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.
ABSTRACT
Our objectives were to compare the cellular and molecular effects of aggregating bovine embryonic vs. somatic cell nuclear transfer (ECNT vs. SCNT) embryos and to determine whether aggregation can improve cattle cloning efficiency. We reconstructed cloned embryos from: 1) morula-derived blastomeres, 2) six adult male ear skin fibroblast lines, 3) one fetal female lung fibroblast line (BFF), and 4) two transgenic clonal strains derived from BFF. Embryos were cultured either singularly (1X) or as aggregates of three (3X). In vitro-fertilized (IVF) 1X and 3X embryos served as controls. After aggregation, the in vitro development of ECNT but not that of SCNT or IVF embryos was strongly compromised. The inner cell mass (ICM), total cell (TC) numbers, and ICM:TC ratios significantly increased for all the aggregates. The relative concentration of the key embryonic transcript POU5F1 (or OCT4) did not correlate with these increases, remaining unchanged in the ECNT and IVF aggregates and decreasing significantly in the SCNT aggregates. Overall, the IVF and 3X ECNT but not the 1X ECNT embryos had significantly higher relative POU5F1 levels than the SCNT embryos. High POU5F1 levels correlated with high in vivo survival, while no such correlation was noted for the ICM:TC ratios. Development to weaning was more than doubled in the ECNT aggregates (10/51 or 20% vs. 7/85 or 8% for 3X vs. 1X, respectively; P < 0.05). In contrast, the SCNT and IVF controls showed no improvement in survival. These data reveal striking biological differences between embryonic and somatic clones in response to aggregation.
