Comparative transcriptome analysis of in vivo‐ and in vitro‐produced porcine blastocysts by small amplified RNA‐Serial analysis of gene expression (SAR‐SAGE)

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 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.

Transcriptional profiling by RNA-Seq of peri-attachment porcine embryos generated by a variety of assisted reproductive technologies

“…Significant differences in the experimental platforms, statistical analyses, and embryo stages might all be invoked as reasons behind this seeming discrepancy. In support of the suggestion that the experimental platform can have profound influences on experimental results even in very similarly conceived experimental designs, Miles et al (48) used serial analysis of gene expression to assess relative transcript abundance in porcine blastocysts and found Ͼ900 transcripts that were putatively differentially expressed in embryos generated in vitro vs. in vivo. Also, as the genes presented herein as differentially expressed are only those that were shown to be different across all gestation stages, it is highly unlikely that asynchrony between embryo types is responsible for the high number of aberrantly expressed genes.…”

Effects of oviductal fluid on the development, quality, and gene expression of porcine blastocysts produced in vitro

“…This hypothesis should be further investigated but it is not unusual for genes that are involved with maintaining the integrity of the mitochondrial respiratory chain (e.g. ATP5B and COX7B) to be downregulated in poor quality blastocysts compared with good quality blastocysts in the pig (Miles et al 2008).…”

Long term effects of ART: What do animals tell us?

“…Comparison of in vivo developed to IVP pig blastocysts by small amplified RNA‐serial analysis evidenced 938 differentially expressed tag sequences, the majority of which were associated with cellular metabolism GO function (followed by cellular transport, regulation of physiological process, and signal transduction). Deeper analysis of metabolism‐involved differentially expressed transcripts evidenced a reduced oxidative phosphorylation in IVP blastocysts, so that defects in mitochondrial function and in energy production could be responsible for their reduced developmental ability (Miles et al, ). Focusing on the effect of in vitro culture only and comparing in vitro cultured to in vivo developed blastocysts obtained from the contralateral horn of the same artificially inseminated gilts, 1170 differentially expressed genes were identified, with an upregulation of genes involved in mRNA transcription, nucleotide and DNA metabolism, amino acid metabolism, lipid metabolism and a down‐regulation of genes involved in carbohydrate metabolism, proteolysis, protein glycosylation ATP binding, lipid, fatty acid and steroid metabolism (Bauer et al, ).…”