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Sample GSM2218678 Query DataSets for GSM2218678
Status Public on Jun 28, 2016
Title blastocysts IVC BASAL vs IVC HIGH COMBI_rep1
Sample type genomic
 
Channel 1
Source name blastocysts BASAL IVC
Organism Bos taurus
Characteristics tissue: day 7.5 embryo
nefa concentration: BASAL
nefa exposure stage: in vitro cultured embryo
Treatment protocol In the IVM experiment, oocytes were exposed to the following conditions: 1) BASAL: physiological NEFA concentrations (72 μM total NEFA containing 28 μM stearic acid (SA), 23 μM palmitic acid (PA) and 21 μM oleic acid (OA)). 2) HIGH COMBI: a combination of elevated NEFA concentrations equivalent to those measured in the follicular fluid during severe lipolytic conditions (425 μM total NEFA, containing 75 μM SA, 150 μM PA and 200 μM OA). In the IVC experiment, embryos were exposed to the following conditions: 1) BASAL: physiological NEFA concentrations (72 μM total NEFA containing 28 μM SA, 23 μM PA, and 21 μM OA). 2) HIGH COMBI: a combination of elevated NEFA concentrations equivalent to those measured in serum under high lipolytic conditions (720 μM total NEFA, containing 280 μM SA, 230 μM PA, and 210 μM OA).
Growth protocol Grade I cumulus oocyte complexes (COCs) were matured in groups of 50-60 in 500 μl serum-free maturation medium containing TCM199 supplemented with fatty acid-free 0.75% BSA, 0.4 mM glutamine, 0.2 mM sodium pyruvate, 0.1 mM cysteamine, 50 mg/ml gentamycin and murine epidermal growth factor (mEGF, 20 ng/ml) for 24 h in humidified air with 5% CO2 at 38.5°C. In the IVM experiment, oocytes were randomly divided in equal groups between treatment-specific maturation media. In the IVC experiment, oocytes were matured in serum-free maturation medium as mentioned above. After IVM, COCs were co-incubated in groups of 100-120 with spermatozoa at a final concentration of 106/ml for 22 h at 38.5°C in 500 μl fertilization medium (containing 114 mM NaCl, 3.1 mM KCl, 0.3 mM Na2HPO4, 2.1 mM CaCl2-2H2O, 0.4 mM MgCl2-6H2O, 25 mM bicarbonate, 1 mM pyruvate, 36 mM lactate, 2 μL/ml phenol red, 6 mg/mL fatty acid-free BSA, 50 μg/mL gentamycin and 0.72U/mL heparin) in a humidified 5% CO2 incubator. Presumptive zygotes were cultured in groups of 25 ± 4. During the IVM experiment, the presumptive zygotes were cultured in 50 μl droplets of mSOF medium with a mineral oil overlay (modular incubator: 38.5 °C, 5% CO2, 5% O2 and 90% N2) until the day of analysis. During the IVC experiment, the presumptive zygotes were incubated in a reduced surface 96-well dish containing 75 µl medium without mineral oil overlay (modular incubator: 38.5°C, 5% CO2, 5% O2 and 90% N2). The mSOF medium contained 108 mM NaCl, 7.2 mM KCl, 1.2 mM KH2PO4, 0.8 mM MgSO4.7H2O, 0.6mM sodium lactate, 25 mM NaHCO3, 0.0266 mM phenol red, 0.73 mM sodium pyruvate, 1.78 mM CaCl2.2H2O, 0.34 mM trisodium citrate, 2.755 mM myoinositol, 3% v/v BME 50x, 1% v/v MEM 100x, 0.4 mM glutamine, 5% fetal bovine serum and 50 μg/mL gentamycin. For the IVC experiment, NEFAs were added to the mSOF medium at concentrations according to the treatment.
Extracted molecule genomic DNA
Extraction protocol Genomic DNA (gDNA) and total RNA were extracted from pools of 10 blastocysts (per treatment of each replicate) using the Allprep DNA/RNA micro kit (Qiagen) according to the manufacturer's protocol.
Label Cy3
Label protocol 2µg amplified gDNA was labelled using with either Cy-3 or Cy-5 dyes using ULS Fluorescent gDNA labelling kit (Kreatech Biotechnology) according to the manufacturer's protocol.
 
Channel 2
Source name blastocysts HIGH COMBI IVC
Organism Bos taurus
Characteristics tissue: day 7.5 embryo
nefa concentration: HIGH COMBI
nefa exposure stage: in vitro cultured embryo
Treatment protocol In the IVM experiment, oocytes were exposed to the following conditions: 1) BASAL: physiological NEFA concentrations (72 μM total NEFA containing 28 μM stearic acid (SA), 23 μM palmitic acid (PA) and 21 μM oleic acid (OA)). 2) HIGH COMBI: a combination of elevated NEFA concentrations equivalent to those measured in the follicular fluid during severe lipolytic conditions (425 μM total NEFA, containing 75 μM SA, 150 μM PA and 200 μM OA). In the IVC experiment, embryos were exposed to the following conditions: 1) BASAL: physiological NEFA concentrations (72 μM total NEFA containing 28 μM SA, 23 μM PA, and 21 μM OA). 2) HIGH COMBI: a combination of elevated NEFA concentrations equivalent to those measured in serum under high lipolytic conditions (720 μM total NEFA, containing 280 μM SA, 230 μM PA, and 210 μM OA).
Growth protocol Grade I cumulus oocyte complexes (COCs) were matured in groups of 50-60 in 500 μl serum-free maturation medium containing TCM199 supplemented with fatty acid-free 0.75% BSA, 0.4 mM glutamine, 0.2 mM sodium pyruvate, 0.1 mM cysteamine, 50 mg/ml gentamycin and murine epidermal growth factor (mEGF, 20 ng/ml) for 24 h in humidified air with 5% CO2 at 38.5°C. In the IVM experiment, oocytes were randomly divided in equal groups between treatment-specific maturation media. In the IVC experiment, oocytes were matured in serum-free maturation medium as mentioned above. After IVM, COCs were co-incubated in groups of 100-120 with spermatozoa at a final concentration of 106/ml for 22 h at 38.5°C in 500 μl fertilization medium (containing 114 mM NaCl, 3.1 mM KCl, 0.3 mM Na2HPO4, 2.1 mM CaCl2-2H2O, 0.4 mM MgCl2-6H2O, 25 mM bicarbonate, 1 mM pyruvate, 36 mM lactate, 2 μL/ml phenol red, 6 mg/mL fatty acid-free BSA, 50 μg/mL gentamycin and 0.72U/mL heparin) in a humidified 5% CO2 incubator. Presumptive zygotes were cultured in groups of 25 ± 4. During the IVM experiment, the presumptive zygotes were cultured in 50 μl droplets of mSOF medium with a mineral oil overlay (modular incubator: 38.5 °C, 5% CO2, 5% O2 and 90% N2) until the day of analysis. During the IVC experiment, the presumptive zygotes were incubated in a reduced surface 96-well dish containing 75 µl medium without mineral oil overlay (modular incubator: 38.5°C, 5% CO2, 5% O2 and 90% N2). The mSOF medium contained 108 mM NaCl, 7.2 mM KCl, 1.2 mM KH2PO4, 0.8 mM MgSO4.7H2O, 0.6mM sodium lactate, 25 mM NaHCO3, 0.0266 mM phenol red, 0.73 mM sodium pyruvate, 1.78 mM CaCl2.2H2O, 0.34 mM trisodium citrate, 2.755 mM myoinositol, 3% v/v BME 50x, 1% v/v MEM 100x, 0.4 mM glutamine, 5% fetal bovine serum and 50 μg/mL gentamycin. For the IVC experiment, NEFAs were added to the mSOF medium at concentrations according to the treatment.
Extracted molecule genomic DNA
Extraction protocol Genomic DNA (gDNA) and total RNA were extracted from pools of 10 blastocysts (per treatment of each replicate) using the Allprep DNA/RNA micro kit (Qiagen) according to the manufacturer's protocol.
Label Cy5
Label protocol 2µg amplified gDNA was labelled using with either Cy-3 or Cy-5 dyes using ULS Fluorescent gDNA labelling kit (Kreatech Biotechnology) according to the manufacturer's protocol.
 
 
Hybridization protocol one Cy3 labeled DNA sample and one Cy5 DNA sample were combined and hybridized to one area of a 2x444,000 Agilent EmbryoGene microarray slide. 
Scan protocol Slides were scanned using Agilent’s High-Resolution Scanner (Agilent Technologies, CA, USA).
Description NEFA IVC MET repl1
Data processing The Limma Bioconductor package was used to apply loess intra-array normalization followed by Quantile inter-array scale normalization to draw the intensities. Normalized intensities were then fitted to a linear model and Bayesian statistics of differential expression were obtained. Probes that exhibited a P-value < 0.05 and an absolute log2 fold-change of at least 1.5 were considered differentially methylated.
The file Normalized_data_all_data.txt contains the lowess normalized log2 ratio representing test/reference.
 
Submission date Jun 27, 2016
Last update date Jun 28, 2016
Contact name Karolien LJ Desmet
E-mail(s) [email protected]
Organization name University of Antwerp
Department Veterinary Sciences
Lab Gamete Research Centre
Street address Universiteitsplein 1
City Wilrijk
State/province Antwerpen
ZIP/Postal code 2610
Country Belgium
 
Platform ID GPL18384
Series (2)
GSE83766 Exposure of bovine oocytes and embryos to elevated non-esterified fatty acid concentrations: integration of epigenetic and transcriptomic signatures in resultant blastocysts [DNA methylation]
GSE83768 Exposure of bovine oocytes and embryos to elevated non-esterified fatty acid concentrations: integration of epigenetic and transcriptomic signatures in resultant blastocysts

Supplementary file Size Download File type/resource
GSM2218678_lame_19_area_A.txt.gz 23.0 Mb (ftp)(http) TXT
Processed data are available on Series record
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