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Title: COMPARATIVE TRANSCRIPTOME ANALYSIS OF IN VIVO AND IN VITRO PORCINE BLASTOCYSTS BY SMALL AMPLIFIED RNA SERIAL ANALYSIS OF GENE EXPRESSION (SAR-SAGE)

Author
item Miles, Jeremy
item Blomberg, Le Ann
item KRISHER, REBECCA - PURDUE UNIVERSITY
item EVERTS, ROBIN - UNIV OF ILLINOIS
item Sonstegard, Tad
item Van Tassell, Curtis - Curt
item Zuelke, Kurt

Submitted to: Molecular Reproduction and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/19/2007
Publication Date: 6/1/2008
Citation: Miles, J.R., Blomberg, L.A., Krisher, R.L., Everts, R.E., Sonstegard, T.S., Van Tassell, C.P., Zuelke, K.A. 2008. Comparative transcriptome analysis of in vivo- and in vitro- produced porcine blastocysts by small amplified RNA-serial analysis of gene expression (SAR-SAGE). Molecular Reproduction and Development. 75:976-88.

Interpretive Summary: Production of embryos in vitro is an important biotechnology with enormous potential for research and commercial applications. Unfortunately, despite several advancements in the in vitro production of embryos, the overall efficiency of in vitro embryo production in pigs remains extremely low, even lower than other livestock species. In vitro-produced pig embryos develop a number of abnormal phenotypes compared with their in vivo-produced counterparts. We hypothesized that the altered phenotypes observed following the production of pig embryos in vitro are a result of global mRNA expression (transcriptome) deviations between in vitro-produced and in vivo-produced porcine embryos. Therefore, the objective of this study was to establish and compare transcriptome profiles from in vitro-produced and in vivo-produced porcine embryos using small amplified RNA-serial analysis of gene expression (SAR-SAGE). The SAR-SAGE analysis identified significant deviations in transcriptome profiles between porcine embryos produced in vivo or in vitro. There was a significant depression in the overall mRNA expression in in vitro-produced pig embryos compared with embryos generated in vivo. A number of compromised biological processes, particularly mitochondrial mechanisms, were identified that may begin to shed light on potential molecular pathways that are disrupted during in vitro production. Thus, the results present a physiological explanation as to why in vitro-produced porcine embryos have a decreased developmental competence and display abnormal phenotypes. Furthermore, this in-depth analysis of transcriptomes from in vitro-produced and in vivo-produced porcine embryos provides insight on potential markers of competence and potential pathways that can be targeted for the improvement of in vitro production methods.

Technical Abstract: Production of embryos in vitro has enormous potential for research and commercial applications. Unfortunately, in vitro production of porcine embryos is extremely inefficient. Despite the characterization of distinct phenotypes, little is known about the molecular mechanisms and altered physiological processes that account for poor development of in vitro- produced embryos. The objective of the current study was to compare global gene expression patterns from in vivo- (IVO) and in vitro-produced (IVP) embryos using SAR-SAGE. Whole-cell RNA from pools of Day 6 IVO and IVP blastocysts was used to construct SAR-SAGE libraries. Sequence analysis of the IVO and IVP libraries yielded a total of 98,771 and 98,408 tags, respectively. A total of 20,029 and 23,453 unique putative transcripts were detected in the IVO and IVP libraries, respectively. Statistical analyses of SAGE tag frequencies between the IVO and IVP libraries indicated that 938 and 193 tags were differentially expressed at a P < 0.05 and P < 0.001 level of significance, respectively, suggesting significant deviations in transcriptome profiles from IVO and IVP embryos. Categorization of differentially expressed transcripts into functional groupings indicated significant deviation in gene expression from IVP blastocysts compared with IVO blastocysts for a number of biological processes including cellular metabolism, organization and response to stress. Real-time PCR confirmed differential expression for several transcripts from IVO and IVP blastocysts. These results demonstrate compromised gene expression from IVP blastocysts compared with IVO blastocysts for a number of biological processes, particularly processes involved in mitochondrial function; thereby providing potential target pathways for improvement of IVP methods.