In order to treat the early embryos, 10 µM of S-(5′-Adenosyl)-L-methionine chloride dihydrochloride, SAM (Sigma-Aldrich, Oakville, Canada# A7007) were freshly prepared by dissolving in SOF medium for each IVF trial and were quickly aliquoted into 100 µL and kept frozen at -80 until being used for each SAM-treatment. For control, only SOF medium without SAM were prepared freshly and aliquots of 100 µL were frozen at -80 until being used for control group during each treatment. The treatment was initiated at ~ 72 hpf (8-cell stage) of in vitro produced bovine embryo. To do that, for each IVF trial, only the 8-cell early embryos were collected and randomly divided equally into SAM-treated and control groups. The SAM treatment were performed at the final concentration of 2 µM SAM through adding 2 µL of 38.5°C frozen-thawed prepared aliquots of SAM (10 µM) into 8 µL of SOF droplets. For control group, similar treatment were performed with 2 µL of 38.5°C frozen-thawed prepared aliquot of SOF medium. The treatment was carried out every three hr, four times/day until blastocyst collection. For each treatment the embryos were transferred into a new SOF droplet (8 µL) and then 2 µL of SAM (10 µM) or SOF were added for SAM-treated or control group, respectively. For each IVF trial both groups were simultaneously treated and transferred into new droplets. Only expanded-blastocyst-stage embryos were collected, in four pools of ten embryos (n=40) per each group for parallel DNA methylome and transcriptome study.
Growth protocol
Bovine blastocyst production in synthetic oviduct fluid (SOF) media was performed with some modifications. Dairy cattle ovaries were collected from a commercial slaughterhouse and were transported to the laboratory in saline (0.9% NaCl) containing 1% Antibiotic-Antimycotic 100X (Gibco. 15240, Invitrogen, Burlington, ON, CAN) agent. Cumulus oocyte complexes (COCs) were aspirated from 2–6 mm follicles within 2 h of reception. Only COCs with at least five layers of cumulus without fragmented cytoplasm, pyknotic cumulus, pale nuclei and abnormal morphology were selected for maturation. The collected COCs were washed three times in HEPES-buffered Tyrode’s medium (TLH) containing with 1.5 % bovine serum albumin (Fraction V, Sigma-Aldrich, Oakville, Canada), 200 µM/mL pyruvate and 50 µg/mL of gentamycin as the washing medium. Pools of 10 COCs were placed in 50 µL droplets of maturation medium comprising TCM199 (Gibco 11150–059; Invitrogen, Burlington, ON, CAN), 0.6% bovine serum albumin (Sigma fraction V), 200 µM/mL pyruvate, 50 µg/mL of gentamycin, 0.1 µg/mL of follicle stimulating hormone (FSH) (Gonal-f, Serono Canada Inc.,Mississauga, QC, CAN) and 1µg/mL Estradiol (E2) covered with filtered mineral oil and incubated for 24 h at 38.5°C with 5% CO2, 20% O2 and high humidity. Matured COCs were washed three time in washing medium (TLH) and were transferred (n=5) to 50 µL fertilization medium drops comprising modified Tyrode’s lactate medium, supplemented with 0.6% bovine serum albumin (Sigma fraction V), 200 µM/mL pyruvate and 50 µg/mL gentamycin, covered with filtered mineral oil. Then 2 µL of a solution containing 1 mM hypotaurine, 2 mM penicillamine and 250 mM epinephrine was added to the COCs-containing fertilization droplets. In vitro fertilization was performed through insemination of the COCs with 25 x 106 sperm/mL. Briefly, the cryo-preserved Holstein bulls semen obtained from L'Alliance Boviteq Inc (Saint-Hyacinthe, QC, Canada) were thawed (37 °C water bath for 30 s) and washed through discontinuous Percoll gradient (45%-90% Percoll ; 700 × g for 30 min RT). After sperm motility assessment and the appropriate concentration adjustment the sperm were added to the COCs-contained fertilization drops and incubated for 15 –18 h at 38.5°C with 5% CO2, 20% O2 and high humidity. Zygotes and unfertilized COCs were mechanically denuded by repeated pipetting and washed three times in TLH to ensure complete removal of cumulus cells and sperm. For standard culture conditions, groups of ten embryos were placed in 10 µL droplets of modified synthetic oviduct fluid (SOF) under filtered mineral oil. Sequential SOF media were used as following: the embryos were first placed in SOF#1 (6 mM lactate, 0.2 mM glucose). The embryo culture dishes were incubated 38.5°C with 6.5% CO2, 5% O2 and high humidity. Embryos were then transferred to 10 µL droplets of SOF#2 (1 mM lactate, 0.5 mM glucose) 96 h post-fertilization (hpf) and finally to 10 µL droplets of SOF#3 (1 mM lactate, 2.5 mM glucose) 144 hpf.
Extracted molecule
total RNA
Extraction protocol
Extraction of total RNA from the collected EB embryos was carried out using the AllPrep DNA/RNA Mini Kit (Qiagen Canada Inc., Mississauga, ON, Canada) according to the manufacture manual and the samples were eluted in 30 µL for extracted RNA. Quantity and quality of each pool of extracted RNA was verified on a 2100 Bioanalyzer (Agilent Technologies, Mississauga, On, Canada). The extracted total RNA from blastocysts were appropriately labeled and immediately frozen at – 80 °C until use.
Label
Cy3
Label protocol
Extracted RNA from each sample (n=10 Blst.) of both SAM-treated (four replicates) and non-treated (four replicates) groups were amplified through in vitro transcription by T7 RNA amplification using the RiboAmp HS Plus RNA Amplification Kit (Molecular Devices) and the anti-sense RNA (aRNA) was produced. Then the quantity of the aRNA were measured using NanoDrop ND-1000 (Nano- Drop Technologies, Wilmington, DE, USA) and 2 µg of aRNA were labeled (Cy3 and Cy5) using the ULS Fluorescent Labelling Kit for Agilent arrays (Kreatech Diagnostics, Amsterdam, The Netherlands). Following purification with Pico-Pure RNA Isolation Kit (Molecular Devices, Sunnyvale, CA, USA) the labeling efficiency was evaluated using the Nano-Drop ND-1000 (Nano- Drop Technologies, Wilmington, DE, USA). Similar to DNA methylome analysis, four samples (10 Blst./sample) (biological replicates) of SAM-treated and control groups were hybridized on EmbryoGENE’s bovine transcriptome 44k Agilent microarray slides (V2) (Agilent Technologies) using a dye-swap design (technical replicates) for a total of eight arrays.
In order to treat the early embryos, 10 µM of S-(5′-Adenosyl)-L-methionine chloride dihydrochloride, SAM (Sigma-Aldrich, Oakville, Canada# A7007) were freshly prepared by dissolving in SOF medium for each IVF trial and were quickly aliquoted into 100 µL and kept frozen at -80 until being used for each SAM-treatment. For control, only SOF medium without SAM were prepared freshly and aliquots of 100 µL were frozen at -80 until being used for control group during each treatment. The treatment was initiated at ~ 72 hpf (8-cell stage) of in vitro produced bovine embryo. To do that, for each IVF trial, only the 8-cell early embryos were collected and randomly divided equally into SAM-treated and control groups. The SAM treatment were performed at the final concentration of 2 µM SAM through adding 2 µL of 38.5°C frozen-thawed prepared aliquots of SAM (10 µM) into 8 µL of SOF droplets. For control group, similar treatment were performed with 2 µL of 38.5°C frozen-thawed prepared aliquot of SOF medium. The treatment was carried out every three hr, four times/day until blastocyst collection. For each treatment the embryos were transferred into a new SOF droplet (8 µL) and then 2 µL of SAM (10 µM) or SOF were added for SAM-treated or control group, respectively. For each IVF trial both groups were simultaneously treated and transferred into new droplets. Only expanded-blastocyst-stage embryos were collected, in four pools of ten embryos (n=40) per each group for parallel DNA methylome and transcriptome study.
Growth protocol
Bovine blastocyst production in synthetic oviduct fluid (SOF) media was performed with some modifications. Dairy cattle ovaries were collected from a commercial slaughterhouse and were transported to the laboratory in saline (0.9% NaCl) containing 1% Antibiotic-Antimycotic 100X (Gibco. 15240, Invitrogen, Burlington, ON, CAN) agent. Cumulus oocyte complexes (COCs) were aspirated from 2–6 mm follicles within 2 h of reception. Only COCs with at least five layers of cumulus without fragmented cytoplasm, pyknotic cumulus, pale nuclei and abnormal morphology were selected for maturation. The collected COCs were washed three times in HEPES-buffered Tyrode’s medium (TLH) containing with 1.5 % bovine serum albumin (Fraction V, Sigma-Aldrich, Oakville, Canada), 200 µM/mL pyruvate and 50 µg/mL of gentamycin as the washing medium. Pools of 10 COCs were placed in 50 µL droplets of maturation medium comprising TCM199 (Gibco 11150–059; Invitrogen, Burlington, ON, CAN), 0.6% bovine serum albumin (Sigma fraction V), 200 µM/mL pyruvate, 50 µg/mL of gentamycin, 0.1 µg/mL of follicle stimulating hormone (FSH) (Gonal-f, Serono Canada Inc.,Mississauga, QC, CAN) and 1µg/mL Estradiol (E2) covered with filtered mineral oil and incubated for 24 h at 38.5°C with 5% CO2, 20% O2 and high humidity. Matured COCs were washed three time in washing medium (TLH) and were transferred (n=5) to 50 µL fertilization medium drops comprising modified Tyrode’s lactate medium, supplemented with 0.6% bovine serum albumin (Sigma fraction V), 200 µM/mL pyruvate and 50 µg/mL gentamycin, covered with filtered mineral oil. Then 2 µL of a solution containing 1 mM hypotaurine, 2 mM penicillamine and 250 mM epinephrine was added to the COCs-containing fertilization droplets. In vitro fertilization was performed through insemination of the COCs with 25 x 106 sperm/mL. Briefly, the cryo-preserved Holstein bulls semen obtained from L'Alliance Boviteq Inc (Saint-Hyacinthe, QC, Canada) were thawed (37 °C water bath for 30 s) and washed through discontinuous Percoll gradient (45%-90% Percoll ; 700 × g for 30 min RT). After sperm motility assessment and the appropriate concentration adjustment the sperm were added to the COCs-contained fertilization drops and incubated for 15 –18 h at 38.5°C with 5% CO2, 20% O2 and high humidity. Zygotes and unfertilized COCs were mechanically denuded by repeated pipetting and washed three times in TLH to ensure complete removal of cumulus cells and sperm. For standard culture conditions, groups of ten embryos were placed in 10 µL droplets of modified synthetic oviduct fluid (SOF) under filtered mineral oil. Sequential SOF media were used as following: the embryos were first placed in SOF#1 (6 mM lactate, 0.2 mM glucose). The embryo culture dishes were incubated 38.5°C with 6.5% CO2, 5% O2 and high humidity. Embryos were then transferred to 10 µL droplets of SOF#2 (1 mM lactate, 0.5 mM glucose) 96 h post-fertilization (hpf) and finally to 10 µL droplets of SOF#3 (1 mM lactate, 2.5 mM glucose) 144 hpf.
Extracted molecule
total RNA
Extraction protocol
Extraction of total RNA from the collected EB embryos was carried out using the AllPrep DNA/RNA Mini Kit (Qiagen Canada Inc., Mississauga, ON, Canada) according to the manufacture manual and the samples were eluted in 30 µL for extracted RNA. Quantity and quality of each pool of extracted RNA was verified on a 2100 Bioanalyzer (Agilent Technologies, Mississauga, On, Canada). The extracted total RNA from blastocysts were appropriately labeled and immediately frozen at – 80 °C until use.
Label
Cy5
Label protocol
Extracted RNA from each sample (n=10 Blst.) of both SAM-treated (four replicates) and non-treated (four replicates) groups were amplified through in vitro transcription by T7 RNA amplification using the RiboAmp HS Plus RNA Amplification Kit (Molecular Devices) and the anti-sense RNA (aRNA) was produced. Then the quantity of the aRNA were measured using NanoDrop ND-1000 (Nano- Drop Technologies, Wilmington, DE, USA) and 2 µg of aRNA were labeled (Cy3 and Cy5) using the ULS Fluorescent Labelling Kit for Agilent arrays (Kreatech Diagnostics, Amsterdam, The Netherlands). Following purification with Pico-Pure RNA Isolation Kit (Molecular Devices, Sunnyvale, CA, USA) the labeling efficiency was evaluated using the Nano-Drop ND-1000 (Nano- Drop Technologies, Wilmington, DE, USA). Similar to DNA methylome analysis, four samples (10 Blst./sample) (biological replicates) of SAM-treated and control groups were hybridized on EmbryoGENE’s bovine transcriptome 44k Agilent microarray slides (V2) (Agilent Technologies) using a dye-swap design (technical replicates) for a total of eight arrays.
Hybridization protocol
Hybridizations were performed according to the microarray manufacturer's instructions (Agilent Technologies, Santa Clara, USA). Briefly, 1 µg of labeled sample (in 40 µL) was mixed with 158 µL of hybridization master mix containing 25 µL of bovine Cot-1 DNA (1.0 mg/mL, Bovine Hybloc competitor DNA, Applied Genetics Laboratories, Melbourne, FL, USA), 2.6 µL of Agilent 100x Blocking Agent and 130 µL of Agilent 2x HI-RPM Hybridization Buffer (Agilent Technologies, Santa Clara, USA). Samples were held at 95 °C for 3 min and at 37 °C for 30 min followed by addition of 65 µL of Agilent-CGHBlock (final volume 260 µL). The samples were loaded onto the microarray and hybridization was carried out in a hybridization oven (Shel Lab, Cornelius, OR, USA) for 40 h at 65 °C and 20 rpm. Washing was carried out according to the microarray manufacturer's instructions
Scan protocol
Slides were scanned with the PowerScanner (Tecan, Männedorf, Switzerland)
Description
Biological replicate 3 of 4. (SAM-treated expanded blastocysts vs. non-treated expanded blastocysts)
Data processing
Intensity values were obtained from slide images using the Array-Pro Analyzer 6.3 software (MediaCybernetics, Bethesda, MD, USA). Raw intensities were subjected to within-array normalization(loess) followed by between-array normalization (quantile) using the limma library from Bioconductor.
Submission date
Nov 27, 2015
Last update date
Feb 29, 2016
Contact name
Habib A. Shojaei Saadi
Organization name
Laval University
Lab
Laboratory of Functional Genomics of Early Embryonic Development
