Michael T. Cairns

Use of microarray technology to assess the time course of liver stress response after confinement exposure in gilthead sea bream (Sparus aurata L.)

BackgroundSelection programs for growth and stress traits in cultured fish are fundamental to the improvement of aquaculture production. The gilthead sea bream (Sparus aurata) is the main aquacultured species in the Mediterranean area and there is considerable interest in the genetic improvement of this species. With the aim of increasing the genomic resources in gilthead sea bream and identifying genes and mechanisms underlying the physiology of the stress response, we developed a cDNA microarray for gilthead sea bream that is enriched by suppression substractive hybridization with stress and immunorelevant genes. This microarray is used to analyze the dynamics of gilthead sea bream liver expression profile after confinement exposure.ResultsGroups of confined and control juvenile fish were sampled at 6, 24, 72 and 120 h post exposure. GeneSpring analyses identified 202 annotated genes that appeared differentially expressed at least at one sampling time (P < 0.05). Gene expression results were validated by quantitative PCR of 10 target genes, and K-means clustering of differently expressed genes identified four major temporal gene expression profiles. Set 1 encompassed a rapid metabolic readjustment with enhanced uptake and intracellular transport of fatty acids as metabolic fuels. Set 2 was associated with a wide variety of tissue repair and remodeling processes that were mostly mediated by the stress response of the endoplasmic reticulum (ER). Sets 3 and 4 encompassed the re-establishment of cellular homeostasis with increased intracellular trafficking and scavenging of reactive oxygen species (ROS), accompanied by a bidirectional regulation of the immune system and a general decline of ROS production.ConclusionsCollectively, these findings show the complex nature of the adaptive stress response with a clear indication that the ER is an important control point for homeostatic adjustments. The study also identifies metabolic pathways which could be analyzed in greater detail to provide new insights regarding the transcriptional regulation of the stress response in fish.

Dietary vegetable oils do not alter the intestine transcriptome of gilthead sea bream (Sparus aurata), but modulate the transcriptomic response to infection with Enteromyxum leei

BackgroundStudies conducted with gilthead sea bream (Sparus aurata L.) have determined the maximum dietary replacement of fish meal and oil without compromising growth or product quality. The present study aimed to analyze the effect of the nutritional background on fish health and fish fed plant protein-based diets with fish oil (FO diet) or a blend of vegetable oils (66VO diet) were exposed for 102 days to the intestinal myxosporean parasite Enteromyxum leei, and the intestine transcriptome was analyzed with a customized oligo-microarray of 7,500 annotated genes.ResultsInfection prevalence was high and similar in the two diet groups, but the outcome of the disease was more pronounced in fish fed the 66VO diet. No differences were found in the transcriptome of both diet control groups, whereas the number of differentially expressed genes in infected groups was considerable. K-means clustering of these differentially expressed genes identified four expression patterns that reflected the progression of the disease with the magnitude of the fold-change being higher in infected 66VO fish. A positive correlation was found between the time of infection and the magnitude of the transcriptional change within the 66VO group, being higher in early infected animals. Within this diet group, a strong up-regulation of many components of the immune specific response was evidenced, whereas other genes related to complement response and xenobiotic metabolism were down-regulated.ConclusionsThe high replacement of fish oil by vegetable oils in practical fish feeds did not modify the intestine transcriptome of gilthead sea bream, but important changes were apparent when fish were exposed to the myxosporean E. leei. The detected changes were mostly a consequence rather than a cause of the different disease progression in the two diet groups. Hence, the developed microarray constitutes an excellent diagnostic tool to address changes associated with the action of intestinal pathogens, but lacks a prognostic value to predict in advance the different susceptibility of growing fish to the current pathogen.

Molecular profiling of the gilthead sea bream (Sparus aurata L.) response to chronic exposure to the myxosporean parasite Enteromyxum leei

The aim of the present work was to investigate the transcriptome response of gilthead sea bream (Sparus aurata) after challenge with the myxosporean Enteromyxum leei, a wide-spread enteric parasite causing heavy economic losses in Mediterranean sparid farms. This parasite causes severe desquamative enteritis which usually leads to death of the fish, and there are no preventative or curative treatments for this enteromyxosis. After 113 days of exposure to parasite-contaminated effluent, fish were classified into three cohorts: control fish not exposed to parasite, those that were exposed and infected, and those that were exposed but not infected. In order to detect target genes that may be candidates for infective status or resistance, a cDNA microarray containing 18,490 cDNA clones enriched in genes differentially expressed after infection was hybridised with head kidney and intestine samples. In infected fish, 371 and 373 genes were differentially regulated at the >1.5-fold level in intestine and head kidney respectively, whereas in non-infected fish 175 and 501 genes were differentially regulated in these tissues, respectively. A global marked gene down-regulation was evident in infected fish, mainly in genes involved in the immune and acute phase response particularly complement and mannose binding lectin. Microarray analysis demonstrated a complex interplay between host and/or parasite derived proteases and protease inhibitors, apoptosis, cell proliferation and antioxidant defence genes in exposed fish. In the head kidney of non-infected fish a marked depression of genes involved in the acute phase response was evident. By contrast, in the intestine of non-infected fish, interferon-stimulated and MHC class II genes involved in antigen processing and presentation were up-regulated, possibly indicating that an active immune response at the local level is important to avoid infection with or proliferation of the parasite.

Acute phase gene expression in rainbow trout (Oncorhynchus mykiss) after exposure to a confinement stressor: A comparison of pooled and individual data

This study set out to investigate whether differential expression of genes for acute phase proteins in rainbow trout (Oncorhynchus mykiss) could be induced by confinement stress, a non-invasive method of activating the neuroendocrine stress response. In addition, a second objective was to assess the variation in gene expression between individual fish within the population of stressed fish in an attempt to identify APP genes having uniform and consistent changes in expression during stress. The liver was chosen for this investigation as it is the primary site of acute phase protein synthesis. Relative expression of the eight genes including transferrin, fibrinogen-like protein 2 (flp2), alpha-1-anti-proteinase-like protein (alpha1-antiprot), leukocyte cell-derived chemotaxin 2 (LECT2), pentraxin, serum amyloid A (SAA), haptoglobin (Hp), and differentially regulated trout protein 1 (DRTP1) was analysed by quantitative real-time PCR (qPCR) over 5 experimental time points spanning the course of a week. The results showed that the expression of three genes, SAA, haptoglobin and DRTP1, were most altered as a result of exposure to confinement stress. A correlation was identified between the expression of haptoglobin and DRTP1. Gene expression analyses in individual fish found that the transcript levels of haptoglobin and DRTP1 genes varied much less between individuals than was the case for SAA. The increase of haptoglobin and DRTP1 gene expression and its uniformity in response to stress make these genes potential biomarkers for stress in trout.

Effect of systemic progesterone concentration on the expression of progesterone-responsive genes in the bovine endometrium during the early luteal phase

Increasing evidence indicates an association between the concentration of systemic progesterone during the early luteal phase of the oestrous cycle and embryo survival rate in cattle. We examined the relationship between the concentration of systemic progesterone on Days 4 to 8 post-ovulation and expression of progesterone receptor (PGR), oestrogen receptor +/- (ESR1) and retinol-binding protein (RBP) mRNA in the bovine endometrium. Heifers were blood sampled from the day of ovulation (Day 0) to Day 8 post-ovulation. On Day 4, animals were divided into low progesterone control (LC) and high progesterone control (HC) groups based on their plasma progesterone concentrations. Half of each group was supplemented with exogenous progesterone resulting in two further groups, low progesterone supplemented (LS) and high progesterone supplemented (HS). Endometrial tissues were recovered from all groups on Day 6 or Day 8 and gene expression was analysed following Northern blotting. Increasing progesterone concentrations were associated with decreased PGR and ESR1 expression. Duration-dependent effects of progesterone supplementation on ESR1 were evident and there was an effect of systemic progesterone concentrations between Day 0 and Day 4 on the expression of RBP at Days 6 and 8. Such progesterone-responsive changes in uterine gene expression are likely to affect embryo development.

Functional Genomics of Stress Responses in Fish

Our understanding of the mechanisms underlying stress responses in fish remains fragmentary. However, new insights into these mechanisms and their biological significance have been provided by investigation at the transcriptional level. Microarray technology has allowed the unbiased analysis of the transcriptome, providing a potentially system-wide overview of stress responses. In this review, we present recently published transcriptomic studies on stress responses in fish exposed to a range of environmental, xenobiotic, social, and aquacultural stressors. Overall, these studies highlight the complexity of transcript patterns, have identified new genes whose expression is significantly modified after exposure to stressors, and have revealed both common and tissue-specific expression signatures. Some shortcomings can be identified, including lack of information on the longer-term compensatory or adaptive phases of the stress response, limitations on gene annotation, and the use of pooled mRNA preparations, which masks variation between individuals. Nonetheless, although the functional genomic analysis of stress responses in fish is still in its infancy, rapid growth in the number of studies and continued advances in technology and database content will inevitably lead to a fuller understanding of the processes involved and to the identification of novel stress indicators with diagnostic or predictive value.

Functional analysis of muscle gene expression profiles associated with tenderness and intramuscular fat content in pork

Warner-Bratzler shear force (WBSF) and % intramuscular fat content (IMF) are objective meat quality measurements that are significantly correlated with aspects of palatability such as tenderness, flavour and juiciness. Using cDNA microarrays, Musculus longissimus transcriptomic profiles at slaughter were compared in samples displaying lower or higher IMF (n=8) and WBSF values on day 1 post mortem (n=8). 101 identified genes were differentially expressed in relation to WBSF, while 160 genes were associated with differences in IMF. Reduced expression of protein synthesis genes and enhanced expression of genes involved in protein degradation were associated with lower WBSF values on day 1. Pathways including oxidative phosphorylation and the citrate cycle were significantly associated with higher IMF. Many lipid oxidation and fatty acid metabolism pathway genes were down-regulated in high IMF tissue, suggesting a suppression of fatty acid turnover in muscle with higher fat content. Identified genes provide targets for the discovery of novel genetic variation influential on pork palatability.

Analysis of stress-induced hepatic gene expression in rainbow trout (Oncorhynchus mykiss) selected for high- and low-responsiveness to stress

The production and welfare of intensively reared fish would be improved by reducing stress responsiveness. One approach to achieving this goal is selective breeding utilising stress-responsive genes as direct genetic markers of the desirable trait. As a first step in this process, microarray analysis has been carried out on liver tissues of rainbow trout selectively bred for high (HR) or low (LR) responsiveness to a stressor. Microarray hybridizations provided gene expression profiles for pooled samples of fish confined for 6 h, 24 h and 168 h and for individual fish (168 h only). 161 genes were shown to be differentially regulated in HR and LR fish during confinement exposure and eight of these gene expression profiles were validated by quantitative PCR. Genes of particular interest included intelectin-2 precursor which showed greater than 100-fold higher expression in HR fish compared to LR fish irrespective of whether the fish were confined or not; interferon inducible transmembrane protein 3 which was differentially stress-induced between the two lines; and hepatic pro-opiomelanocortin B (POMC B) which was upregulated during stress in HR fish but downregulated in LR fish. All these offer potential as direct markers of low stress responsiveness in a marker-assisted selection scheme.

Effects of combined progesterone and 17β-estradiol treatment on the transcriptome of cultured human myometrial smooth muscle cells

A transcriptomic analysis of cultured human uterine smooth muscle cells (hUtSMCs) was performed to examine gene expression profiles in smooth muscle in an environment containing the two major steroid hormones that regulate the human myometrium in physiological states associated with estrous, pregnancy, labor, and pathophysiological states such as leiomyoma and endometrial cancer. hUtSMCs were treated with progesterone (P4) and 17β-estradiol (E2) individually and in combination, in the presence and absence of RU486 (mifepristone). Transcription of many genes was modulated in the presence of P4 or E2 alone, but almost six times more genes were transcriptionally modulated in the presence of the P4/E2 hormone combination. In total 796 annotated genes were significantly differentially expressed in the presence of both P4 and E2 relative to their expression in untreated cells. Functional withdrawal of P4 by addition of RU486 effectively reversed almost all transcriptional changes caused by P4/E2 treatment. Gene ontology analysis of differentially expressed genes revealed a strong association between P4/E2 treatment and downregulated expression of genes involved in cell communication, signal transduction, channel activity, inflammatory response, and differentiation. Upregulated processes included cell survival, gene transcription, steroid hormone biosynthesis, muscle development, insulin receptor signaling, and cell growth.

Transcriptomic effects of estradiol treatment on cultured human uterine smooth muscle cells.

Contractility of the myometrial smooth muscle cells during the estrous cycle and pregnancy is modulated by estrogen but the temporal expression of estrogen (relative to progesterone) and the type of contraction involved are distinctly different in pregnancy and estrous. This in vitro cell culture study investigated the global gene expression profile of human uterine smooth muscle cells (hUtSMCs) following 17β-estradiol (E2) treatment. In response to E2 treatment 540 genes, many of which have not been previously described as estrogen responsive, were identified as significantly differentially expressed. These genes are involved in biological processes that include muscle contraction, cell migration and adhesion, apoptosis and phosphorylation. Evidence from this study suggests that 17β-estradiol may have effects that are contrary to an overall contraction phenotype. The hUtSMC in vitro culture system is a useful model to investigate steroid effects on smooth muscle cells and may provide additional clues as to how smooth muscle cells behave in vivo.