 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 28, 2016 |
| Title |
blastocysts IVM BASAL vs IVM HIGH COMBI_rep4 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
blastocysts BASAL IVM
|
| Organism |
Bos taurus |
| Characteristics |
tissue: day 7.5 embryo
nefa concentration: BASAL
nefa exposure stage: in vitro matured oocyte
|
| 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.
|
| |
|
| Channel 2 |
| Source name |
blastocysts HIGH COMBI IVM
|
| Organism |
Bos taurus |
| Characteristics |
tissue: day 7.5 embryo
nefa concentration: HIGH COMBI
nefa exposure stage: in vitro matured oocyte
|
| 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.
|
| |
|
| |
| 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 IVM MET repl4
|
| 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 |
| GSM2218677_slide_88_area_A.txt.gz |
23.2 Mb |
(ftp)(http) |
TXT |
| Processed data are available on Series record |
|
|
|
|
 |
