283 TRANSCRIPTIONAL ANALYSIS OF BIOPSIES DERIVED FROM IN VITRO-PRODUCED BOVINE BLASTOCYSTS IN RELATION TO PREGNANCY SUCCESS AFTER TRANSFER TO RECIPIENTS

Abstract

Early embryonic mortality is a recognized cause of reproductive failure in cattle leading to the loss of a large number of potential calves, retarded genetic progress, and significant loss of money and time in rebreeding cows. The purpose of this work was to address the relationship between the transcriptional profile of embryos, using the microarray technique, and pregnancy success based on blastocyst biopsies taken prior to transfer to recipients. Biopsies (3 to 40% of the intact embryo) were taken from IVP Day 7 blastocysts (n = 98) and 60 to 70% was transferred to recipients (2-year-old Simmental heifers) after re-expansion. Based on the success of pregnancy, biopsies were pooled in 3 groups: those resulting in no pregnancy (G1), resorption (G2), and calf delivery (G3). Gene expression analysis of these groups of biopsies was performed using a home-made bovine pre-implantation-specific array (with 219 clones) and bovine cDNA array (BlueChip) (with 2000 clones) (Sirard et al. 2005 Reprod. Fertil. Dev. 17, 47–57). Approximately 3 µg of amplified RNA from pooled biopsies (10 biopsies each) was used as a template in reverse transcription reactions incorporating amino-modified dUTPs into labeled cDNA using the CyScribe^TM post-labelling kit (Amersham Bioscience, Freiburg, Germany). The synthesized cDNAs from all 3 groups were differentially labeled using Cy3 and Cy5 dyes. Slides were scanned using a GenePix 4000B scanner and images were analyzed using GenePix Pro Version 4.0 software (Axon Instruments, Union City, CA, USA). Data were analyzed using Significant Analysis for Microarray (SAM) software (http//www.stst.stanford.edu/tips/SAM). Quantitative real-time PCR was used to confirm the differentially expressed genes revealed by microarray experiments. Results revealed that 52 genes were differentially regulated between G1 and G3, and 58 genes were differentially regulated between G2 and G3. Biopsies resulting in calf delivery were enriched with genes necessary for implantation (COX-2 and CDX2), signal transduction (PLAU), polyamine biosynthesis (ODC1), response to oxidative stress (TXN), growth factor (BMP15), and placenta-specific 8 (PLAC8). Biopsies that ended up with resorption were enriched with transcripts involving protein phosphorylation (KRT8) plasma membrane (OCLN), and glucose metabolism (PGK1, AKR1B1). Biopsies resulting in no pregnancy were enriched with transcripts involving inflammatory cytokines (TNF), protein amino acid binding (EEF1A1), transcription factors (MSX1, PTTG1), glucose metabolism (PGK1, AKR1B1), and CD9, which is an inhibitor of implantation. In conclusion, we generated direct candidates of blastocyst-specific genes that determine the fate of the embryo after transfer.