Human embryos: Supernumerary human embryos were obtained from patients at the Boston IVF Clinic through informed consent with the approval of the Internal Review Board of Harvard University. Embryos thus obtained were cultured in a two-step culture system (Sage/Biopharma, CT) in micro-drops at 37? C and 5% CO2 under oil (Sage, CT) as described previously. Embryos from each stage were segregated into three groups. Each group was considered to be a biologic replicate: embryos within each replicate were pooled for further analysis. The blastocyst embryos were graded according to the Gardner scale. Mouse embryos: Briefly, female mice were impregnated, and embryos were flushed from the oviducts. Individual embryos were morphologically staged by light microscopy. Bovine embryos: Ovaries were washed and 2-6 mm follicles were aspirated. Only oocytes surrounded by several cell layers of dense cumulus were selected for culture. Oocytes were washed three times in TL-HEPES (0.22mM sodium pyruvate, 3mg/ml BSA, pH 7.4), and we placed maturation media that had been equilibrated in 5% carbon dioxide and air (39°C, high humidity). The maturation medium consisted of TC199 supplemented with 3ml of bovine LH and FSH (Sioux Biochemical), 0.25mM sodium pyruvate, and 10% fetal calf serum. After 20 hours of incubation in maturation media, oocytes were removed, washed in TL-HEPES and placed in groups of 10 oocytes each into 44 μl microdrops of IVF-Talp (Biowhittaker, Walkersburg, MD) supplemented with 0.22mM sodium pyruvate, and 6 mg/ml essentially fatty acid-free BSA. Live sperm were separated by centrifugation in a percoll gradient (Sigma, P1985). Eggs were fertilized by adding one million/ml of sperm, 2.0μg heparin (Sigma H-3125), pencillamine, hypotaurine and epinephrine (Sigma-P-4875, H-1384, E-4250) to each drop of fertilization media. Oocytes were removed from the fertilization media after 18-22 hours, washed in TL-HEPES, placed in a 1.5 ml tube, and vortexed for 2 min to remove cumulus cells. Twenty-five cumulus-free oocytes were placed in each 50μl drop of SOF culture medium, supplemented with 8 mg/ml of fatty acid-free BSA, nonessential and essential amino acids and pyruvate. The IVF conditions were optimized for each species. To minimize the influence of IVF conditions to cross-species comparison, we subsequently based the comparisons on either relative expression changes or co-expression patterns.
Extracted molecule
total RNA
Extraction protocol
Total RNA extraction was carried out using the mirVana RNA extraction kit according to the manufacturers protocols (Applied Biosciences/Ambion, Austin, TX ).
Label
biotin
Label protocol
RNA was prepared utilizing three rounds of amplification and hybridized to Affymetrix GeneChipâ Bovine Genome Expression Arrays (Affymetrix, Inc., Santa Clara, CA) utilizing Ambion’s MessageAmp TM II aRNA Amplification and MessageAmpTM II-Biotin Enhanced Kits (Applied Biosystems, Foster City, CA) according to the manufacturers’ instructions.
Hybridization protocol
Following a 16-hour hybridization the chip was washed and stained utilizing an Affymetrix GeneChipâ Fluidics Station 450 and Affymetrix GeneChipâ Command Console Software. Initial staining utilized streptavidin-conjugated phycoerythrin dye (Invitrogen Corporation, Carlsbad, CA). This stain was enhanced with biotinylated goat anti-streptavidin antibody (Vector Laboratories, Burlingame, CA), then counterstained with the conjugated phycoerythrin.
Scan protocol
The chip was scanned with an Affymetrix GeneChipâ Scanner model 3000 7G Plus. Files of the fluorescence intensities were utilized to generate corresponding probe expression levels utilizing Affymetrix Expression Console version 1.1, and quality control values were checked.
Description
Gene expression data from embryo at 16 cell stage
Data processing
The data were analyzed with dChip. Normalization was under default settings.
