cell type: Cumulus cells from cumulus-oocyte complexes treated with: none (untreated control) in vitro culture time: 3 h
Treatment protocol
Culture of COCs in control conditions (see growth protocol below).
Growth protocol
Ovaries of slaughtered gilts were collected at a local abattoir and transported to the laboratory in a thermos at 37°C. The contents of the medium size follicles (3-6 mm in diameter) was aspirated by a syringe connected with a 20 G needle, pooled in a Petri-dish and washed twice with PBS. COCs were isolated from the diluted aspirate and washed in PBS. Only COCs surrounded by compact multi-layered cumulus were selected for experiments. About 30 COCs were cultured in 0.5 ml of medium M-199 (#31150; Gibco, Life Technologies, Rockville, USA) supplemented with 0.91 mM sodium pyruvate, 0.57 mM cysteine, 4.2 mM Hepes, antibiotics and 5% fetal calf serum (FCS; #F2442). The COCs were cultured in 4-well dishes (Nunclon, Roskilde, Denmark) at 38.5 °C in a humidified atmosphere of 5% CO2 in the air for 3 h.
Extracted molecule
total RNA
Extraction protocol
At the end of the culture period, about 150 COCs were transferred in a Petri dish with 2 ml of PBS supplemented with 5 % FCS. The cumulus cells were stripped of oocytes mechanically by pipetting. The oocytes were removed and PBS with a suspension of the separated cumulus cells was transferred in a microtube and briefly centrifuged in a microcentrifuge. The supernatant was discarded and the pellet of cumulus cells was lysed in 300 µl of RTL buffer (Qiagen, Hilden, Germany) and stored at −80 °C. Total RNA from all nine samples was isolated using an RNeasy PLUS mini kit (Qiagen) according to the manufacturer’s instructions. RNA integrity was verified by using RNA 6000 Nano LabChip by Agilent 2100 Bioanalyzer. Only RNA samples with resulting RNA integrity number (RIN) between 8.2–9.1 were used for amplification of aRNA.
Label
AlexaFluor 555
Label protocol
aRNA was prepared with one-round amplification using AminoAllyl MessageAmp II Kit (Ambion, Austin, Texas, USA). Time of in vitro transcription was 14 h. Five µg of aRNA were conjugated with either Alexa Fluor 555 or 647 dyes (Invitrogen, Carlsbad, California, USA). Labeled aRNAs were purified using PicoPure RNA isolation kit (Arcturus) according to the manufacturer’s instructions; 2 µg of each labeled aRNA (Alexa Fluor 555 and Alexa Fluor 647 conjugated aRNA samples according to the experimental design) were mixed together and stored at −80 °C.
cell type: Cumulus cells from cumulus-oocyte complexes treated with: AREG/EREG in vitro culture time: 3 h
Treatment protocol
The maturation of COCs was stimulated by the addition of recombinant human AREG (100 ng/ml, Sigma) and EREG (100 ng/ml; R&D Systems, Minneapolis, MN, USA) into the culture medium.
Growth protocol
Ovaries of slaughtered gilts were collected at a local abattoir and transported to the laboratory in a thermos at 37°C. The contents of the medium size follicles (3-6 mm in diameter) was aspirated by a syringe connected with a 20 G needle, pooled in a Petri-dish and washed twice with PBS. COCs were isolated from the diluted aspirate and washed in PBS. Only COCs surrounded by compact multi-layered cumulus were selected for experiments. About 30 COCs were cultured in 0.5 ml of medium M-199 (#31150; Gibco, Life Technologies, Rockville, USA) supplemented with 0.91 mM sodium pyruvate, 0.57 mM cysteine, 4.2 mM Hepes, antibiotics and 5% fetal calf serum (FCS; #F2442). The COCs were cultured in 4-well dishes (Nunclon, Roskilde, Denmark) at 38.5 °C in a humidified atmosphere of 5% CO2 in the air for 3 h.
Extracted molecule
total RNA
Extraction protocol
At the end of the culture period, about 150 COCs were transferred in a Petri dish with 2 ml of PBS supplemented with 5 % FCS. The cumulus cells were stripped of oocytes mechanically by pipetting. The oocytes were removed and PBS with a suspension of the separated cumulus cells was transferred in a microtube and briefly centrifuged in a microcentrifuge. The supernatant was discarded and the pellet of cumulus cells was lysed in 300 µl of RTL buffer (Qiagen, Hilden, Germany) and stored at −80 °C. Total RNA from all nine samples was isolated using an RNeasy PLUS mini kit (Qiagen) according to the manufacturer’s instructions. RNA integrity was verified by using RNA 6000 Nano LabChip by Agilent 2100 Bioanalyzer. Only RNA samples with resulting RNA integrity number (RIN) between 8.2–9.1 were used for amplification of aRNA.
Label
AlexaFluor 647
Label protocol
aRNA was prepared with one-round amplification using AminoAllyl MessageAmp II Kit (Ambion, Austin, Texas, USA). Time of in vitro transcription was 14 h. Five µg of aRNA were conjugated with either Alexa Fluor 555 or 647 dyes (Invitrogen, Carlsbad, California, USA). Labeled aRNAs were purified using PicoPure RNA isolation kit (Arcturus) according to the manufacturer’s instructions; 2 µg of each labeled aRNA (Alexa Fluor 555 and Alexa Fluor 647 conjugated aRNA samples according to the experimental design) were mixed together and stored at −80 °C.
Hybridization protocol
Microarray slides were rehydrated for 10 sec above water bath at 50 °C, dried on the heat block for 5 sec at 65 °C and cooled for 1 min at room temperature. This procedure was repeated 4 times. UV cross-linking was performed by exposing rehydrated slides to 180mJ of UV radiation. After crosslinking the slides were washed in 0.1% SDS with constant gently mixing for 5 min followed by rinsing in ddH2O and 3 min incubation in 100% ethanol with constant gently mixing, all steps at room temperature. Slides were dried by centrifugation for 4 min at 200 g. Just before use the microarray slides were pre-hybridized at 42 °C for 1 h in pre-hybridization buffer (5xSSC, 0.1%SDS, 1%BSA) using Tecan HS400Pro hybridization station (TECAN, Austria). Pre-mixed labeled aRNA probes were fragmented using Fragmentation buffer (Ambion Austin, Texas, USA) according to the manufacturer’s instructions. After fragmentation, probes were denatured for 5 min at 90 °C, mixed with 130 µl of SlideHyb Glass Array Hybridization Buffer # 1 (Ambion Austin, Texas, USA) preheated to 68 °C and hybridized for 15 h at 42 °C in the Tecan HS400Pro to pre-hybridized microarray slides. After hybridization, slides were washed and dried automatically in Tecan HS400Pro instrument and stored in dark until scanned (the same day).
Scan protocol
Slides were scanned using High-Resolution Microarray Scanner (Agilent) at 5 µm resolution on 20-bit dynamic range setting. Agilent Feature Extraction Software (v10.7) was used for image analysis.
Description
biological replicate 2 (dye-swap)
Data processing
Microarray data background correction, normalization and statistical inference of changes in gene expression were performed using Bioconductor package Limma in R statistical environment. Briefly, spot and background median signals were imported into R data-frame and normex background correction was applied. After print-tip loess within array normalization A-quantile normalization was used to make signal intensities more comparable across arrays.
Gene expression analysis of pig cumulus-oocyte complexes stimulated in vitro with follicle stimulating hormone or epidermal growth factor-like peptides
Data table header descriptions
ID_REF
VALUE
log2 ratios of Ch2/Ch1 after print-tip loess within array and A-quantile between array normalization
