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. Author manuscript; available in PMC: 2017 Oct 1.
Published in final edited form as: Nat Cell Biol. 2017 Mar 6;19(4):282–291. doi: 10.1038/ncb3485

Figure 4.

Figure 4

Nuclear remodelling is necessary for normal mitotic segregation. (a) Schematic of interphase transfer. To prevent chromosomal condensation, G0/G1 donor nuclei were transferred into interphase oocytes (G1 → G1, top panel). As controls, mitotic donor nuclei were transferred into G1 oocytes (M → G1, middle panel) and G1 donor nuclei into MII oocytes (G1 → MII, bottom panel), respectively. Grey boxes indicate somatic chromosome condensation. (b) DNA replication in NT embryos incubated with EdU (green) for 1 h at the indicated times after activation. (c) Immunostaining for H3K9me3 distribution and γH2AX in NT embryos at G2 (13 h postactivation). (d) Mitotic chromosome segregation following G1 → G1 NT. (e) Arrested G1 → G1 NT embryo. (f) Quantification of chromosomal segregation errors after transfer of interphase nuclei from ESCs, T cells or cumulus cells at the indicated cell cycle stages. The bars represent the mean from n=54 (M → MII, ESCs), 40 (M → G1, ESCs), 44 (G1 → G1, ESCs), 124 (M → MII, T cells), 42 (M → G1, T cells), 37 (G1 → G1, T cells), 48 (G1 → MII, cumulus) and 75 (G1 → G1, cumulus) number of embryos. (g) Immunostaining at the first mitosis in a cumulus cell NT embryo using oocytes activated immediately after transfer. (h) Percentage of embryos with abnormal chromosomal segregation at the first mitosis when activated at the indicated time points after transfer of naive T cells. n = 28 (2–3 h), 5 (1 h) and 7 (0 h) embryos. (i) Schematic of NT at G1 of the 2-cell stage. (j,k) Replicated DNA (EdU labelling) in blastomeres of control parthenotes (j) and 2-cell NT embryos (k) at 12 h post-transfer. Insets show nuclei at greater magnification and arrowheads point to somatic heterochromatin. (l) Immunostaining for H3K9me3 in 2-cell G1 NT embryos. (m) Percentage of EdU-positive blastomeres in parthenotes and G1 2-cell NT embryos at the indicated time points after the first cleavage. n=4 (parthenotes, 3 h), 8 (NT, 3 h), 12 (parthenotes, 24 h), 38 (NT, 24 h), 11 (parthenotes, 48 h) and 30 (NT, 48 h) blastomeres. (n) Abnormal cleavage of 2-cell NT embryo. (Parth, parthenote). Scale bars, 10 μm.