At Day 5 after insemination, 5 µl of KSOM-BE2 or 5 µl of KSOM-BE2 containing 100 ng/ml recombinant bovine CSF2 (a gift from Novartis, Basle Switzerland) were added to each drop to achieve a final CSF2 concentration of 0 or 10 ng/ml. Transfer into recipients: Morula, blastocyst and expanded blastocyst stage conceptuses classified as Grade 1 were harvested on Day 7 after insemination were transfered to thirty-five multiparous lactating Holstein cows were used as recipients. A total of 4 replicates were completed between June and October 2009 with 5-11 recipients per replicate. For each replicate, eligible cows were synchronized for embryo transfer as described previously. Conceptus recovery: At Day 15 after expected ovulation, the ovary ipsilateral to the uterine horn that received the conceptus was examined using ultrasonography to confirm the presence of the corpus luteum. Cows that did not have a visible CL were not flushed. Conceptuses were recovered transcervically by flushing the uterine horn ipsilateral to the CL was flushed with Dulbecco’s phosphate-buffered saline (DPBS) with 1% (v/v) polyvinyl alcohol (PVA). The flushings recovered from the first 60 ml flush were centrifuged at 1000xg for 10 min and stored at -20oC for analysis of antiviral activity. Conceptuses were washed once in DPBS-PVA and the conceptus was dissected to produce a piece of tissue containing the embryonic disc and some nearby extraembryonic membranes (termed here as embryonic disc) and two pieces of extraembryonic membranes that were on either end of the embryonic disc. Tissues were immediately snap frozen separately in liquid nitrogen.
Growth protocol
Production of conceptuses: Cumulus oocyte complexes (COCs) from ovaries from a mixture of beef and dairy cattle were collected in Tissue Culture Medium-199 (TCM-199) with Hank’s salts without phenol red supplemented with 2% (v/v) bovine steer serum containing 2 U/ml heparin, 100 U/ml penicillin-G, 0.1 mg/ml streptomycin, and 1 mM glutamine. Oocytes were allowed to mature for 20-22 h in groups of 10 in 50 µl microdrops of TCM-199 with Earle’s salts supplemented with 10% (v/v) bovine steer serum, 2 µg/ml estradiol 17-µ, 20 µg/ml bovine follicle stimulating hormone, 22 µg/ml sodium pyruvate, 50 µg/ml gentamicin sulfate, and 1 mM glutamine. Matured oocytes were then washed in HEPES-TALP and transferred in groups of 50 to four-well plates containing 600 µL of IVF-TALP supplemented with 25 µL PHE (0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 µM epinephrine in 0.9% [w/v] NaCl), and fertilized with 30 µL Percoll-purified spermatozoa (~ 1x106 sperm cells). Embryos were cultured at 38.5oC in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 (v/v). At Day 5 after insemination, 5 µl of KSOM-BE2 or 5 µl of KSOM-BE2 containing 100 ng/ml recombinant bovine CSF2 (a gift from Novartis, Basle Switzerland) were added to each drop to achieve a final CSF2 concentration of 0 or 10 ng/ml. Transfer into recipients: Morula, blastocyst and expanded blastocyst stage conceptuses classified as Grade 1 were harvested on Day 7 after insemination were transfered to thirty-five multiparous lactating Holstein cows were used as recipients. A total of 4 replicates were completed between June and October 2009 with 5-11 recipients per replicate. For each replicate, eligible cows were synchronized for embryo transfer as described previously. Conceptus recovery: At Day 15 after expected ovulation, the ovary ipsilateral to the uterine horn that received the conceptus was examined using ultrasonography to confirm the presence of the corpus luteum. Cows that did not have a visible CL were not flushed. Conceptuses were recovered transcervically by flushing the uterine horn ipsilateral to the CL was flushed with Dulbecco’s phosphate-buffered saline (DPBS) with 1% (v/v) polyvinyl alcohol (PVA). The flushings recovered from the first 60 ml flush were centrifuged at 1000xg for 10 min and stored at -20oC for analysis of antiviral activity. Conceptuses were washed once in DPBS-PVA and the conceptus was dissected to produce a piece of tissue containing the embryonic disc and some nearby extraembryonic membranes (termed here as embryonic disc) and two pieces of extraembryonic membranes that were on either end of the embryonic disc. Tissues were immediately snap frozen separately in liquid nitrogen.
Extracted molecule
total RNA
Extraction protocol
Microarray analysis was performed using a subset of 8 samples of embryonic disc and 8 samples of extraembryonic membranes from filamentous conceptuses (4 per treatment). Total cellular RNA was extracted with the RNeasy Plus Micro kit (Qiagen-Inc) following manufacturer’s instructions. Concentration of the input RNA was determined by Nanodrop 2000 spectrophotometer and RNA integrity was determined by use of the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only samples that showed high RNA integrity (RIN > 7) were used for the microarray hybridization and quantitative PCR analysis. Extracted RNA was stored at -80oC until microarray analysis.
Label
Cy3
Label protocol
samples of RNA (1.5-3.0 pg/µl) were concentrated to 5 µl using Savant SpeedVac (Therno Scientific) at low heat and amplified into ss-cDNA using the NuGEN WT-Ovation Pico RNA amplification System (NuGen Technologies, Inc, San Carlos, CA, USA) following manufacturer's instructions. Amplified ss-cDNA was purified using Zymo Spin IIC columns (Zymo Research, Orange, California, USA) and stored at -20 oC overnight. Product yield and purity were determined by the Nanodrop 1000 spectrophotometer assuming that 1 absorbance unit at 260 nm of ss-cDNA is equal to 33 µg/ml. All 260/230 values were greater than 2. Aliquots containing 2 µg of ss-cDNA were labeled with the Agilent Genomic DNA Enzymatic Labeling kit to incorporate cyanine 3- or 5-labeled CTP. For half the replicates, ss-cDNA from control embryos was labeled with Cy3 and ss-cDNA from CSF2-treated embryos was labeled with Cy5. For the other replicates, control embryos were labeled with Cy 5 and CSF2-treated embryos with Cy3.
Hybridization protocol
Hybridizations were set up using 2 µg of each sample (Cy3 and Cy5 components) for a total of 4 µg per array. Volumes were brought to 44 µl with Diethylpyrocarbonate (DEPC)-water and then 11 µl of Agilent 10x GE Blocking Agent was added. The mixtures were incubated at 98oC for 3 minutes and then cooled to room temperature for 5 minutes before adding 55 µl of Agilent 2x Hi-RPM Hybridization buffer. Tubes were flash-spinned on a microfuge and lightly vortexed before loading 100 µl of the hybridization mixture onto each array. Hybridization was carried out for 17 h at 65°C and 10 rpm in a SureHyb gasket slide (Agilent). Washing and scanning procedures were carried out using standard Agilent guidelines for gene expression microarray processing.
Scan protocol
At the end of hybridization, microarray slides were sequentially washed using standard Agilent guidelines for gene expression microarray processing. Microarray slides were scanned immediately using an Agilent G2505B scanner.
Description
in vitro produced embryos were transfered into receptors cows at Day 7 of development and recovered at Day 15 of embryo development - extraembryonic membranes were isolated for global gene expression analysis
Data processing
Images were extracted and pre-processed using the Agilent Feature Extraction Software v 9.5 with default analysis parameters for the initial extraction, signal quantifications, and scaling of the generated data. The software produces background adjustment and normalizations for the dye of individual genes. The intensity of each spot was summarized as the median pixel intensity. All the generated values were then transformed to log2. The Lowess method was used for intensity normalization within each array. JMP® Genomics 3.1 for SAS® 9.1.3 software (SAS Inst., Inc., Cary, NC) was used for data global normalization and identification of differentially expressed genes. The PROC ANOVA procedure was used for simultaneous comparisons and the quantile method for intensity normalization. The model included replicate and treatment. Replicate (array) was considered random and treatment was considered fixed. Correction for false discovery rate was performed by the Benjamini and Hochberg method (1995). Only genes with median pixel intensity of at least 2.8 were considered to be expressed. Genes with a 1.5-fold difference and false discovery rate ≤ 0.01 were considered differentially expressed
raw data is green channel in US83800208_252364710013_S01_GE2-v5_10_Apr08_1_4.txt
