134 TRANSCRIPTOME PROFILE OF BOVINE BLASTOCYSTS DERIVED FROM ALTERNATIVE IN VIVO AND IN VITRO CULTURE CONDITIONS AT SPECIFIC PHASES OF EARLY EMBRYONIC DEVELOPMENT

Abstract

An understanding of gene expression patterns due to altered environmental conditions during different time points of the pre-implantation period would improve our knowledge on regulation of embryonic development and improve success of embryo culture. The aim of this study was to examine the effect of alternative in vivo and in vitro culture conditions at specific phases of early embryonic development on transcriptome profile of bovine blastocysts. Using nonsurgical endoscopic oviducal transfer technology, 5 different blastocyst groups were produced. The first 2 groups were matured in vitro and then either transferred after maturation or after in vitro fertilization to synchronized recipients. The other 3 groups were matured, fertilized and cultured in vitro until 4-cell, 16-cell and morula stage before transfer. Blastocysts from each group were collected by uterine flushing at Day 7 and pooled in groups of 10. Complete in vitro (IVP) and in vivo blastocysts were produced and used as controls. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group vs the in vivo control group to examine the transcriptome profile of blastocysts. Compared with the in vivo control group, clear dramatic shifts were found in the number of differentially expressed genes (DEG, fold change ≥2) at 2 different time points. The first shift occurred for blastocyst groups that were transferred after in vitro fertilization and before embryonic genome activation (EGA). The second shift occurred for blastocyst groups that were transferred after EGA, as well as for the IVP group. Ontological classification of DEG showed that the more time spent under in vitro conditions, the higher the percentage of DEG involved in cell death and apoptotic processes. Moreover, lipid metabolism was the most significant process affected in the blastocysts transferred after in vitro maturation and blastocysts transferred at 16-cell stage. Most DEG involved in this process were down-regulated. Pathway analysis revealed that signalling pathways were the dominant pathways in all groups except the group that was transferred after in vitro maturation. That group showed significant down-regulation for genes involved in retinoic acid receptors activation pathways. These results showed that fertilization and EGA were the most critical developmental stages influenced by in vitro culture conditions and subsequently affect blastocyst quality, as measured in terms of gene expression patterns. Moreover, we identified molecular mechanisms and pathways that were influenced by altered culture conditions. These findings will enable the examination of strategies for modifying in vitro culture conditions at critical stages that will allow more efficient production of developmentally competent blastocysts.