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RNA-Seq analysis uncovers transcriptomic variations between morphologically similar in vivo- and in vitro-derived bovine blastocysts.

Driver AM, Pe├▒agaricano F, Huang W, Ahmad KR, Hackbart KS, Wiltbank MC, Khatib H - BMC Genomics (2012)

Bottom Line: There were also 395 novel transcribed units found, of which 45 were differentially expressed (FDR < 0.05).Using GO enrichment analysis, multiple biological pathways were found to be significantly enriched for differentially expressed genes (FDR < 0.01), including cholesterol and sterol synthesis, system development, and cell differentiation.Thus, our results support that IVF may influence at the transcriptomic level and that morphology is limited in full characterization of bovine preimplantation embryos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Dairy Science, University of Wisconsin, Madison, WI 53706, USA.

ABSTRACT

Background: A valuable tool for both research and industry, in vitro fertilization (IVF) has applications range from gamete selection and preservation of traits to cloning. Although IVF has achieved worldwide use, with approximately 339,685 bovine embryos transferred in 2010 alone, there are still continuing difficulties with efficiency. It is rare to have more than 40% of fertilized in vitro cattle oocytes reach blastocyst stage by day 8 of culture, and pregnancy rates are reported as less than 45% for in vitro produced embryos. To investigate potential influences in-vitro fertilization (IVF) has on embryonic development, this study compares in vivo- and in vitro-derived bovine blastocysts at a similar stage and quality grade (expanded, excellent quality) to determine the degree of transcriptomic variation beyond morphology using RNA-Seq.

Results: A total of 26,906,451 and 38,184,547 fragments were sequenced for in vitro and in vivo embryo pools, respectively. We detected expression for a total of 17,634 genes, with 793 genes showing differential expression between the two embryo populations with false discovery rate (FDR) < 0.05. There were also 395 novel transcribed units found, of which 45 were differentially expressed (FDR < 0.05). In addition, 4,800 genes showed evidence of alternative splicing, with 873 genes displaying differential alternative splicing between the two pools (FDR < 0.05). Using GO enrichment analysis, multiple biological pathways were found to be significantly enriched for differentially expressed genes (FDR < 0.01), including cholesterol and sterol synthesis, system development, and cell differentiation.

Conclusions: Thus, our results support that IVF may influence at the transcriptomic level and that morphology is limited in full characterization of bovine preimplantation embryos.

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Comparison of changes in four differentially expressed genes from the RNA-Seq results (light grey) vs. qRT-PCR (dark grey). Bars above the X-axis denote genes with higher expression in the in vitro embryos while bars below the X-axis denote genes with higher expression in the in vivo embryos.
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Figure 1: Comparison of changes in four differentially expressed genes from the RNA-Seq results (light grey) vs. qRT-PCR (dark grey). Bars above the X-axis denote genes with higher expression in the in vitro embryos while bars below the X-axis denote genes with higher expression in the in vivo embryos.

Mentions: For the validation of RNA-Seq results, four genes showing high levels of significance (CFL1, MYL7, DUS6, and B4GALT4) were selected and normalized to GBG5 via qRT-PCR in the individual embryo aRNA samples. Two genes, CFL1 and MYL7, showed higher expression in the in vitro embryos with an average of 79.5- and 25.5-fold differences, respectively, when measured with qRT-PCR and 18- and 60-fold differences, respectively, using RNA-Seq (Figure 1). The other two genes, DUS6 and B4GALT4, showed higher expression in the in vivo embryos, with an average of 15- and 4.6-fold differences, respectively using qRT-PCR and 21- and 18-fold difference, respectively, with RNA-Seq (Figure 1). Thus, these four genes showed similar patterns of mRNA abundance in RNA-Seq analysis and qRT-PCR.


RNA-Seq analysis uncovers transcriptomic variations between morphologically similar in vivo- and in vitro-derived bovine blastocysts.

Driver AM, Pe├▒agaricano F, Huang W, Ahmad KR, Hackbart KS, Wiltbank MC, Khatib H - BMC Genomics (2012)

Comparison of changes in four differentially expressed genes from the RNA-Seq results (light grey) vs. qRT-PCR (dark grey). Bars above the X-axis denote genes with higher expression in the in vitro embryos while bars below the X-axis denote genes with higher expression in the in vivo embryos.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368723&req=5

Figure 1: Comparison of changes in four differentially expressed genes from the RNA-Seq results (light grey) vs. qRT-PCR (dark grey). Bars above the X-axis denote genes with higher expression in the in vitro embryos while bars below the X-axis denote genes with higher expression in the in vivo embryos.
Mentions: For the validation of RNA-Seq results, four genes showing high levels of significance (CFL1, MYL7, DUS6, and B4GALT4) were selected and normalized to GBG5 via qRT-PCR in the individual embryo aRNA samples. Two genes, CFL1 and MYL7, showed higher expression in the in vitro embryos with an average of 79.5- and 25.5-fold differences, respectively, when measured with qRT-PCR and 18- and 60-fold differences, respectively, using RNA-Seq (Figure 1). The other two genes, DUS6 and B4GALT4, showed higher expression in the in vivo embryos, with an average of 15- and 4.6-fold differences, respectively using qRT-PCR and 21- and 18-fold difference, respectively, with RNA-Seq (Figure 1). Thus, these four genes showed similar patterns of mRNA abundance in RNA-Seq analysis and qRT-PCR.

Bottom Line: There were also 395 novel transcribed units found, of which 45 were differentially expressed (FDR < 0.05).Using GO enrichment analysis, multiple biological pathways were found to be significantly enriched for differentially expressed genes (FDR < 0.01), including cholesterol and sterol synthesis, system development, and cell differentiation.Thus, our results support that IVF may influence at the transcriptomic level and that morphology is limited in full characterization of bovine preimplantation embryos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Dairy Science, University of Wisconsin, Madison, WI 53706, USA.

ABSTRACT

Background: A valuable tool for both research and industry, in vitro fertilization (IVF) has applications range from gamete selection and preservation of traits to cloning. Although IVF has achieved worldwide use, with approximately 339,685 bovine embryos transferred in 2010 alone, there are still continuing difficulties with efficiency. It is rare to have more than 40% of fertilized in vitro cattle oocytes reach blastocyst stage by day 8 of culture, and pregnancy rates are reported as less than 45% for in vitro produced embryos. To investigate potential influences in-vitro fertilization (IVF) has on embryonic development, this study compares in vivo- and in vitro-derived bovine blastocysts at a similar stage and quality grade (expanded, excellent quality) to determine the degree of transcriptomic variation beyond morphology using RNA-Seq.

Results: A total of 26,906,451 and 38,184,547 fragments were sequenced for in vitro and in vivo embryo pools, respectively. We detected expression for a total of 17,634 genes, with 793 genes showing differential expression between the two embryo populations with false discovery rate (FDR) < 0.05. There were also 395 novel transcribed units found, of which 45 were differentially expressed (FDR < 0.05). In addition, 4,800 genes showed evidence of alternative splicing, with 873 genes displaying differential alternative splicing between the two pools (FDR < 0.05). Using GO enrichment analysis, multiple biological pathways were found to be significantly enriched for differentially expressed genes (FDR < 0.01), including cholesterol and sterol synthesis, system development, and cell differentiation.

Conclusions: Thus, our results support that IVF may influence at the transcriptomic level and that morphology is limited in full characterization of bovine preimplantation embryos.

Show MeSH