Expression of genes associated with BMP signaling pathway in porcine oocytes before and after IVM – a microarray approach

Collected oocytes were exposed to two Brilliant Cresyl Blue (BCB) tests and divided into two groups. The first group (before IVM) included oocytes graded as BCB-positive (BCB⁺) and not subjected to further IVM. The second group (after IVM) included BCB⁺ oocytes which were then in vitro matured, and graded as BCB⁺ after IVM.

A total of 45 pubertal crossbred Landrace gilts bred on a commercial local farm were used in this study. They had a mean age of 155 days (range 140–170 days) and a mean weight of 100 kg (95–120 kg). All animals were housed under identical conditions and fed the same forage (depending on age and reproductive status). All experiments were approved by the Local Ethic Committee.

The ovaries and reproductive tracts were recovered at slaughter and transported to the laboratory within 40 min. at 38 °C in 0.9% NaCl. To provide optimal conditions for subsequent oocyte maturation and fertilization in vitro, the ovaries of each animal were placed in a 5% fetal bovine serum solution (FBS; Sigma-Aldrich Co., St. Louis, MO, USA) in PBS. Single large follicles (>5 mm) were opened by puncturing with a 5 ml syringe and 20-G needle in a sterile Petri dish, and COCs were recovered. The COCs were washed three times in modified PBS supplemented with 36 μg/ml pyruvate, 50 μg/ml gentamycine, and 0.5 mg/ml BSA (Sigma-Aldrich, St. Louis, MO, USA). COCs were selected under an inverted microscope Zeiss, Axiovert 35 (Lübeck, Germany), counted, and morphologically evaluated using the scale suggested by Jackowska et al. Only COCs of grade I possessing homogeneous ooplasm and uniform, compact cumulus cells were considered for further use, resulting in a total of 300 grade I oocytes (3 x n = 50 before IVM group, 3 x n = 50 after IVM group).

Brilliant Cresyl Blue (BCB) test, which measures activity of glucose-6-phosphate (G6PDH) enzyme, was used for assessment of oocytes’ quality and maturity [20]. The G6PDH enzyme converts BCB stain from blue to colorless. In oocytes that completed the growth activity of the enzyme decreases and the stain cannot be reduced, resulting in blue oocytes (BCB⁺). To perform the BCB staining test, oocytes were washed twice in modified Dulbecco’s Phosphate Buffered Saline (DPBS) commercially supplemented with 0.9 mM calcium, 0.49 mM magnesium, 0.33 mM pyruvate, and 5.5 mM glucose (Sigma-Aldrich, St. Louis, MO, USA), and additionally with 50 IU/ml penicillin, 50 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 0.4% Bovine Serum Albumin (BSA) [w/v] (Sigma-Aldrich, St. Louis, MO, USA). They were then treated with 13 μM BCB (Sigma-Aldrich, St. Louis, MO) diluted in DPBS at 38.5 °C, 5% CO₂ for 90 min. After treatment, the oocytes were transferred to DPBS and washed twice. During washing, the oocytes were examined under an inverted microscope and classified as stained blue (BCB ⁺ ) or colorless (BCB⁻). Only the granulosa cell-free BCB⁺ oocytes were used for subsequent molecular analysis (before IVM group) or IVM followed by second BCB test and molecular analysis (after IVM group).

After the first BCB test, the BCB⁺ COCs were subjected to IVM. Immature oocytes have compact cumulus cell layers that required removal for further oocyte evaluation. Thus, COCs were first incubated with bovine testicular hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) for 2 min at 38^o C to separate cumulus and granulosa cells. Cells were then removed by vortexing the BCB⁺ oocytes in 1% sodium citrate buffer followed by mechanical displacement using a small-diameter glass micropipette (Nichiryo, Nishikata, Japan). The COCs were cultured in Nunclon™Δ 4-well dishes (Thermo Fisher Scientific, Waltham, MA, USA) in 500 μl standard porcine IVM culture medium: TCM-199 (tissue culture medium) with Earle’s salts and L-glutamine (Gibco BRL Life Technologies, Grand Island, NY, USA), supplemented with 2.2 mg/ml sodium bicarbonate (Nacalai Tesque, Inc., Kyoto, Japan), 0.1 mg/ml sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), 10 mg/ml BSA (Bovine Serum Albumin) (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mg/ml cysteine (Sigma-Aldrich, St. Louis, MO, USA), 10% (v/v) filtered porcine follicular fluid, and gonadotropin supplements at final concentrations of 2.5 IU/ml hCG (human Chorionic Gonadotropin) (Ayerst Laboratories, Inc., Philadelphia, PA, USA) and 2.5 IU/ml eCG (equine Chorionic Gonadotropin) (Intervet, Whitby, ON, Canada). Wells were covered with a mineral oil overlay and cultured for 44 h at 38 °C under 5% CO₂. After cultivation, the BCB staining test was performed, and BCB⁺ oocytes were used for further experiments.

Total RNA was extracted from samples using TRI Reagent (Sigma, St Louis, MO, USA) and RNeasy MinElute cleanup Kit (Qiagen, Hilden, Germany). The amount of total mRNA was determined from the optical density at 260 nm, and the RNA purity was estimated using the 260/280 nm absorption ratio (higher than 1.8) (Nano Drop spectrophotometer, Thermo Scientific, ALAB, Poland). The RNA integrity and quality were checked on a Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA). The resulting RNA integrity numbers (RINs) were between 8.5 and 10 with an average of 9.2 (Agilent Technologies, Inc., Santa Clara, CA, USA). The RNA in each sample was diluted to a concentration of 100 ng/μl with an OD260/OD280 ratio of 1.8/2.0. From each RNA sample, 500 ng of RNA was taken. The remaining amount of isolated RNA was used for the RT-qPCR study.

The Affymetrix procedure was previously described by Trejter et al. [21]. Total RNA (100 ng) from each pooled sample was subjected to two rounds of sense cDNA amplification (Ambion® WT Expression Kit). The cDNA was used for biotin labeling and fragmentation by Affymetrix Gene Chip® WT Terminal Labeling and Hybridization (Affymetrix). Biotin-labeled fragments of cDNA (5.5 μg) were hybridized to Affymetrix® Porcine Gene 1.1 ST Array Strip (48 °C/20 h), where the expression profile of 12,258 porcine transcripts was examined. Microarrays were then washed and stained according to the technical protocol using the Affymetrix Gene Atlas Fuidics Station. The array strips were scanned employing Imaging Station of the Gene Atlas System. Preliminary analysis of the scanned chips was performed using Affymetrix Gene Atlas TM Operating Software. The quality of the gene expression data was checked according to quality control criteria provided by the software. Obtained CEL files were imported into downstream data analysis software.

All analyses were performed using Bioconductor software based on the statistical R programming language. For background correction, normalization, and summation of raw data, the Robust Multiarray Averaging (RMA) algorithm implemented in “affy” package of Bioconductor was applied. Biological annotation was taken from Bioconductor “oligo” package where the annotated data frame object was merged with a normalized data set, leading to a complete gene data table. Statistical significance of the analyzed genes was performed by moderated t-statistics from the empirical Bayes method. Obtained p-values were corrected for multiple comparisons using the Benjamini and Hochberg’s false discovery rate. Selection of significantly changed gene expression values (differentially expressed genes) was based on a p-value below 0.05 and an expression fold higher than |2|. The homogeneity of analyzed groups was checked by a principal component analysis (PCA) algorithm incorporated in “rgl” Bioconductor package.

Differentially expressed genes were subjected to the selection of genes associated with BMP signaling pathways. Differentially expressed gene lists (separated for up- and down-regulated) were uploaded to DAVID software (Database for Annotation, Visualization and Integrated Discovery) where differentially expressed genes belonging to “BMP signaling pathway” gene ontology biological process group (GO BP) were obtained [22]. Expression data of these genes were subjected to a hierarchical clusterization procedure and presented as a heatmap.

Interactions between differentially expressed genes/proteins belonging to “BMP signaling pathway” ontology group were investigated by STRING10 software (Search Tool for the Retrieval of Interacting Genes) [23]. List of gene names was used as query for interaction prediction. Search criteria were based on co-occurrences of genes/proteins in scientific texts (text mining), co-expression, and experimentally observed interactions. The results of this analysis generated a gene/protein interaction network where the intensity of the edges reflects the strength of the interaction score. In addition to interaction prediction, STRING also performs functional enrichments of GO terms based on previously uploaded gene sets from “BMP signaling pathway” GO BP term.

RT-qPCR analysis was performed in order to validate microarray results, using both the same RNA samples used for PCR and microarray profiling experiments.

Total RNA was isolated from oocytes before or after IVM. The RNA samples were re-suspended in 20 μl of RNase-free water and stored in liquid nitrogen. RNA samples were treated with DNase I and reverse-transcribed (RT) into cDNA. RT-qPCR was conducted in a Light Cycler real-time PCR detection system (Roche Diagnostics GmbH, Mannheim, Germany) using SYBR® Green I as a detection dye, and target cDNA was quantified using the relative quantification method. The relative abundance of CHRDL1, FST, TGFβR3, SMAD4, and ID1transcripts in each sample was standardized to the internal standards. For amplification, 2 μl of cDNA solution was added to 18 μl of QuantiTect® SYBR® Green PCR (Master Mix Qiagen GmbH, Hilden, Germany) and primers (Table 1). One RNA sample of each preparation was processed without the RT-reaction to provide a negative control for subsequent PCR.