82 EFFECT OF HIGH FETAL CALF SERUM CONCENTRATION IN THE GENE EXPRESSION PATTERN OF IN VITRO PRODUCED BOVINE EMBRYOS

Abstract

Even though FCS provides energy substrates, amino acids, vitamins, growth factors, and heavy-metal chelators, its supplementation has been associated with several embryo abnormalities such as mitochondrial degeneration, metabolic deviations, excessive lipid accumulation, and decreased embryo survival after cryopreservation. The aim of the present study was to evaluate the effect of high FCS concentration in the gene expression pattern of in vitro-produced bovine embryos. Slaughterhouse ovaries were used to obtain oocytes (N = 360), which were matured and fertilized in vitro (Day 0). Presumptive zygotes were divided in 2 culture media: with low (SOFaa with 0.5% BSA and 2.5% FCS) or high (SOFaa with 0.5% BSA and 10% FCS) FCS concentration. Cleavage was evaluated on Day 3. Embryo development was evaluated after 7 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 was performed using the PicoPure RNA isolation Kit (Applied Biosystems®, Foster City, CA, USA). Extracted RNA was evaluated through 2100-Bioanalyzer (Agilent Technologies®, Palo Alto, CA, USA) and DNAse treated (Qiagen®, Valencia, CA, USA). RiboAmp RNA Amplification Kit (Applied Biosystems®) was used to amplify the RNA (T7 RNA polymerase-catalysed amplification reaction). The aRNA output was evaluated through NanoDrop ND-1000 (NanoDrop Technologies®, Wilmington, DE, USA). A biotin-labelled cRNA and fragmented cRNA were obtained through 3′IVT Express Kit (Affymetrix®, Santa Clara, CA, USA) to perform the hybridization (N = 3 per group) 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. The data from in vitro embryo production were analysed through the PROC GLM (SAS Institute Inc., Cary, NC, USA). Cleavage rate (81.4 ± 1.5 and 85.5 ± 1.4) and blastocyst production (41.8 ± 2.4 and 47.2 ± 2.8) were not different (P > 0.05) between low and high FCS concentrations, respectively. A total of 40 genes were differentially expressed between low and high FCS concentration. A total of 28 genes were annotated, with 37 genes up-regulated and 3 genes down-regulated by high FCS concentration. The associated network functions of gene expression, RNA damage and repair, and post-transcriptional modification; and cell-to-cell signalling and interaction were generated by Ingenuity Pathway Analysis® (Redwood City, CA, USA). Differentially expressed genes involved in carbohydrate metabolism (GAPVD1, MGAT4A), lipid metabolism (ELOVL5), cellular assembly and organisation (EZR, LRP2), and cell death and survival (DRT8) were identified. In conclusion, high FCS supplementation was associated with different expression profiles of genes regulating carbohydrate and lipid metabolism, cellular assembly and organisation, and cell death and survival.

The authors acknowledge support from FAPESP and LNBio-CNPEM.