Nuclear landscapes were studied during preimplantation development of bovine embryos, generated either by in vitro fertilization (IVF), or generated as cloned embryos by somatic cell nuclear transfer (SCNT) of bovine fetal fibroblasts, using 3-dimensional confocal laser scanning microscopy (3D-CLSM) and structured illumination microscopy (3D-SIM). Nuclear landscapes of IVF and SCNT embryonic nuclei were compared with each other and with fibroblast nuclei. We demonstrate that reprogramming of fibroblast nuclei in cloned embryos requires changes of their landscapes similar to nuclei of IVF embryos. On the way toward the 8-cell stage, where major genome activation occurs, a major lacuna, enriched with splicing factors, was formed in the nuclear interior and chromosome territories (CTs) were shifted toward the nuclear periphery. During further development the major lacuna disappeared and CTs were redistributed throughout the nuclear interior forming a contiguous higher order chromatin network. At all stages of development CTs of IVF and SCNT embryonic nuclei were built up from chromatin domain clusters (CDCs) pervaded by interchromatin compartment (IC) channels. Quantitative analyses revealed a highly significant enrichment of RNA polymerase II and H3K4me3, a marker for transcriptionally competent chromatin, at the periphery of CDCs. In contrast, H3K9me3, a marker for silent chromatin, was enriched in the more compacted interior of CDCs. Despite these striking similarities, we also detected major differences between nuclear landscapes of IVF and cloned embryos. Possible implications of these differences for the developmental potential of cloned animals remain to be investigated. We present a model, which integrates generally applicable structural and functional features of the nuclear landscape.
Keywords: 3D-CLSM, 3-dimensional confocal laser scanning microscopy; 3D-SIM, 3-dimensional structured illumination microscopy; B23, nucleophosmin B23; BTA, Bos taurus; CDC, chromatin domain cluster; CT, chromosome territory; EM, electron microscopy; ENC, embryonic nuclei with conventional nuclear architecture; ENP, embryonic nuclei with peripheral CT distribution; H3K4me3; H3K4me3, histone H3 with tri-methylated lysine 4; H3K9me3; H3K9me3, histone H3 with tri-methylated lysine 9; H3S10p, histone H3 with phosphorylated serine 10; IC, interchromatin compartment; IVF, in vitro fertilization; MCB, major chromatin body; PR, perichromatin region; RNA polymerase II; RNA polymerase II-S2p, RNA polymerase II with phosphorylated serine 2 of its CTD domain; RNA polymerase II-S5p, RNA polymerase II with phosphorylated serine 5 of its CTD domain; SC-35, splicing factor SC-35; SCNT, somatic cell nuclear transfer.; bovine preimplantation development; chromatin domain; chromosome territory; embryonic genome activation; in vitro fertilization (IVF); interchromatin compartment; major EGA, major embryonic genome activation; somatic cell nuclear transfer (SCNT).