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Spatial separation of parental genomes in preimplantation mouse embryos.

Mayer W, Smith A, Fundele R, Haaf T - J. Cell Biol. (2000)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Genetik, 14195 Berlin, Germany.

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.

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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.
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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.

Mentions: The germ-cell line in male mice was labeled with the halogenated thymidine analogue BrdU, as described previously (Ito et al. 1988). This BrdU-substituted DNA was detected in embryos of the next generation by anti-BrdU immunofluorescence staining and served as a cytological marker for the paternal genome in interphase nuclei of the zygote and cleaving mouse embryo. Fig. 1 a shows a mouse egg upon fertilization. The condensed sperm nucleus contained BrdU in both DNA strands of the paternal chromosomes and, therefore, was heavily labeled with the anti-BrdU antibody, whereas the activated female pronucleus was devoid of label. Decondensation of the sperm chromatin was followed by formation of the male pronucleus. Both the male and female pronuclei swelled and became apposed in the center of the zygote. The male pronucleus exhibited a nearly uniform punctate BrdU staining (Fig. 1 b). Since no localized (partial) labeling of the male pronucleus was seen in >20 one-cell embryos analyzed at 10 and 18 h after fertilization, we conclude that the entire male genome was more or less uniformly substituted with BrdU. The smaller female pronuclei always remained BrdU negative, demonstrating the specificity of the technique. Following a very short G2 phase of 1 h (Howlett 1986) and breakdown of the pronuclear envelopes, the two chromosome sets remained completely separated, forming a single diploid nucleus (Fig. 1 c). Even in the absence of colcemid, highly condensed metaphase chromosomes could be observed at ∼20–22 h after fertilization (Fig. 1 d). It is striking that the disruptive mitotic process did not lead to an intermingling of the two chromatin sets.


Spatial separation of parental genomes in preimplantation mouse embryos.

Mayer W, Smith A, Fundele R, Haaf T - J. Cell Biol. (2000)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169371&req=5

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.
Mentions: The germ-cell line in male mice was labeled with the halogenated thymidine analogue BrdU, as described previously (Ito et al. 1988). This BrdU-substituted DNA was detected in embryos of the next generation by anti-BrdU immunofluorescence staining and served as a cytological marker for the paternal genome in interphase nuclei of the zygote and cleaving mouse embryo. Fig. 1 a shows a mouse egg upon fertilization. The condensed sperm nucleus contained BrdU in both DNA strands of the paternal chromosomes and, therefore, was heavily labeled with the anti-BrdU antibody, whereas the activated female pronucleus was devoid of label. Decondensation of the sperm chromatin was followed by formation of the male pronucleus. Both the male and female pronuclei swelled and became apposed in the center of the zygote. The male pronucleus exhibited a nearly uniform punctate BrdU staining (Fig. 1 b). Since no localized (partial) labeling of the male pronucleus was seen in >20 one-cell embryos analyzed at 10 and 18 h after fertilization, we conclude that the entire male genome was more or less uniformly substituted with BrdU. The smaller female pronuclei always remained BrdU negative, demonstrating the specificity of the technique. Following a very short G2 phase of 1 h (Howlett 1986) and breakdown of the pronuclear envelopes, the two chromosome sets remained completely separated, forming a single diploid nucleus (Fig. 1 c). Even in the absence of colcemid, highly condensed metaphase chromosomes could be observed at ∼20–22 h after fertilization (Fig. 1 d). It is striking that the disruptive mitotic process did not lead to an intermingling of the two chromatin sets.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Genetik, 14195 Berlin, Germany.

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.

Show MeSH
Related in: MedlinePlus