|
| Status |
Public on Apr 08, 2017 |
| Title |
High_Insulin_4 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
High insulin (10ug/ml)
|
| Organism |
Bos taurus |
| Characteristics |
tissue: blastocysts
|
| Treatment protocol |
Bovine insulin (I5500, Sigma-Aldrich, Stockholm, Sweden) was added to the medium during the IVM period of 22 hours, at either 0.1 µg/mL (INS0.1) or 10 µg/mL (INS10). The two different insulin concentrations INS10 and INS0.1 were used in this study in purpose to investigate a potential dose-dependent effect of insulin during oocyte maturation.
|
| Growth protocol |
Briefly, abattoir-derived cumulus oocyte complexes (COCs) (n=882) were matured in groups of 30-40 in vitro for 22 h in 500 µl serum-free maturation medium (IVM). IVM medium, consisted of bicarbonate-buffered TCM199 (M2154) supplemented with 0.68 mM L-glutamine (G8540), 0.5 mg/mL FSH and 0.1 mg/mL LH (Stimufol; PARTNAR Animal Health, Port Huron, Canada), 50 mg/mL gentamicin and 0.4% w/v BSA. Bovine insulin (I5500) was added to the medium at either 0.1 µg/mL (INS0.1) or 10 µg/mL (INS10). A control group without insulin supplementation (INS0) was run in parallel during each production week, medium. Matured oocytes were fertilized in vitro with sperm from a dairy bull with proven field and in vitro fertility and presumed zygotes were incubated under humidified atmosphere of 5 % CO₂, 5% O₂ and 90 % N₂ at 38.5°C in 500 μl modified synthetic oviductual fluid per well until Day 8. Resulting blastocysts (BC8) were evaluated (developmental stage and quality grade) and washed three times in PBS-PVA before individually frozen at -80°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
gDNA was extracted from pools of 10 blastocysts using the AllPrep DNA/RNA micro kit (Qiagen). The methylation analysis was performed using EmbryoGENE DNA methylation array (EDMA) platform. The methodology have been thoroughly describef in (Saadi et al., 2014). Briefly, 10ng of DNA sample and spike-in control were cut in ≈160bp fragment using MSEI restriction enzyme. MseLig 12 and MseLig 21 adapters were added to the fragmented gDNA sites. Sample were the digested using methyl sensitive restriction enzyme HpaII, HinP1I and AciI (FastDigesttm). After cleavage by methyl sensitive enzyme, the gDNA was purified by ethanol precipitation and dissolve in nuclease free water. Selective amplification of methylated fragments was performed using two rounds of ligation-mediated PCR (LM-PCR). The PCR products were resolved on 0.8% agarose gel to assess the quality. Then the previously added linker were removed.
|
| Label |
Cy3
|
| Label protocol |
For each sample, 2 μg of DNA was labeled using the Universal Linkage System (ULS) labeling kit (Kreatech Biotechnology) according to the manufacturer’s instructions with minor modifications: 1 μL of Cy-ULS dye was added per 1 μg of genomic DNA adjusted with 10× labeling buffer. The labelling mixture was then held for 30 min at 85°C in a thermocycler, followed by 3 min on ice. Non-reacted ULS-Cy3/5 was removed by purification using a QIAquick PCR Purification Kit and samples were eluted in 23.5 μL nuclease-free water. A 1.5 μL aliquot was used to determine DNA concentration and dye incorporation using the ND- 1000 NanoDrop.
|
| |
|
| Channel 2 |
| Source name |
control, no treatment
|
| Organism |
Bos taurus |
| Characteristics |
tissue: blastocysts
|
| Treatment protocol |
Bovine insulin (I5500, Sigma-Aldrich, Stockholm, Sweden) was added to the medium during the IVM period of 22 hours, at either 0.1 µg/mL (INS0.1) or 10 µg/mL (INS10). The two different insulin concentrations INS10 and INS0.1 were used in this study in purpose to investigate a potential dose-dependent effect of insulin during oocyte maturation.
|
| Growth protocol |
Briefly, abattoir-derived cumulus oocyte complexes (COCs) (n=882) were matured in groups of 30-40 in vitro for 22 h in 500 µl serum-free maturation medium (IVM). IVM medium, consisted of bicarbonate-buffered TCM199 (M2154) supplemented with 0.68 mM L-glutamine (G8540), 0.5 mg/mL FSH and 0.1 mg/mL LH (Stimufol; PARTNAR Animal Health, Port Huron, Canada), 50 mg/mL gentamicin and 0.4% w/v BSA. Bovine insulin (I5500) was added to the medium at either 0.1 µg/mL (INS0.1) or 10 µg/mL (INS10). A control group without insulin supplementation (INS0) was run in parallel during each production week, medium. Matured oocytes were fertilized in vitro with sperm from a dairy bull with proven field and in vitro fertility and presumed zygotes were incubated under humidified atmosphere of 5 % CO₂, 5% O₂ and 90 % N₂ at 38.5°C in 500 μl modified synthetic oviductual fluid per well until Day 8. Resulting blastocysts (BC8) were evaluated (developmental stage and quality grade) and washed three times in PBS-PVA before individually frozen at -80°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
gDNA was extracted from pools of 10 blastocysts using the AllPrep DNA/RNA micro kit (Qiagen). The methylation analysis was performed using EmbryoGENE DNA methylation array (EDMA) platform. The methodology have been thoroughly describef in (Saadi et al., 2014). Briefly, 10ng of DNA sample and spike-in control were cut in ≈160bp fragment using MSEI restriction enzyme. MseLig 12 and MseLig 21 adapters were added to the fragmented gDNA sites. Sample were the digested using methyl sensitive restriction enzyme HpaII, HinP1I and AciI (FastDigesttm). After cleavage by methyl sensitive enzyme, the gDNA was purified by ethanol precipitation and dissolve in nuclease free water. Selective amplification of methylated fragments was performed using two rounds of ligation-mediated PCR (LM-PCR). The PCR products were resolved on 0.8% agarose gel to assess the quality. Then the previously added linker were removed.
|
| Label |
CY5
|
| Label protocol |
For each sample, 2 μg of DNA was labeled using the Universal Linkage System (ULS) labeling kit (Kreatech Biotechnology) according to the manufacturer’s instructions with minor modifications: 1 μL of Cy-ULS dye was added per 1 μg of genomic DNA adjusted with 10× labeling buffer. The labelling mixture was then held for 30 min at 85°C in a thermocycler, followed by 3 min on ice. Non-reacted ULS-Cy3/5 was removed by purification using a QIAquick PCR Purification Kit and samples were eluted in 23.5 μL nuclease-free water. A 1.5 μL aliquot was used to determine DNA concentration and dye incorporation using the ND- 1000 NanoDrop.
|
| |
|
| |
| Hybridization protocol |
Hybridizations were performed according to the microarray manufacturer’s instructions (Agilent Technologies). Briefly, 1.4 μg of labeled sample (in 40 μL) was mixed with 158 μL of hybridization master mix containing 25 μL of Bovine Hybloc competitor DNA (1 mg/ml) , (Applied Genetics Laboratories), 2.6 μL of Agilent 100x Blocking Agent and 130 μL of Agilent 2× HI-RPM Hybridization Buffer (Agilent Technologies). 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) 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) and analyzed with Array-Pro Analyzer 6.3 software (MediaCybernetics).
|
| Description |
Biological replicats 4 of 4
|
| Data processing |
The array data analysis methods and downstream analysis pipelines are described in detail in (Saadi et al., 2014). Briefly, after Loess normalization, quantile inter-array scale normalization was performed and fitted to a linear model and thus, Bayesian statistics of differential expression were obtained. Thus, differentially methylated probes were identified using linear models for microarray data (LIMMA).
|
| |
|
| Submission date |
Apr 07, 2017 |
| Last update date |
Apr 08, 2017 |
| Contact name |
Marc-André Sirard |
| E-mail(s) |
[email protected]
|
| Organization name |
Université Laval
|
| Department |
Sciences Animales
|
| Street address |
Offfice 2732, 2440 Hochelaga Blvd.
|
| City |
Québec City |
| State/province |
Quebec |
| ZIP/Postal code |
G1V 0A6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL18384 |
| Series (2) |
| GSE97515 |
Exposure of bovine oocytes to elevated insulin concentration (10 ug/ml) during in vitro maturation changes DNA methylation pattern in Day-8-blastocysts – an epigenetic study. |
| GSE97517 |
Exposure of bovine oocytes to insulin concentration (0.1 and 10 ug/ml) during in vitro maturation changes DNA methylation pattern in Day-8-blastocysts - an epigenetic study |
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