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First published online 21 April 2004
doi: 10.1242/dev.01116

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Regulation of histone H3 lysine 9 methylation in oocytes and early pre-implantation embryos

Honglin Liu^*,, Jin-Moon Kim and Fugaku Aoki

Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan

View larger version (41K): [in a new window]   Fig. 1. Asymmetric histone H3 methylation of lysine 9 in the parental genomes of one-cell mouse embryos. Mouse GV-stage (GV) and MII-stage (MII) oocytes, one-cell embryos collected 3, 6 and 12 hours after in vitro fertilization (IVF 3, 6, 12 h) (A), and parthenogenetic and androgenetic one-cell embryos (B), were subjected to immunocytochemistry with antibody to methylated histone H3 Lys 9 (MeH3/K9). The antibody was localized with FITC-conjugated secondary antibodies (green), and DNA was stained with DAPI (blue). The parthenogenetic embryos were prepared by exposing MII-stage oocytes to 7% ethanol for 6 minutes, and collected for immunocytochemistry 6 hours later. The androgenetic embryos were prepared by fertilizing enucleated oocytes in vitro, and collected for immunocytochemistry 6 hours after fertilization. Arrows indicate the male (m) and female (f) pronuclei. pb, polar body. The experiments were conducted at least three times and similar results were obtained. More than 25 images were obtained for each type of embryo and representative examples are shown.

View larger version (54K): [in a new window]   Fig. 2. Histone H3 lysine 9 (H3/K9) methylation patterns in two-cell mouse embryos that were produced by fertilization, parthenogenesis and androgenesis. Mouse two-cell embryos that were collected 32 hours after in vitro fertilization (A), and parthenogenetic and androgenetic two-cell embryos (B), were subjected to immunocytochemistry with the antibody to methylated H3/K9 (MeH3K9). The antibody was localized with FITC-conjugated secondary antibodies (green), and DNA was stained with DAPI (blue). The parthenogenetic embryos were prepared by exposing MII-stage oocytes to 7% ethanol for 6 minutes, and collected for immunocytochemistry 32 hours later. The androgenetic embryos were prepared by fertilizing enucleated oocytes in vitro, and collected for immunocytochemistry 32 hours after fertilization. The experiments were conducted four times and similar results were obtained. More than 25 images were obtained for each type of embryo and representative examples are shown.

View larger version (12K): [in a new window]   Fig. 3. Changes in the methylation levels of H3/K9 during the first and second cell cycles of mouse embryos. The embryos were collected at 6, 12, 20 and 30 hours after insemination, designated as samples one-cell G1, one-cell G2, two-cell G1 and two-cell G2, respectively, and subjected to immunocytochemistry using the antibody against methylated H3/K9. The intensity of fluorescence was analyzed semi-quantitatively, as described in Materials and methods. The nuclei of diploid parthenogenetic two-cell G2 embryos were used as controls in each experiment, and the averaged value of the fluorescence intensity in the controls was arbitrarily set at 50%. The fluorescence intensity of each sample was expressed relative to this value. Data were accumulated from two independent experiments, and were expressed on the basis of DNA content. The numbers of blastomeres examined were: 14, 53, 90 and 38 for the one-cell G1, one-cell G2, two-cell G1 and two-cell G2 stages, respectively. The columns and bars represent mean±s.e.m.

View larger version (49K): [in a new window]   Fig. 4. Changes in the methylation levels of H3/K9 during early pre-implantation development in the mouse. Embryos that were prepared by in vitro fertilization (IVF), parthenogenesis (Partheno) and androgenesis (Andro) were examined for H3/K9 methylation using immunocytochemistry. The diploid parthenogenetic embryos were prepared by activating MII-stage oocytes by treatment with 10 mM Sr2+ in CZB for 10 minutes, followed by an additional 6-hour incubation in CZB that contained 5 µg/ml cytochalasin B to prevent extrusion of the polar bodies from the oocytes. The diploid androgenetic embryos were prepared by collecting the one-cell embryos with two pronuclei after in vitro fertilization of the enucleated oocytes. (A) Immunofluorescent confocal micrographs of mouse embryos. The two-, four- and eight-cell embryos were collected 30, 40 and 50 hours, respectively, after in vitro fertilization or parthenogenetic activation. As not all of the nuclei in some of the four-cell embryos could be aligned, only three nuclei are shown. (B) Semi-quantification of methylated histone H3/K9 in the pre-implantation mouse embryos. Mouse one-, two-, four- and eight-cell embryos were collected 12, 20, 40 and 50 hours, respectively, after in vitro fertilization or parthenogenetic activation. The diploid parthenogenetic two-cell embryos were used as control embryos in each experiment, and the averaged value of the fluorescence intensity in the control embryos was arbitrarily set at 100%. The fluorescence intensity observed for each sample was expressed relative to this value. Data were accumulated from at least two independent experiments, and were expressed on the basis of the blastomere. The numbers of blastomeres examined were: 8, 18, 32 and 47 for the androgenetic one-, two-, four- and eight-cell embryos, respectively; 22, 66, 47 and 70 for the parthenogenetic one-, two-, four- and eight-cell embryos, respectively; and 53, 90, 56 and 76 for the in vitro-fertilized one-, two-, four- and eight-cell embryos, respectively. The columns and bars represent mean±s.e.m.

View larger version (80K): [in a new window]   Fig. 5. H3/K9 methylation in male pronuclei that were transplanted into enucleated GV- or MII-stage oocytes. The reconstructed oocytes were produced by transferring the male pronucleus 6 hours after insemination into enucleated oocytes at (A) the GV stage (NT-GV) or (B) the MII stage (NT-MII), and were used for immunocytochemistry following culture for 3 hours with (for NT-GV) or without (for NT-MII) 0.2 mM IBMX. The antibody that recognizes the methylated lysine 9 on histone H3 (MeH3K9) was localized with FITC-conjugated secondary antibodies (green), and DNA was stained with DAPI (blue). Intact oocytes at the GV stage (GV) and MII stage (MII) that had not been enucleated are shown as controls. The reconstruction experiments for the NT-GV and NT-MII oocytes were conducted four and three times, respectively. In total, 23 NT-GV and 18 NT-MII oocytes were reconstructed successfully and examined for histone methylation. Similar patterns were observed for each type of reconstructed oocyte; representative examples are shown.

View larger version (48K): [in a new window]   Fig. 6. Involvement of protein synthesis and gene expression in H3/K9 methylation in mouse embryos. (A) Cycloheximide (50 µg/ml) was added at the time of insemination, and H3/K9 methylation (MeH3K9) was examined 12 hours post-insemination. (B) -Amanitin (25 µg/ml) was added 4 hours after activation of the androgenetic embryos, and H3/K9 methylation was examined 32 hours post-activation. The polyclonal antibody that recognizes methylated H3/K9 was localized with FITC-conjugated secondary antibodies (green), and the DNA was stained with DAPI (blue). The experiments using cycloheximide and -amanitin were conducted three and two times, respectively, and similar results were obtained. 21, 12, 18 and 11 embryos were examined in the cycloheximide (+), cycloheximide (-), -amanitin (+) and -amanitin (-) groups, respectively, and representative examples are shown.

View larger version (72K): [in a new window]   Fig. 7. Relationship between H3/K9 and DNA cytosine methylation in the male pronucleus following transplantation into GV-stage or MII-stage oocytes. Reconstructed oocytes were produced by transferring male pronuclei into enucleated GV-stage oocytes, followed by a 3-hour culture to retrieve meiosis (NT-GVB) or to inhibit meiotic resumption in the presence of IBMX (NT-GV). Alternatively, the male pronuclei were transferred into enucleated MII-stage oocytes (NT-MII), followed by culture for 3 hours in the presence of nocodazole to prevent spontaneous activation. The antibodies directed against H3/K9 methylation (MeH3K9) and DNA cytosine methylation (5-MeC) were detected using the cy3-conjugated secondary antibody (red) and the FITC-conjugated secondary antibody (green), respectively. The reconstruction experiments for the NT-GVB, NT-GV and NT-MII oocytes were conducted three times. In total, 18 NT-GVB, 20 NT-GV and 18 NT-MII oocytes were reconstructed successfully and examined for histone methylation. Similar patterns were observed for each type of reconstructed oocytes; representative examples are shown.

View larger version (43K): [in a new window]   Fig. 8. Schematic of the hypothetical mechanism that generates different H3/K9 methylation patterns, so that the different parental origins of genomes are distinguished during early pre-implantation development. In oocytes, H3/K9 methylase actively catalyzes the protein in the maternal genomes, but not in the cytoplasmic pool. After fertilization, the methylase is inactivated, and H3/K9 in the paternal genome, which is incorporated from the cytoplasmic pool, remains undermethylated. The methylase is still present but inactivated by proteins that are newly synthesized after fertilization. Thus, asymmetric H3/K9 methylation is established in the parent genomes. This asymmetric methylation is maintained until the two-cell stage, because of the lack of methylase and demethylase activities. However, the methylation level decreases gradually in a passive fashion after each DNA replication. When the DNA is replicated, the pre-existing nucleosomal structure is disrupted at the replication fork, and the core histones are dissociated from the DNA. The histones from the nucleoplasmic pool are sequestered together with the histones from the pre-existing nucleosomes, and amalgamate into a single structure. Thus, in the nascent chromatin, pre-existing histones are diluted with histones that are incorporated from the nucleoplasm after DNA replication (Wolffe, 1998). At the four-cell stage, H3/K9 methylase is activated, and catalyzes the proteins in the genomes of both parental origins.

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