183 EFFECT OF WELL IN WELL CULTURE OF BOVINE EMBRYOS ON GENE EXPRESSION PROFILE

Abstract

To culture embryos in small groups, the well in well culture system (miniwells harboring 1 single embryo within the well) has been developed previously. In this work, we aimed to examine the effects of the microenvironment provided by well in well culture and embryo density on the relative abundance of transcripts in the resulting embryos. Cumulus–oocyte complexes (COCs) were aspirated from small follicles (2 to 8 mm), and COCs were cultured in 400 μL of modified TCM (TCM-199, Sigma, Taufkirchen, Germany) supplemented with 12% heat-inactivated estrous cow serum and 10 μg mL^–1 of FSH (FSH-p, Sheering, Kenilworth, NJ, USA) for 24 h at 39°C in a humidified atmosphere with 5% CO₂ in air. Fertilization was performed in Fert-TALP supplemented with 1 μg mL^–1 of heparin. Zygotes were allocated randomly in 2 groups, namely: well in well culture (16 miniwells of 0.7 diameter and deepness each containing 1 embryo per well) and group of 16 (group culture of 16 embryos per well). Six pools each containing 20 Day 7 blastocysts derived from the first 2 groups were used to investigate large-scale gene expression analysis using BlueChip cDNA-Array. Three pools each containing 5 blastocysts were used for Array data validation by real-time PCR using primers specific to 5 selected genes (ATP5, PLAC8, KRT8, S100A10, and ZP3). During validation in vivo-derived bovine blastocysts were included to be used as standard. Significance Analysis of Microarray identified 75 transcripts differentially expressed between the 2 groups. Blastocysts derived from well in well culture were found to be enriched with genes regulating different molecular functions including structural constituent of ribosome (RPS29), protein binding (Cul1), calcium ion binding (S100A10, NPTX2), nitric oxide synthase regulator activity (HSPCA), and RNA polymerase II transcription factor activity (UHRF1). However, blastocysts derived from group of 16 culture were found to be enriched with genes involved in oxidoreductase activity (ALOX15, AKR1B), cytochrome-c oxidase activity (COX7A2), hydrogen ion transporting ATP synthase activity (ATP5O), transcription (PTTG1), and cell redox homeostasis (TXN). According to their biological process, genes enriched in blastocysts derived from well in well culture belong to small molecule transport and signal transduction, whereas most downregulated genes have a metabolic function. Comparison of the transcript abundance of the 5 selected genes in the 3 embryo groups showed that the expression of ATP5, PLAC8, and KRT8 in embryos from well in well culture resembles to the relative abundance in blastocyst derived from in vivo culture. However, with respect to the expression of S100A10 and ZP3 genes, blastocysts derived from group culture showed similarity with embryos derived from in vivo. In conclusion, microenvironment affects the gene expression pattern of the resulting embryos.