Microarray Analysis of In Vitro-Produced Bovine Embryos with Phenazine Ethosulfate.

Abstract

Phenazine ethosulfate (PES) is a metabolic regulator that inhibits fat acids synthesis and favors the pentose-phosphate pathway. In previous study, PES supplementation increased embryo cryotolerance and quality. Microarrays have become a valuable high throughput technology that efficiently describes transcripts abundance of samples. The aim of the present study was to evaluate the global gene express pattern of in vitro produced bovine embryos after the supplementation of phenazine ethosulfate (PES) in the culture media. Slaughterhouse ovaries were used to obtain oocytes (N=200), which were matured and fertilized in vitro (Day 0). Presumptive zygotes were divided in two culture media: PES supplementation (SOFaa with 0.5% BSA, 2.5% FCS, and 0.3μM PES) and Control group (SOFaa with 0.5% BSA and 2.5% FCS). Embryo development was evaluated after seven days under standard culture conditions (at 38.5°C in atmosphere of 5%O2, 5%CO2 and 90%N2). The produced blastocysts were placed in PBS solution and washed five times. A single blastocyst was frozen in a minimal volume of PBS and stored at −80°C until RNA extraction. Total RNA extraction of a single blastocyst was performed using the PicoPure RNA isolation Kit (Arcturus - Applied Biosystems). Following extraction, RNA samples were DNAse treated (Qiagen) and RiboAmp RNA Amplification Kit (Applied Biosystems) was used to linearly amplify the mRNA fraction of total RNA using cDNA as template in a T7 RNA polymerase-catalysed amplification reaction. The aRNA output was evaluated through NanoDrop ND-1000 (NanoDrop Technologies) and 2100-Bioanalyzer (Agilent Technologies®). A biotin-labelled cRNA and fragmented cRNA were obtained through 3'IVT Express Kit (Affymetrix) to perform the hybridization (N=7) using GeneChip Bovine Genome Array (Affymetrix). Following hybridization, probe arrays were washed, stained and scanned. Microarray data analysis was performed in the software FlexArray 1.6.1.1. Genes with a fold change of at least 1.5 and a probability of P=0.05 were considered differentially expressed. Cleavage was 83.4±1.3 versus 85.3±1.4% (respectively for PES supplementation and Control group; P>0.05). Blastocyst production was 43.1±2.2 and 42.3±2.4 (respectively for PES supplementation and Control group; P>0.05). The mean value of aRNA produced from a single bovine embryo after amplification was 1.5±0.3 and 1.8±0.2μg (respectively for PES supplementation and Control group) with a distribution size of 200–1500 nucleotides and A260:A280 ratio of 1.8–2.1. A total of 32 genes were differentially expressed between PES supplementation and control group. A total of 29 genes were annotated, with 3 genes upregulated and 29 genes downregulated by PES supplementation. Genes involved in mitochondrial metabolic process (ATP5G1, ATP6V0A4, ATP5B AND NDUFB10), oxidative stress (SOD2) and apoptosis (BAG2) were downregulated by PES supplementation. Therefore, PES acted by altering the mitochondrial metabolism, oxidative stress and apoptosis. Acknowledgements: FAPESP and LNBio - National Laboratory of Biosciences/MCT.