233 ASSOCIATION OF THE BOVINE 2-CELL-STAGE BLASTOMERE TRANSCRIPTOME PROFILE WITH THE INDIVIDUAL DEVELOPMENTAL POTENTIAL OF THE SISTER BLASTOMERE

Abstract

Transcriptome profiling has been used to identify genes related to developmental competence in bovine embryos and oocytes for several years. However, the direct relationship between the transcriptome profile and developmental potential of the same pre-implantation embryo is missing. Therefore, in the present study, one blastomere of a 2-cell-stage embryo was taken as a biopsy and immediately snap-frozen for transcriptome analysis; the sister blastomere was cultured individually in a well-of-the-well culture system. Frozen individual blastomeres taking the form of 2-cell-stage embryos were pooled together, depending on the developmental destination of the sister blastomeres. Accordingly, three groups were defined: 1) embryos that did not cleave after separation (2CB), 2) embryos arrested before embryonic genome activation (8CB), and 3) embryos that reached the blastocyst stage (BL). On the basis of these developmental phenotypes, blastomeres were pooled and used for transcriptome analysis, using a bovine EmbryoGene microarray platform (Agilent Technologies, Santa Clara, CA, USA). Results revealed 632 genes to be differentially regulated (fold change, P ≥ 1.5, P ≤ 0.05; FDR, P ≤ 0.1) between competent (BL group) and incompetent 2-cell-stage embryos (2CB) as well as 150 genes between the BL group and 8CB. Seventy-seven genes were commonly differentially regulated. Functional annotation analyses of differentially regulated genes indicated those genes to be involved in the protein ubiquitination pathway on the one hand and in the oxidative stress response, including oxidoreductase, peroxidase, and antioxidant activity, as well in oxidative phosphorylation (e.g. NDUFS1, MAPK14, CAT, PRDX1, and PRDX6) on the other hand. Furthermore, selected candidate genes known to function as direct and indirect scavengers of reactive oxygen species (ROS) were analysed for their expression in an independent model for developmental competence; namely, early- and late-cleaved 2-cell-stage embryos. Moreover, ROS detection was performed, showing higher accumulation of ROS in late-cleaved 2-cell-stage embryos, whereas lower ROS levels were detected in early-cleaved 2-cell-stage embryos associated with higher expression of ROS scavengers. The overall findings of the present study indicate the potential of using blastomere biopsies at 2-cell-stage embryos for molecular analysis to understand the molecular mechanisms associated with the further developmental competence of early-stage embryos.