Double cytoplast embryonic cloning improves in vitro but not in vivo development from mitotic pluripotent cells in cattle

Sarah Jane Appleby; Pavla Misica-Turner; Fleur Catherine Oback; Arindam Dhali; Zachariah Louis McLean; Björn Oback

doi:10.3389/fgene.2022.933534

Double cytoplast embryonic cloning improves in vitro but not in vivo development from mitotic pluripotent cells in cattle

Front Genet. 2022 Sep 28:13:933534. doi: 10.3389/fgene.2022.933534. eCollection 2022.

Authors

Sarah Jane Appleby^{1

2}, Pavla Misica-Turner¹, Fleur Catherine Oback¹, Arindam Dhali¹, Zachariah Louis McLean^{1

2}, Björn Oback^{1

2

3}

Affiliations

¹ Animal Biotech, AgResearch, Hamilton, New Zealand.
² School of Science, University of Waikato, Hamilton, New Zealand.
³ School of Medical Sciences, University of Auckland, Auckland, New Zealand.

Abstract

Cloning multiple animals from genomically selected donor embryos is inefficient but would accelerate genetic gain in dairy cattle breeding. To improve embryo cloning efficiency, we explored the idea that epigenetic reprogramming improves when donor cells are in mitosis. We derived primary cultures from bovine inner cell mass (ICM) cells of in vitro fertilized (IVF) embryos. Cells were grown feeder-free in a chemically defined medium with increased double kinase inhibition (2i⁺). Adding recombinant bovine interleukin 6 to 2i⁺ medium improved plating efficiency, outgrowth expansion, and expression of pluripotency-associated epiblast marker genes (NANOG, FGF4, SOX2, and DPPA3). For genotype multiplication by embryonic cell transfer (ECT) cloning, primary colonies were treated with nocodazole, and single mitotic donors were harvested by mechanical shake-off. Immunofluorescence against phosphorylated histone 3 (P-H3) showed 37% of nocodazole-treated cells in metaphase compared to 6% in DMSO controls (P < 1 × 10^-5), with an average of 53% of P-H3-positive cells expressing the pluripotency marker SOX2. We optimized several parameters (fusion buffer, pronase treatment, and activation timing) for ECT with mitotic embryonic donors. Sequential double cytoplast ECT, whereby another cytoplast was fused to the first cloned reconstruct, doubled cloned blastocyst development and improved morphological embryo quality. However, in situ karyotyping revealed that over 90% of mitotic ECT-derived blastocysts were tetraploid or aneuploid with extra chromosomes, compared to less than 2% in the original ICM donor cells. Following the transfer of single vs. double cytoplast embryos, there was no difference between the two methods in pregnancy establishment at D35 (1/22 = 5% vs. 4/53 = 8% for single vs. double ECT, respectively). Overall, post-implantation development was drastically reduced from embryonic mitotic clones when compared to somatic interphase clones and IVF controls. We conclude that mitotic donors cause ploidy errors during in vitro development that cannot be rescued by enhanced epigenetic reprogramming through double cytoplast cloning.

Keywords: bovine; cell cycle; cloning; embryo (animal); mitotic; pluripotent.