Demethylation of the zygotic paternal genome (original) (raw)
Previous molecular analysis1,2,3 of digested genomic DNA from mature germ cells indicated that ovulated mouse oocytes are globally undermethylated compared with the sperm genome. A gradual genome-wide demethylation seems to occur during pre-implantation development, leading to indistinguishable alleles at most gene loci, but not at those that are imprinted1,2.
Intermediate amounts of methylation could be caused by a combination of undermethylated maternal and methylated paternal DNA3, although they could be the result of highly dynamic and sometimes opposing demethylation or de novo methylation processes in parental genomes4. To investigate this, we analysed the global methylation of the paternal and maternal genomes in mouse pre-implantation embryos by using immunofluorescence against 5-methylcytosine (MeC). Because most MeC is found in various repeat-DNA families4,5, antibody staining mainly reflects the density of MeC in interspersed repetitive sequences.
At 3 hours (Fig. 1a) and 6 hours (Fig. 1b) after fertilization, both the paternal and maternal chromosomes stained equally intensely with anti-MeC antibody. Because the ovulated oocytes are globally undermethylated, this finding suggests that there is rapid de novo methylation of egg chromatin shortly after fertilization1. After 8 hours, the paternal pronuclei were decondensed and so were much larger than the maternal pronuclei. Unexpectedly, all paternal pronuclei analysed after 8 hours showed very little methylation (Fig. 1c).
Figure 1: Differential demethylation of parental chromatin in the early mouse embryo.
a–e, Anti-5-methylcytosine (MeC) immunofluorescence of mouse one-cell embryos. a, Zygote 3 h after fertilization with intense MeC labelling of both pronuclei (>10). Numbers in parentheses indicate the number of embryos analysed. b Paternal and maternal pronuclei at 6 h (>10). c, Undermethylated paternal pronucleus at 8 h (>20). The smaller female pronucleus remains methylated. d, Aphidicolin-treated one-cell embryo displaying demethylation of the male pronucleus (>20). e, First metaphase (>5). f–j, Controls. Anti-DNA immunofluorescence of one-cell embryos demonstrates that both chromatin sets are accessible to antibody molecules. f, 3 h (>5). g, 6 h (>5). h, 8 h (>5). i, Aphidicolin treatment (>5). j, First metaphase (2). k,l, MeC staining of two-cell embryos at 22 h (>20) (k) and 32 h (>20) (l) shows that the paternal and maternal compartments have different methylation levels. m, Four-cell embryo at 45 h (>10). The MeC-staining intensity of the maternal half of the nucleus is weaker than in two-cell embryos. Scale bar, 10 μm.
To prevent DNA replication, one-cell embryos were collected after 6 hours and cultured for a further 10 hours in the presence of aphidicolin (at 2 μg ml−16. The unreplicated paternal pronuclei also became MeC-negative (Fig. 1d). This demethylation may be associated with transient hyperacetylation of histone H4 (ref. 7), because both replication and transcription are initiated earlier in the male pronucleus8, which is less condensed.
At first metaphase, we observed two differentially methylated and spatially separated chromosome sets9 (Fig. 1e). To exclude the possibility that differential MeC staining was due to changes in the accessibility of paternal DNA, we stained mouse embryos with anti-DNA antibody10: male and female pronuclei produced equally intense immunofluorescence (Fig. 1f–j). Two-cell embryos in phases G1 and G2 of the cell cycle were prepared at 22 and 32 hours. Most interphase nuclei displayed highly localized MeC staining (Fig. 1k,l), reflecting the compartmentalization of the two genomes, which may bring about the differential epigenetic reprogramming of the two genomes.
The amount of global methylation of the maternal genome was largely maintained from the early pronuclear to the two-cell stage, but four-cell embryos 45 hours after fertilization had a much lower MeC density over the maternal half of the nucleus (Fig. 1m). Interphase nuclei of 16- and 32-cell embryos had equivalently low methylation of paternal and maternal DNA (data not shown). Thus, in contrast to the very rapid and active demethylation of the paternal pronucleus, gradual demethylation of the maternal genome occurred passively during the second and third cleavage stages by a replication-dependent mechanism9, which may involve the loss of maintenance methylase activity. The second polar body remained methylated throughout pre-implantation development.
Our results provide a dramatic demonstration of the loss of DNA modification after fertilization. Active zygotic demethylation of the paternal genome has important implications for the understanding of genomic imprinting, X-chromosome inactivation, mammalian cloning and in vitro fertilization.