Transcriptomic response of skeletal muscle to lipopolysaccharide in the gilthead seabream (Sparus aurata)
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ABSTRACT:
ORGANISM(S): Sparus aurata
SUBMITTER: Josep Planas
PROVIDER: E-GEOD-36339 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Training at sustainable swimming speeds can produce changes in fish skeletal muscle that are important for aquaculture due to their growth-potentiating effects. Such changes may be even more relevant when fish are fed diets containing an increasing proportion of carbohydrates as an energy source. We evaluated the effects of moderate-intensity sustained swimming on the transcriptomic response of red and white muscle in rainbow trout fed a carbohydrate-rich diet using microarray and qPCR. Analysis of the red and white muscle transcriptome revealed significant changes in the expression of a large number of genes (395 and 597, respectively), with a total of 218 differentially expressed genes (DEGs) common for both muscles. A large number of the genes involved in glucose use and energy generation, contraction, development, synthesis and catabolism of proteins were up-regulated in red and white muscle. Additionally, DEGs in both muscles were involved in processes of defense response and apoptosis. Skeletal muscle contraction activates a transcriptional program required for the successful adaptation of both muscles to the changing demands imposed by swimming conditions. Future studies should further clarify the mechanisms involved in the adaptation of both tissues to exercise and assess possible benefits of such conditions for cultured fish. Total RNA from pooled red and white skeletal muscle samples of individual rainbow trout from each group (resting fish, n=8; swimming fish, n=8) was labeled with Cy3-dUTP and Cy5-dUTP (GE Healthcare, Barcelona, Spain). We used a dye swap experimental design and each cDNA from a pooled RNA sample was hybridized to two microarrays. For the first slide, cDNAs from resting and swimming fish were labeled with Cy5 and Cy3, respectively, and for the second array dye assignment was reversed. Therefore, samples from individual fish within each group were pooled and expression values shown represent the means of 6 M-CM-^W 2 = 12 technical replicates. A total of four slides were used in this study
2014-05-01 | E-GEOD-47141 | biostudies-arrayexpress
Project description:Metabolic processes and sexual maturation closely interact during the long-distance reproductive migration of many fish species to their spawning grounds. In the present study, we have for the first time used exercise experimentally to investigate the effects on sexual maturation in rainbow trout. Pubertal autumn-spawning seawater-raised female rainbow trout Oncorhynchus mykiss (n=26; 50-cm, 1.5-kg) were rested or swum at a near optimal speed of 0.75 body-lengths per second in a 6,000 L swim-flume under natural reproductive conditions (16 °C fresh-water, starvation, 8h-light:16h-dark photoperiod). Fish were sampled after arrival and subsequently after 10 days (resting or swimming 307 km) and 20 days (resting or swimming 636 km). Ovarian development was significantly reduced in the swimmers. Analysis of the expression of key factors in the reproductive axis included pituitary kiss1-receptor, lh and fsh and ovarian lh-receptor, fsh-receptor, aromatase and vitellogenin-receptor (vtgr). Swimmers had lower pituitary lh and ovarian vtgr expression than resters. Furthermore, the number of late vitellogenic oocytes was lower in swimmers than in resters, probably resulting from the lower vtgr expression, and vitellogenin plasma levels were higher. Therefore, swimming exercise suppresses oocyte development possibly by inhibiting vitellogenin uptake. Transcriptomic changes that occurred in the ovary of exercised fish were investigated using a salmonid cDNA microarray platform. Protein biosynthesis and energy provision were among the sixteen functional categories that were all down-regulated in the ovary. Down-regulation of the transcriptomic response in the ovary illustrates the priority of energy reallocation and will save energy to fuel exercise. A swimming-induced ovarian developmental suppression at the start of vitellogenesis during long-term reproductive migration may be a strategy to avoid precocious muscle atrophy. In order to simulate the natural reproductive conditions of anadromous salmonids, experiments were performed with sea water-raised rainbow trout, Oncorhynchus mykiss. Resters and swimmers were starved during the experiment, seawater was replaced by fresh water and photoperiod was changed. These changes were expected to affect reproductive parameters but as âbackgroundâ effect because both resters as swimmers were experiencing these conditions at the same time in the same set-up. Any additional effects in swimming fish would thus be caused by swimming only. Pubertal autumn spawning rainbow trout (n=26; ~50 cm, ~1.5 kg) were purchased from a Danish exporter (Frederiksvaerk Aleexport) where they had been raised for 2 years in freshwater followed by 4 months in sea water cages at 10 â°. They were transferred by truck within 16 h to Spakenburg (Spakenburg Paling, The Netherlands) and subsequently in the same water in a similar holding tank to the swim-flume at Leiden University (The Netherlands). The next day, 5 randomly chosen fish were sampled as âstartâ-group while others were randomly divided into a ârestâ-group (n=10) and a âswimâ-group (n=11). During the following 4 days, the brackish 10 â° water was stepwise replaced by freshwater at 16 °C and photoperiod was changed from 16L:8D to 8L:16D. Fish were then acclimatized for two days to their new conditions. Subsequently, swimmers were first swum at a speed of 0.33 body-lengths per second (BL/s) which was increased the next day to the final, near optimal speed of 0.75 BL/s (0.34 m/s or 29.4 km/day). Fish were sampled after 10 days (5 resters and 5 swimmers) and 20 days (5 resters and 6 swimmers). At sampling, fish were anesthetized using oil of cloves that was commercially purchased from a drugstore (diluted 1:10 in ethanol and used at a dosage of 1.5 ml/l). Fish were euthanized by decapitation and dissected for the ovary which was flash frozen in liquid nitrogen. RNA was isolated from pituitary and ovary samples according to the TRIZOL reagent protocol by Invitrogen (Baro, Spain). Isolated RNA was DNAse treated using RQ1 DNAse Promega (Madison, USA) and reverse transcribed using Superscript IIITM (Baro, Spain) according to the manufacturerâs protocol. Microarray analyses were performed on ovarian tissue for 20-days-swimmers vs. 20-days-resters. A rainbow trout cDNA microarray platform was used that was previously validated and described by Koskinen et al., 2004ab, Krasnov et al., 2005, MacKenzie et al., 2006ab, 2008 and Djordjevic et al., 2009, and deposited in GEO under accession number GPL1212, the original cDNA microarray (SFA2.0 chip), and GPL6154; the updated 1.8 K platform. This platform compromises 1818 genes representing 366 functional categories. Total RNA was extracted from flash frozen samples from experimental fish of the 20-days-swim group (n=5) and 20-days-rest group (n=5) like described before. Total RNA corresponding to pooled samples from the swim or rest groups was labeled with Cye3 and Cye5-dCTP (GE Healthcare, Barcelona, Spain) using SuperScript III reverse transcriptase (Invitrogen, Baro, Spain) and oligo(dT) primer. In our experience, reverse labeling combined with multiple replication of spots provide robust normalization and high statistical power of analyses. cDNA was purified with MicroconYM30 (Millipore). The slides were pretreated with 1% BSA(fraction V), 20 à SSC, 10% SDS (30 min at 50°C) and washed with 2 à SSC (3 min) and 0.2 à SSC (3 min) and hybridized overnight in cocktail containing 50 à Denhardt's, 20 à SSC, 10% SDS, 10μg/μl polyadenylate and 10 μg/μl yeast tRNA. All chemicals were from Sigma-Aldrich. Scanning was performed with ScanArray 5000 and images were processed with QuantArray (GSI Luminomics). The measurements in spots were filtered by criteria I/B ⥠3 and (I-B)/(SI + SB) ⥠0.6, where I and B are the mean signal and background intensities and SI, SB are the standard deviations. After subtraction of mean background, LOWESS normalization was performed. To assess differential expression of genes, the normalized log intensity ratios (expression ratio) were analyzed with Student's t-test (P <0.05). Validation of the microarray was performed by Q-PCR of 6 differentially expressed genes (DEGs) as described above. The used primers were either published (Alpha-globin I-1: Donate Jimeno, 2008) or designed, again by using the Genamics software.
2010-06-24 | E-GEOD-20131 | biostudies-arrayexpress
Project description:The aquaculture industry has confronted severe economic losses due to infectious diseases in the last years. Piscirickettsiosis or Salmonid Rickettsial Septicaemia (SRS) is the bacterial disease caused by Piscirickettsia salmonis. This Gram-negative, non-motile, cellular pathogen has the ability to infect, survive, replicate, and propagate in salmonid monocytes/macrophages generating a systemic infection characterized by the colonization of several organs including kidney, liver, spleen, intestine, brain, ovary and gills. In this study, we attempted to determine whether global gene expression differences can be detected in different genetic groups of Atlantic salmon as a result of Piscirickettsia salmonis infection. Moreover, we sought to characterize the fish transcriptional response in order to reveal the mechanisms that might confer resistance in Atlantic salmon to an infection with Piscirickettsia salmonis. In doing so, after challenging with Piscirickettsia salmonis, we selected the families with the highest (HS) and the lowest (LS) recorded susceptibility for gene expression analysis using 32K cGRASP microarrays. Our results revealed in LS families expression changes are linked to iron depletion, as well as, low contents of iron in kidney cells and low bacterial load, indicated that the iron-withholding strategy of innate immunity is part of the mechanism of resistance against Piscirickettsia salmonis. This information contributes to elucidate the underlying mechanisms of resistance to Piscirickettsia salmonis infection in Atlantic salmon and to identify new candidate genes for selective breeding programmes. Forty full-sibling families of Atlantic salmon (Salmo salar) were infected by intraperitoneal injection with 0.2 mL Piscirickettsia salmonis (PS889, isolated from Oncorhynchus kisutch, 1M-CM-^W104 PFU/mL). After forty days, the fishes were harvested and the cumulative mortality (dead fish / total fish) for each family was calculated. For the second challenge, the six families with the highest cumulative mortality levels were considered of relatively high susceptibility (HS) and the six families with the lowest cumulative mortality levels were considered of relatively low susceptibility (LS) to the infection. Five control and five infected fish from three HS and three LS families were analyzed. For each HS and LS family, pools of RNA from control and infected fish were prepared separately and were reverse transcribed. Four slides were for each used family hybridized including two dye-swaped slides. Labeled samples were hybridized on a 32K cDNA microarray, developed at the Consortium for Genomics Research on All Salmonids Project (cGRASP), GEO accession number: GPL8904.
2013-01-04 | E-GEOD-43255 | biostudies-arrayexpress
Project description:Hibernation is energy saving adaptation involving suppression of activity to survive in highly seasonal environments. Immobility and disuse generate muscle loss in most mammalian species. In contrast to other mammals, bears and ground squirrels demonstrate limited muscle atrophy over the physical inactivity of winter hibernation. This suggests that hibernating mammals have adaptive mechanisms to prevent disuse muscle atrophy. To identify common transcriptional program underlying molecular mechanisms preventing muscle loss, we conducted a large-scale gene expression screening in hind limb muscles comparing hibernating and summer active black bears and arctic ground squirrels by the use of custom 9,600 probe cDNA microarrays. The molecular pathway analysis showed an elevated proportion of overexpressed genes involved in all stages of protein biosynthesis and ribosome biogenesis in muscle of both species during hibernation that implies induction of translation at different hibernation states. The induction of protein biosynthesis likely contributes to attenuation of disuse muscle atrophy through prolonged periods of immobility and starvation. This adaptive mechanism allows hibernating mammals to maintain full musculoskeletal function and preserve mobility during and immediately after hibernation, thus promoting survival. The lack of directional changes in genes of protein catabolic pathways does not support the importance of metabolic suppression for preserving muscle mass during winter. Coordinated reduction of multiply genes involved in oxidation reduction and glucose metabolism detected in both species is consistent with metabolic suppression and lower energy demand in skeletal muscle during inactivity of hibernation. Black bears sampled during winter hibernation were compared with the animals sampled during summer. Muscle tissue were hybridized on a custom 12,800 cDNA probe nylon membrane microarray platform . Six hibernating and six summer active bears were studied in the experiment.
2014-10-16 | E-GEOD-62220 | biostudies-arrayexpress
Project description:Hibernation is energy saving adaptation involving suppression of activity to survive in highly seasonal environments. Immobility and disuse generate muscle loss in most mammalian species. In contrast to other mammals, bears and ground squirrels demonstrate limited muscle atrophy over the physical inactivity of winter hibernation. This suggests that hibernating mammals have adaptive mechanisms to prevent disuse muscle atrophy. To identify common transcriptional program underlying molecular mechanisms preventing muscle loss, we conducted a large-scale gene expression screening in hind limb muscles comparing hibernating and summer active black bears and arctic ground squirrels by the use of custom 9,600 probe cDNA microarrays. The molecular pathway analysis showed an elevated proportion of overexpressed genes involved in all stages of protein biosynthesis and ribosome biogenesis in muscle of both species during hibernation that implies induction of translation at different hibernation states. The induction of protein biosynthesis likely contributes to attenuation of disuse muscle atrophy through prolonged periods of immobility and starvation. This adaptive mechanism allows hibernating mammals to maintain full musculoskeletal function and preserve mobility during and immediately after hibernation, thus promoting survival. The lack of directional changes in genes of protein catabolic pathways does not support the importance of metabolic suppression for preserving muscle mass during winter. Coordinated reduction of multiply genes involved in oxidation reduction and glucose metabolism detected in both species is consistent with metabolic suppression and lower energy demand in skeletal muscle during inactivity of hibernation. Arctic ground squirrels sampled during winter hibernation were compared with the animals sampled during summer. Muscle was hybridized on a custom 9,600 probes nylon membrane microarray platform. Ten in late torpor, four in early arousal, then in late arousal were studied in experiments.
2014-10-16 | E-GEOD-62377 | biostudies-arrayexpress
Project description:A high-density oligonucleotide microarray for channel catfish (Ictalurus punctatus) was designed and produced with Maskless Array Synthesizer technology (MAS). The microarray contained ~379,652 24-mer oligonucleotides covering ~18,999 catfish unique sequences. The global expression profiling of the catfish spleens stimulated by lipopolysaccharide (LPS) for 2h, 4h, 8h and 24h was investigated with the microarray. In the spleen samples, 409 genes were identified to be induced or repressed greater than 2-fold by LPS treatment. Self-organizing maps (SOM) clustering analysis was applied to interpret gene expression patterns, and 84 of these genes were clustered into six expression patterns. Real-time RT-PCR was used to verify the microarray results for 9 selected genes representing different expression levels. The results from real-time RT-PCR were positively correlated (R^2 = 0.87) with the results from the microarray. Channel catfish were injected with LPS or PBS carrier. Fish were killed by anesthesia overdose at 2, 4, 8, or 24h post-injection and total RNA was isolated from spleen. The experiment contained two replicate fish per time point. Relative signal intensities for each feature were generated using the Robust Multi-Array Average algorithm and log2 transformed. Transformed data were then processed using quantile normalization, and the average and std. error for each gene were calculated from the 10 perfectly matched oligos only. After detransformation, the ratio (fold change) was calculated using PBS treated samples (control) as the baseline against LPS-treated samples.
2005-11-30 | E-GEOD-3261 | biostudies-arrayexpress
Project description:Farmed fish are unavoidably exposed to husbandry-related stressors which can result in outbreaks of disease, reduced reproductive success and poor growth. Biomarkers are needed for the establishment of selective breeding approaches for the production of stress resistant fish. In this context, microarray analysis has been carried out on liver tissues of two genetic lines of rainbow trout, high responders (HR) and low responders (LR), after confinement for various time periods. Microarray hybridisations provided gene expression profiles for 10 individual fish (5 of the HR line and 5 of the LR line) after 168h of confinment stress. Keywords : stress response, individual variation, fixed time point, comparison of two lines All 10 samples were hybridised against a common reference source that contained equal amounts of all 10 samples. Each sample was hybridised twice against this reference using a dye-swap setup.
2010-11-24 | E-GEOD-25440 | biostudies-arrayexpress
Project description:The study investigated the acute and simultaneous response of the mammary and liver transcriptome to an intra-mammary lipopolysaccharide (LPS) challenge in early-lactating cows, and its consequences on metabolic biomarkers and liver composition. At 7 days of lactation, 7 cows served as controls (CTR) and 7 cows (LPS) received an intra-mammary E. coli LPS challenge. The mammary and liver tissues were sampled at 2.5 h from challenge for transcriptomic profiling. Liver composition was evaluated at 2.5 h and 7 d after challenge and blood biomarkers were analized at -2, 3, 7 and 14 d from challenge. In mammary tissue, the LPS challenge resulted in 189 differentially expressed gene (DEG), with 20 downregulated and 169 upregulated, while in the liver were found 107 DEG with 42 downregulated and 65 upregulated in LPS vs CTR. In the udder, the Dynamic Impact Approach (DIA) highlighted the activation of NOD-like receptor signaling, Toll-like receptor signaling, RIG-I-like receptor signaling and Apoptosis pathways, while in the liver were inhibited the Fatty acid elongation in mitochondria and activated the p53 signaling pathway. The LPS induced the alteration of liver lipid metabolism (rise of total lipid and triglyceride concentration), a systemic inflammation (rise of blood ceruloplasmin and bilirubin) and an increase of body fat mobilization. In cows subjected to intra-mammary LPS, the mammary gland responds activating mechanisms of pathogen recognition. In the liver the response likely depends by mediators coming from udder and affects the liver functionality and mainly the fatty acid metabolism. Fourteen Holstein cows entering their second or greater lactation were used. Cows were housed in a ventilated tie-stall barn and were fed a common lactation diet (Net energy of lactation = 1.69 Mcal/kg DM) as a total mixed ration once daily (0600 h) and milked twice daily (0400 and 1600 h). A bovine oligonucleotide (70-mers) microarray with >13,000 annotated sequences developed at the University of Illinois, was used for transcript profiling. Details on the development, annotation, and use of this microarray have been reported previously by Loor et al., 2007 (http://physiolgenomics.physiology.org/content/32/1/105.abstract). Methods for microarray hybridization and scanning were as reported by Loor et al. (2007). Briefly, slides were hydrated, dried, and placed in a UV Stratalinker 1800 (Stratagene, La Joya, CA) for ~5 min. Slides were washed with 0.2% SDS solution, rinsed with MilliQ (Millipore) H2O, and placed in warm prehybridization soln for 45 min at 42 C. The same amount of Cy3- or Cy5-labelled cDNA from mammary or liver and a reference standard RNA pool (made of different bovine tissues) were co-hybridized using a dye-swap design (i.e., two microarrays per sample). Slides were incubated for 48 h at 45 C prior to scanning. Criteria for evaluation of slide quality included: identification of number of spots with a minimum median signal intensity of 3 SD above background; keeping slides with a minimum of 20,000 spots with minimum median signal intensity of 3 SD above background in both Cy3 and Cy5 channels; and keeping slides with a minimum mean intensity of 400 relative fluorescent units in both Cy3 and Cy5 channels across the entire slide. Data from a total of 112 microarrays were normalized for dye and microarray effects (i.e., Lowess normalization and microarray centering) and used for statistical analysis. Data were analyzed using the Proc MIXED procedure of SAS (SAS, SAS Inst. Inc., Cary, NC). Fixed effects were treatment (LPS challenge, control (no infsuion)) and dye. Random effects included cow and microarray. Raw P values were adjusted using Benjamini and HochbergM-bM-^@M-^Ys false discovery rate (FDR). Differences in relative expression due to treatment were considered significant at an FDR-adjusted P < 0.05. For a more stringent characterization between the two treatments, a 1.5-fold difference in mRNA expression was set as threshold among differentially expressed genes.
2014-12-17 | E-GEOD-64225 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE25440: Individual variation in confinement response at 168h GSE25473: Time course study of confinement stress in HR and LR trout lines Refer to individual Series
2010-11-24 | E-GEOD-25584 | biostudies-arrayexpress
Project description:Farmed fish are unavoidably exposed to husbandry-related stressors which can result in outbreaks of disease, reduced reproductive success and poor growth. Biomarkers are needed for the establishment of selective breeding approaches for the production of stress resistant fish. In this context, microarray analysis has been carried out on liver tissues of two genetic lines of rainbow trout, high responders (HR) and low responders (LR), after confinement for various time periods. Microarray hybridisations provided gene expression profiles for pools of fish (stress: n=5; control: n=3) after 6h, 24h and 168h of confinement stress. Keywords : stress response, pools, time course, comparison of two lines. Samples were hybridised directly to each other in a parallel loop design. Each treatment (LC, LS, HC, HS) was hybidised in a loop of all 3 time points and each treatment loop was interconnected by hybridisations at a specific time. The publication will have this figure.
2010-11-24 | E-GEOD-25473 | biostudies-arrayexpress