ETV2-null porcine embryos survive to post-implantation following incomplete enucleation

in Reproduction
Authors:
Geunho Maeng Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA

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Wuming Gong Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA

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Satyabrata Das Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA

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Demetris Yannopoulos Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA

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Daniel J Garry Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA
Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA
Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota, Minneapolis, Minnesota, USA
NorthStar Genomics, Eagan, Minnesota, USA

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Mary G Garry Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA
Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA
Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota, Minneapolis, Minnesota, USA
NorthStar Genomics, Eagan, Minnesota, USA

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Correspondence should be addressed to M G Garry; Email: garry002@umn.edu
DOI:
https://doi.org/10.1530/REP-19-0382
Volume/Issue:
Volume 159: Issue 5
Page Range:
539–547
Article Type:
Research Article
Online Publication Date:
May 2020
Copyright:
© 2020 Society for Reproduction and Fertility 2020
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Blind enucleation is used in porcine somatic cell nuclear transfer (SCNT) to remove the metaphase II (MII) spindle from the oocyte. Deviation of the MII spindle location, however, leads to incomplete enucleation (IE). Here, we report that the rate of complete enucleation (CE) using the blind method was 80.2 ± 1.7%, although this significantly increased when the polar body-MII deviation was minimized (≦45°). While it is established that IE embryos will not survive to full term, the effect of IE on early stage development is unknown. We have previously demonstrated in mice and pigs that ETV2 deletion results in embryonic lethality due to the lack of hematoendothelial lineages. We observed that ETV2-null cloned embryos derived from blindly and incompletely enucleated oocytes had both WT and mutant sequences at E18 and, using FISH analysis, we observed triploidy. We also compared SCNT embryos generated from either CE or intentionally IE oocytes using the spindle viewer system. We observed a higher in vitro blastocyst rate in the IE versus the CE-SCNT embryos (31.9 ± 3.2% vs 21.0 ± 2.1%). Based on known processes in normal fertilization, we infer that the IE-SCNT embryos extruded the haploid second PB after fusion with donor fibroblasts and formed a near-triploid aneuploid nucleus in each blastomere. These studies demonstrate the peri-implantation survival of residual haploid nuclei following IE and emphasize the need for complete enucleation especially for the analysis of SCNT embryos in the peri-implantation stage and will, further, impact the field of reverse xenotransplantation.

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Print ISSN:
1470-1626
Online ISSN:
1741-7899

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