200 IN VITRO EMBRYO PRODUCTION IS ASSOCIATED WITH DISTINCT ALTERATIONS IN THE TRANSCRIPTOME BETWEEN THE BLASTOCYST STAGE AND THE INITIATION OF ELONGATION IN CATTLE

Abstract

The majority of embryonic loss in cattle occurs before maternal recognition of pregnancy around Day 16 postconception. The origin of the embryo can have a significant impact on the dynamics of embryo mortality. The aim of the present study was to compare transcript profiles between Day 7 (spherical blastocyst) and Day 13 (ovoid conceptus) bovine embryos derived in vitro or in vivo using the bovine Affymetrix microarray. Subsequent mapping of these differentially expressed genes (DEG) into relevant functional groups and pathways using ingenuity pathway analysis would identify the most important pathways involved in conceptus elongation in cattle. All embryos were produced either in vitro or in vivo by superovulation. A proportion of Day 7 blastocysts were snap frozen and the remainder were transferred (n = 10 per recipient) to synchronized heifers, recovered on Day 13, and snap frozen individually. Three pools of Day 7 blastocysts (n = 25 per pool) and Day 13 conceptuses (n = 5 per pool) were used for microarray analysis. A total of 909 and 1806 transcripts were differentially expressed between Day 7 and 13 in in vivo- and in vitro-derived embryos, respectively, of which a core of 465 transcripts was common to both groups. These 465 genes, likely to be crucial for the transition from blastocyst to the initiation of elongation, were associated with 5 canonical pathways, including interleukin (IL)-6 and IL-10 signalling, the endoplasmic reticulum stress pathway, and nitrogen metabolism. The top networks identified genes associated with 1) cellular development, lipid metabolism, and small molecular biochemistry; 2) cell-to-cell signaling; and 3) amino acid metabolism, including transcripts such as MYC, SLC25A12, HSPH1, LXN, ALDH18A1, PMP22, PEG3, CDH2, and Hsp70, which were up-regulated in the conceptus on Day 13. The top canonical pathways among the 444 DEG unique to in vivo embryos were 1) glycerolipid metabolism, 2) steroid biosynthesis, and 3) acute phase response signalling. One of the interaction networks identified was associated with cardiovascular system development and function, gene expression, and organismal development, including transcripts of proteins such as laminins and claudins, which are important for cell communication and morphogenesis during embryonic development. Among the 1341 DEG unique to in vitro-produced embryos, the top pathways were 1) glycerophospholipid metabolism, 2) IL-6 signalling, and 3) endothelial-1 signalling, whereas one of the interaction networks identified was associated with hematological disease, organismal injury, and abnormalities and cellular development, including POU5F1, a transcription factor known to bind to DNA and activate or repress transcription of several genes important for early embryonic development. In conclusion, this analysis has identified genes and pathways crucial to the transition from a spherical blastocyst to an ovoid conceptus as well as those uniquely associated with a greater likelihood of embryonic survival (those unique to in vivo embryos) or loss (those unique to in vitro embryos).