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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.

Show MeSH
Alternative splicing validations for two genes using PCR with cDNA as the template. Lanes A and B correspond to the two fragment sizes for the gene AP2B1 (134 and 92 bp) while G and H represent the two transcripts for ZDHHC16 (97 and 118 bp). Lanes C and I are positive controls (GBG5, 115 bp) while D, E, F, J, K, and L are negative controls.
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Figure 2: Alternative splicing validations for two genes using PCR with cDNA as the template. Lanes A and B correspond to the two fragment sizes for the gene AP2B1 (134 and 92 bp) while G and H represent the two transcripts for ZDHHC16 (97 and 118 bp). Lanes C and I are positive controls (GBG5, 115 bp) while D, E, F, J, K, and L are negative controls.

Mentions: Validation of alternative splicing was confirmed for the genes AP2B1 and ZDHHC16. Both of these genes showed evidence for single exon skipping. As such, primers were designed around this region to confirm the splicing event. The first primer set for AP2B1 had exon inclusion and produced a 134-bp product in comparison to the second primer set excluding the exon which resulted in a 92-bp product (Figure 2). For ZDHHC16, two primers sets were also created, with the first set producing a 97-bp product (transcript that excludes the exon) and the second set producing a 118-bp product that included the skipped exon (Figure 2). Since both transcripts were present in both embryo samples for each gene, although differing in ratio, they were tested in a pool each of in vivo and in vitro embryo cDNA for validation. Overall, RNA-Seq results for alternative splicing were also validated.


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)

Alternative splicing validations for two genes using PCR with cDNA as the template. Lanes A and B correspond to the two fragment sizes for the gene AP2B1 (134 and 92 bp) while G and H represent the two transcripts for ZDHHC16 (97 and 118 bp). Lanes C and I are positive controls (GBG5, 115 bp) while D, E, F, J, K, and L are negative controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Alternative splicing validations for two genes using PCR with cDNA as the template. Lanes A and B correspond to the two fragment sizes for the gene AP2B1 (134 and 92 bp) while G and H represent the two transcripts for ZDHHC16 (97 and 118 bp). Lanes C and I are positive controls (GBG5, 115 bp) while D, E, F, J, K, and L are negative controls.
Mentions: Validation of alternative splicing was confirmed for the genes AP2B1 and ZDHHC16. Both of these genes showed evidence for single exon skipping. As such, primers were designed around this region to confirm the splicing event. The first primer set for AP2B1 had exon inclusion and produced a 134-bp product in comparison to the second primer set excluding the exon which resulted in a 92-bp product (Figure 2). For ZDHHC16, two primers sets were also created, with the first set producing a 97-bp product (transcript that excludes the exon) and the second set producing a 118-bp product that included the skipped exon (Figure 2). Since both transcripts were present in both embryo samples for each gene, although differing in ratio, they were tested in a pool each of in vivo and in vitro embryo cDNA for validation. Overall, RNA-Seq results for alternative splicing were also validated.

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