Spatial separation of parental genomes in preimplantation mouse embryos

Abstract

We have used two different experimental approaches to demonstrate topological separation of parental genomes in preimplantation mouse embryos: mouse eggs fertilized with 5-bromodeoxyuridine (BrdU)-labeled sperm followed by detection of BrdU in early diploid embryos, and differential heterochromatin staining in mouse interspecific hybrid embryos. Separation of chromatin according to parental origin was preserved up to the four-cell embryo stage and then gradually disappeared. In F1 hybrid animals, genome separation was also observed in a proportion of somatic cells. Separate nuclear compartments during preimplantation development, when extreme chromatin remodelling occurs, and possibly in some differentiated cell types, may be associated with epigenetic reprogramming.

Figure 1

Distribution of paternal chromatin in early mouse embryos. BrdU-treated male mice were mated with untreated females and the resulting embryos stained with FITC-conjugated anti-BrdU antibody (green). Nuclei and chromosomes were counterstained with DAPI (blue). a, Highly condensed sperm nucleus and fertilized egg (3). Numbers in parentheses indicate the number of embryos analyzed. b, Male and female pronuclei at 10 h after fertilization (20). The somewhat larger male pronucleus shows a nearly uniform BrdU staining, indicating that the entire sperm DNA is substituted with BrdU. c, After nuclear envelope breakdown the two chromosome sets form a single diploid nucleus (2). d, First metaphase at 20 h after fertilization (5). e, Two-cell embryo during G₁ phase at 22 h (>10). The second polar body remains completely BrdU negative. f, Two-cell embryo during G₂ phase at 32 h (>10). The male chromatin occupies approximately half of the nuclear volume. g, Four-cell embryo and second polar body at 45 h after fertilization (10). At this point, only half of the paternal chromosomes are still labeled with BrdU. h, 32-cell embryo at 78 h (>5). The one or two BrdU-positive sperm DNA strands per nucleus are consistent with random strand-segregation mechanisms. Bars, 10 μm.

Figure 2

Distribution of paternal and maternal centromeres in early mouse embryos derived from matings between (untreated) MSP males and MMU females. Biotinylated MSP genomic DNA and digoxigenated MMU genomic DNA were hybridized together and detected with FITC-avidin and Cy3-conjugated antidigoxin antibody. Chromosomes and nuclei were counterstained with DAPI (blue). a, Well-spread first metaphase of MMU × MSP hybrid embryo. The left image shows DAPI staining and the right image comparative FISH of the same spread. The maternal MMU centromeres exhibit red FISH signals. The paternal centromeres are stained in green. b, Two-cell F1 embryo (>10). Numbers in parentheses indicate the number of embryos analyzed. Maternal and paternal centromere complements remain separated and together occupy approximately half of the nucleus. c, Four-cell embryo showing genome separation and centromere clustering (10). Note the absence of green (paternal centromere) fluorescence in the polar body. d, Eight-cell embryo (5). Separation of the two centromere sets is seen in some, but not all, cells. Bars, 10 μm.

Figure 3

Distribution of paternal (green) and maternal (red) centromeres in somatic cells of MMU × MSP hybrid animal. a, Peritoneal fibroblast nuclei (>500) displaying spatial separation of paternal and maternal heterochromatin blocks. Numbers in parentheses indicate the number of cells analyzed. Bar, 10 μm. b, Random distribution of MMU and MSP chromosomes around representative prometaphase rosettes (>50).