lindane residues in whole body was measured by gas chromatography. Nonspecific immune response was investigated through the determination of sera lysozyme and ceruloplasmin; both were significantly modified by lindane exposure while the immediate stimulating effects of vitamin C were observed. Cellular immunity was investigated by determining the number of B lymphocytes (analyzed by cytofluorometry) and their ability to proliferate with mitogens. One month after exposure to lindane (10 mg/kg) the proportion of Ig+ lymphocytes in head kidney was significantly decreased by the insecticide. Higher levels of vitamin C (2000 mg/kg) led to a significant increase in this parameter. Thus, vitamin C had a compensating effect on the number of Ig+ lymphocytes in exposed fish. Lindane at 10 mg/kg decreased the proliferation of B lymphocytes, but this was not confirmed at 50 mg/kg. Vitamin C stimulated the proliferation for the latter concentration after 2 months of intake. In lindane-exposed fish, the PMA-induced chemiluminescent response of head kidney phagocytic cells was variable from one assay to another, while most of the time vitamin C acted as a stimulant. The humoral response to Yersinia ruckeri was not modified by lindane but was significantly increased by vitamin C for 1 month after the antigen injection and thus 2 months after vitamin intake.

Animals↗