48 research outputs found

    Water quality.

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    <p>Water quality parameters at each caging site along the Bow River, Calgary, Alberta from September 12 to September 30, 2016. Values represent mean ± SEM (n = 8–9).</p

    Stress-Immune-Growth Interactions: Cortisol Modulates Suppressors of Cytokine Signaling and JAK/STAT Pathway in Rainbow Trout Liver

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    <div><p>Chronic stress is a major factor in the poor growth and immune performance of salmonids in aquaculture. However, the molecular mechanisms linking stress effects to growth and immune dysfunction is poorly understood. The suppressors of cytokine signaling (SOCS), a family of genes involved in the inhibition of JAK/STAT pathway, negatively regulates growth hormone and cytokine signaling, but their role in fish is unclear. Here we tested the hypothesis that cortisol modulation of SOCS gene expression is a key molecular mechanism leading to growth and immune suppression in response to stress in fish. Exposure of rainbow trout (<i>Oncorhynchus mykiss</i>) liver slices to cortisol, mimicking stress level, upregulated SOCS-1 and SOCS-2 mRNA abundance and this response was abolished by the glucocorticoid receptor antagonist mifepristone. Bioinformatics analysis confirmed the presence of putative glucocorticoid response elements in rainbow trout SOCS-1 and SOCS-2 promoters. Prior cortisol treatment suppressed acute growth hormone (GH)-stimulated IGF-1 mRNA abundance in trout liver and this involved a reduction in STAT5 phosphorylation and lower total JAK2 protein expression. Prior cortisol treatment also suppressed lipopolysaccharide (LPS)-induced IL-6 but not IL-8 transcript levels; the former but not the latter cytokine expression is via JAK/STAT phosphorylation. LPS treatment reduced GH signaling, but this was associated with the downregulation of GH receptors and not due to the upregulation of SOCS transcript levels by this endotoxin. Collectively, our results suggest that upregulation of SOCS-1 and SOCS-2 transcript levels by cortisol, and the associated reduction in JAK/STAT signaling pathway, may be a novel mechanism leading to growth reduction and immune suppression during stress in trout.</p></div

    The Transcriptomics of Glucocorticoid Receptor Signaling in Developing Zebrafish

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    <div><p>Cortisol is the primary corticosteroid in teleosts that is released in response to stressor activation of the hypothalamus-pituitary-interrenal axis. The target tissue action of this hormone is primarily mediated by the intracellular glucocorticoid receptor (GR), a ligand-bound transcription factor. In developing zebrafish (<i>Danio rerio</i>) embryos, GR transcripts and cortisol are maternally deposited into the oocyte prior to fertilization and influence early embryogenesis. To better understand of the molecular mechanisms involved, we investigated changes in the developmental transcriptome prior to hatch, in response to morpholino oligonucleotide knockdown of GR using the Agilent zebrafish microarray platform. A total of 1313 and 836 mRNA transcripts were significantly changed at 24 and 36 hours post fertilization (hpf), respectively. Functional analysis revealed numerous developmental processes under GR regulation, including neurogenesis, eye development, skeletal and cardiac muscle formation. Together, this study underscores a critical role for glucocorticoid signaling in programming molecular events essential for zebrafish development.</p> </div

    Novel Nongenomic Signaling by Glucocorticoid May Involve Changes to Liver Membrane Order in Rainbow Trout

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    <div><p>Stress-induced glucocorticoid elevation is a highly conserved response among vertebrates. This facilitates stress adaptation and the mode of action involves activation of the intracellular glucocorticoid receptor leading to the modulation of target gene expression. However, this genomic effect is slow acting and, therefore, a role for glucocorticoid in the rapid response to stress is unclear. Here we show that stress levels of cortisol, the primary glucocorticoid in teleosts, rapidly fluidizes rainbow trout (<em>Oncorhynchus mykiss</em>) liver plasma membranes <em>in vitro</em>. This involved incorporation of the steroid into the lipid domains, as cortisol coupled to a membrane impermeable peptide moiety, did not affect membrane order. Studies confirmed that cortisol, but not sex steroids, increases liver plasma membrane fluidity. Atomic force microscopy revealed cortisol-mediated changes to membrane surface topography and viscoelasticity confirming changes to membrane order. Treating trout hepatocytes with stress levels of cortisol led to the modulation of cell signaling pathways, including the phosphorylation status of putative PKA, PKC and AKT substrate proteins within 10 minutes. The phosphorylation by protein kinases in the presence of cortisol was consistent with that seen with benzyl alcohol, a known membrane fluidizer. Our results suggest that biophysical changes to plasma membrane properties, triggered by stressor-induced glucocorticoid elevation, act as a nonspecific stress response and may rapidly modulate acute stress-signaling pathways.</p> </div

    Glucocorticoid receptor signaling is involved in SOCS upregulation.

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    <p>The effect of cortisol and mifepristone either alone or in combination on SOCS-1 (A) and SOCS-2 (B) mRNA abundance in rainbow trout liver. Liver slices were incubated with control media or media containing cortisol (100 ng/ml), mifepristone (MP; 1000 ng/mL; Sigma) or a combination of mifepristone and cortisol for 24 h. Values are plotted as % control and show mean ± S.E.M (n = 6 fish livers); different upper case letters denote significant treatment effects (One way repeated measures ANOVA, p < 0.05).</p

    Pre-exposure to cortisol suppresses LPS signaling.

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    <p>Cortisol modulates LPS-induced IL-6 (A) but not IL-8 (B) mRNA abundance in rainbow trout liver. This is paralleled by an increase in SOCS-2 expression with cortisol exposure (C). Liver slices were pre-incubated with control media or media containing cortisol (100ng/ml) for 24 h after which they were incubated with control media or media containing LPS (30μg/ml) for 6 h. Values are plotted as % control and shown as mean ± S.E.M (n = 7 fish livers); different lower case letters denote significant treatment effects and interactions; * denotes overall cortisol effects (two way repeated measures ANOVA, p < 0.05).</p

    Protein spots identified by tandem mass spectrometric analysis and MASCOT database searching.

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    <p>Spot IDs correspond to labeled spots in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066119#pone-0066119-g001" target="_blank">Figure 1</a>.</p><p>p-values from the 1-way RMANOVA and False Discovery Rate (FDR) for each protein are shown. Fold changes are relative change in protein spot volume from pre-hibernation to hibernation.</p

    Interactome networks of select pathways identified as GR responsive by Ingenuity Pathway Analysis software.

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    <p>The IPA software organized and classified genes to identify important networks that were modulated by GR knockdown. These interactome networks detail the regulatory connections between genes as detailed by the connective arrows. The most strongly affected process was nervous system development at both 24 hpf (A) and 36 hpf (B). Other interesting pathways are DNA replication and energy production at 24 hpf (C), cardiovascular development at 36 hpf (D) and developmental disorders at 36 hpf (E). These final 3 pathways each involve a gene that were quantified by qPCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080726#pone-0080726-g004" target="_blank">Figure 4</a>): <i>f5</i> (C, E), and <i>pomca</i> (D). Single-way arrows indicate one gene regulating another, two-sided arrows indicate co-regulation, looped arrows indicate self-regulation. The shape of each member of the network indicates its cellular location (according to IPA software classifications): Extracellular (diamond); plasma membrane (hexagon); cytosol (square); nucleus (circle); unknown (triangle). The color of each member of the network indicates its mean fold change range: >2 (dark green); 1-2 (light green); unchanged (grey); 0.5-1 (light red), <0.5 (dark red).</p

    Whole-body cortisol content.

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    <p>Whole-body cortisol content in adult fathead minnows sampled in the lab before (0 min) and after a standardized stressor (30, 60, 240 min). For each boxplot, the line within the box represents the median whole-body cortisol concentration (n = 11–12). The box ranges from the first to the third quartile (interquartile range), the whiskers extend to ±1.5 times the interquartile range, and values beyond this are denoted as black dots. The asterisks denote a significant difference between stressed fish and unstressed fish (post-hoc Dunnett’s test; p<0.05).</p

    Impact of bisphenol A (BPA) on embryo development.

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    <p>Phenotypic changes post-hatch in trout embryos (65 dpf) treated with BPA. Rainbow trout oocytes were acutely exposed to either low (30 µg. ml<sup>−1</sup>) or high (100 µg.ml<sup>−1</sup>) BPA for 3 h, fertilized with untreated sperm, water hardened and maintained at 8.5°C. Image clearly shows morphological differences, including smaller size and the presence of yolk (arrow shown) in the BPA groups compared to the control.</p
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