Identification and quantification of differentially represented transcripts in preimplantation bovine embryos.

摘要

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine expressed sequence tag (EST) project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's Exact Test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3,144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P ≤ 0.01) in at least one pairwise comparison. Fifteen of these transcripts were selected for further examination using quantitative real-time PCR. LSMEANS (SAS PROC GLM) were used to determine significant differences in transcript abundance. QRTPCR confirmed that nine of the 15 transcripts were significantly differentially represented in at least one pairwise comparison, while three more of the transcripts exhibited a strong trend (P 0.05) of different abundance levels in at least one pairwise comparison. By further investigating these results, we may be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro-produced and nuclear transfer-derived embryos which more closely follow a normal program of development.