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

Driver AM, Peñagaricano 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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Morphological assessment of embryos at the blastocyst stage with a grade of 7-1 according to IETS standards. A = in vivo blastocyst; B = in vitro blastocyst.
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Figure 3: Morphological assessment of embryos at the blastocyst stage with a grade of 7-1 according to IETS standards. A = in vivo blastocyst; B = in vitro blastocyst.

Mentions: In vivo embryos were collected from non-superovulated lactating Holstein cows (n = 84, multiparous; approximately 77 days after calving) at the University of Wisconsin-Madison. To allow a synchronous follicular development and tight regulation of ovulatory follicle size and time of ovulation, cows were synchronized with the Double OvSynch protocol similar to that described in Souza et al. [30]. Most cows have single ovulation after this protocol and exhibit excellent fertility for lactating dairy cows [30]. Cows were artificially inseminated with semen from one of three high-fertility bulls. The use of a large number of cows and three bulls was to ensure an adequate number of excellent quality embryos produced from a single sire. Cows were evaluated using ultrasonography to determine where ovulation occurred, and seven days post-insemination, the ipsilateral horn(s) underwent a non-surgical uterine horn flushing for embryo recovery as described in Hackbart et al. [31]. Embryos were then graded for embryo quality (1 = excellent or good, 2 = fair, 3 = poor, and 4 = degenerate) and embryo stage (1 = 1-cell, 2 = 2-16 cell, 3 = early morula, 4 = compacting morula, 5 = early blastocyst, 6 = blastocyst, 7 = expanded blastocyst, 8 = hatched blastocyst) according to International Embryo Transfer Society (IETS) standards [32]. For the purpose of this study, we collected blastocyst stage embryos that were expanded and with excellent quality (Figure 3). Upon collection, embryos were placed in RNALater (Ambion, TX) to preserve RNA integrity and frozen at -20°C until RNA extraction.


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

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

Morphological assessment of embryos at the blastocyst stage with a grade of 7-1 according to IETS standards. A = in vivo blastocyst; B = in vitro blastocyst.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Morphological assessment of embryos at the blastocyst stage with a grade of 7-1 according to IETS standards. A = in vivo blastocyst; B = in vitro blastocyst.
Mentions: In vivo embryos were collected from non-superovulated lactating Holstein cows (n = 84, multiparous; approximately 77 days after calving) at the University of Wisconsin-Madison. To allow a synchronous follicular development and tight regulation of ovulatory follicle size and time of ovulation, cows were synchronized with the Double OvSynch protocol similar to that described in Souza et al. [30]. Most cows have single ovulation after this protocol and exhibit excellent fertility for lactating dairy cows [30]. Cows were artificially inseminated with semen from one of three high-fertility bulls. The use of a large number of cows and three bulls was to ensure an adequate number of excellent quality embryos produced from a single sire. Cows were evaluated using ultrasonography to determine where ovulation occurred, and seven days post-insemination, the ipsilateral horn(s) underwent a non-surgical uterine horn flushing for embryo recovery as described in Hackbart et al. [31]. Embryos were then graded for embryo quality (1 = excellent or good, 2 = fair, 3 = poor, and 4 = degenerate) and embryo stage (1 = 1-cell, 2 = 2-16 cell, 3 = early morula, 4 = compacting morula, 5 = early blastocyst, 6 = blastocyst, 7 = expanded blastocyst, 8 = hatched blastocyst) according to International Embryo Transfer Society (IETS) standards [32]. For the purpose of this study, we collected blastocyst stage embryos that were expanded and with excellent quality (Figure 3). Upon collection, embryos were placed in RNALater (Ambion, TX) to preserve RNA integrity and frozen at -20°C until RNA extraction.

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