Dynamic Changes of Nuclear Halo Structure of In Vivo-Fertilized and Cloned Preimplantation Mouse Embryos.

Abstract

Eukaryotic chromosomes are organized into domains through matrix attachment regions playing important roles in transcription, recombination and replication. The non-matrix-associated domains form loops of DNA which appear as a nuclear halo surrounding the nuclear matrix. Little is known regarding DNA looping in mouse preimplantation embryos. In this study we monitored the changes in nuclear halo structures of in vivo fertilized embryos as well as those derived from somatic cell nuclear transfer (SCNT). Nuclear halos were prepared by extraction with 2 M NaCl then visualized with propidium iodide. The halo diameters were measured by confocal microscopy. Embryos fertilized in vivo and cultured in vitro were collected at the pronuclear, 2-, 4- and 8-cell stages. The average diameter of the nuclear halo decreased significantly from the pronuclear to the 8-cell stage (male pronuclei: 29.53± 0.86 μm (n=5); female pronuclei: 20.04±1.00 μm (n=5); 2-cell: 22.88± 0.49 μm (n=10); 4-cell: 16.16±0.31 μm (n=6); 8-cell: 12.67± 0.18 μm (n=56)) showing dynamic changes in halo size occur during mouse early embryo development. Because TSA treatment increases mouse SCNT efficiency, we then examined SCNT embryos at 1 h post donor injection, as well as 6 h and 10 h post chemical activation with and without Trichostatin A (TSA) treatment. Premature Chromosome Condensation was observed at 1 h post cell injection. Nuclear halo extraction procedure significantly increased the size of nuclear halo at 6 and 10 h post-activation. Interestingly, we also found significant decreases in nuclear halo areas after TSA treatment at 6 h post-activation (738.4± 78 μm2 (n=7) vs. 483.4± 42.5μm2 (n=7)). Taken together, DNA looping analysis can be used to access nuclear organization during preimplantation embryo development and to begin to investigate the mechanism of nuclear reprogramming by SCNT.