Comparative transcriptome analysis of in vivo- and in vitro-produced porcine blastocysts by small amplified RNA-serial analysis of gene expression (SAR-SAGE).
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 of in vitro-produced embryos. The objective of this study was to compare global gene expression patterns from in vivo- (IVO) and in vitro-produced (IVP) porcine embryos using small amplified RNA-serial analysis of gene expression (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 98,771 and 98,408 tags, respectively. A total of 20,029 and 23,453 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 a 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 independent IVO and IVP blastocysts. These results demonstrate compromised gene expression in 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.
Miles
Jeremy R
JR
USDA-ARS, Biotechnology and Germplasm Laboratory, Beltsville, Maryland 20705, USA.
Blomberg
Le Ann
LA
Krisher
Rebecca L
RL
Everts
Robin E
RE
Sonstegard
Tad S
TS
Van Tassell
Curtis P
CP
Zuelke
Kurt A
KA
D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D020224:Expressed Sequence Tags; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D066298:In Vitro Techniques; D008297:Male; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012313:RNA / Q000235:genetics; D034421:Sus scrofa / Q000196:embryology* / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D012313:RNA