Fig. 4: Micronucleus extraction from ACS embryos and analysis.

a, b Micronuclei extraction from ACS embryos at the two-cell stage. The arrow shows a micronucleus. c Rate of micronuclei extracted embryos by type of ACS embryos. Blue, embryos from which one micronucleus could be extracted; red, embryos that attempted to extract one micronucleus but simultaneously extracted another micronucleus from another blastomere; black, embryos that could not be extracted because the nucleus was also sucked into the pipette when the micronucleus was taken out. d, e If the micronucleus is located close to the nucleus, when the micronucleus is pulled, the nucleus moves with it and is sucked into the pipette. f–i Embryos from which micronuclei could not be extracted were fixed and stained to examine the tubulin binding and presence or absence of major satellite regions. Circles or arrows indicate micronuclei. Numbers indicate micronucleus ID. When attempting to extract micronuclei, the nucleus also moved (f, g), so micronuclei extraction was stopped, the binding of tubulin to micronuclei (h), and the presence of major satellite regions was examined (i). The right section shows high magnification. j–n When micronucleus could be extracted, those micronuclei were injected into enucleated or intact oocytes and fixed and stained as above. j–l Micronuclei injected into enucleated oocytes. The arrow shows a micronucleus. m Micronucleus in enucleated oocytes derived from IFM embryos. m-2 and m-3 shows a high magnification of the square. n Two micronuclei in enucleated oocyte derived from EPM embryos. n-2 and n-3 shows a high magnification of the square. The arrow shows a micronucleus. Bar, 10 µm. o, p The relationships among DNA bridge, tubulin binding (o), and presence/absence of major satellite regions (p) in micronuclei that could not be extracted from embryo (o-1 or p-1) and micronuclei that could be extracted from embryo (o-2 or p-2) are summarized.