During oocyte growth, chromatin structure is altered globally and gene expression is silenced. To investigate the involvement of epigenetic modifications in the regulation of these phenomena, changes in global DNA methylation and in various histone modifications, i.e. acetylation of H3K9, H3K18, H4K5, and H4K12, and methylation of H3K4 and H3K9, were examined during the growth of mouse oocytes. Immunocytochemical analysis revealed that the signal intensities of all these modifications increased during growth and that fully grown, germinal vesicle (GV)-stage oocytes showed the most modifications. Since acetylation of most of the lysine residues on histones and methylation of H3K4 are associated with active gene expression, the increased levels of these modifications do not seem to be associated with gene silencing in GV-stage oocytes. Given that there are two types of GV-stage oocytes with different chromatin configurations and transcriptional activities, the epigenetic modification statuses of these two types were compared. The levels of all the epigenetic modifications examined were higher in the SN(surrounded nucleolus)-type oocytes, in which highly condensed chromatin is concentrated in the area around the nucleolus and gene expression is silenced than in the NSN(not surrounded nucleolus)-type oocytes, in which less-condensed chromatin does not surround the nucleolus and gene expression is active. In addition, the expression levels of various enzymes that catalyze histone modifications were shown by RT-PCR to increase with oocyte growth. Taken together, the results show that all of the epigenetic modifications increased in a concerted manner during oocyte growth, and suggest that these increases are not associated with gene expression. Reproduction (2007) 133 85-94

The gene expression pattern of differentiated oocytes is reprogrammed into that of totipotent preimplantation embryos before and/or after fertilization. To elucidate the mechanisms of genome reprogramming, we investigated histone H3 lysine 79 dimethylation (H3K79me2) and trimethylation (H3K79me3) in oocytes and preimplantation embryos via immunocytochemistry. In somatic cells and oocytes, H3K79me2 was observed throughout the genome, whereas H3K79me3 was localized in the pericentromeric heterochromatin regions in which there are no active genes. Because H3K79me2 is considered an active gene marker, H3K79 methylation seems to have differing functions depending on the number of methyl groups added on the same residues. Both H3K79me2 and H3K79me3 decreased soon after fertilization, and the hypomethylated state was maintained at interphase (before the blastocyst stage), except for a transient increase in H3K79me2 at mitosis (M phase). H3K79me3 was not detected throughout preimplantation, even at M phase. To investigate the involvement of H3K79me2 in genome reprogramming, somatic nuclei were transplanted into enucleated oocytes. H3K79me2 in these nuclei was demethylated following parthenogenetic activation. However, the nuclei that had been transplanted into the parthenogenetic embryos 7 h after activation were not demethylated. This suggests that the elimination of H3K79 methylation after fertilization is involved in genomic reprogramming.

Zygotes are totipotent cells that have the ability to differentiate into all cell types. It is believed that this ability is lost gradually and differentiation occurs along with the progression of preimplantation development. Here, we hypothesized that the loose chromatin structure is involved in the totipotency of one-cell stage embryos and that the change from loose to tight chromatin structure is associated with the loss of totipotency. To address this hypothesis, we investigated the mobility of eGFP-tagged histone H2B (eGFP-H2B), which is an index for the looseness of chromatin, during preimplantation development based on fluorescent recovery after photobleaching (FRAP) analysis. The highest mobility of eGFP-H2B was observed in pronuclei in 1-cell stage embryos and mobility gradually decreased during preimplantation development. The decrease in mobility between the 1- and 2-cell stages depended on DNA synthesis in 2-cell stage embryos. In nuclear transferred embryos, chromatin in the pseudopronuclei loosened to a level comparable to the pronuclei in 1-cell stage embryos. These results indicated that the mobility of eGFP-H2B is negatively correlated with the degree of differentiation of preimplantation embryos. Therefore, we suggest that highly loosened chromatin is involved in totipotency of 1-cell embryos and the loss of looseness is associated with differentiation during preimplantation development.

Freeze-drying has been frequently used to preserve food and microorganisms at room temperature (RT) for extended periods of time; however, its application to mammalian species is difficult. Here, we developed a method to prolong the stability of freeze-dried (FD) mice spermatozoa at RT for more than one year without using any cryoprotectant agents. Our data showed that maintaining a vacuum in ampoules is critical to ensuring the viability of FD spermatozoa, as the stability of spermatozoa DNA increased when imperfectly vacuumed ampoules were detected using a non-destructive test and eliminated. Finally a large number of healthy offspring were obtained from mice oocytes fertilized with FD spermatozoa stored at RT for more than one year. Although the birth rate from three-month stored spermatozoa was lower than that from one-day stored spermatozoa, no further reduction was observed even in one-year stored spermatozoa. Therefore, FD spermatozoa preserved in this study were highly tolerant to warm temperatures. This method of storage shows a great potential for the preservation of genetic resources of mammalian species, such as genetically-modified mouse strains, without the use of electric power.

The global chromatin configuration is dramatically remodeled during fertilization and early preimplantation development. Although the chromocenters, which are pericentromeric heterochromatin clusters, are observed in the nuclei of oocytes, they disappear after fertilization and then reappear at the four-cell stage. To elucidate the mechanism of this reorganization of heterochromatin, we investigated the expression and nuclear localization of DOT1L, which is involved in the regulation of heterochromatin structure through histone H3 lysine 79 (H3K79) methyltransferase activity, during preimplantation development. The Dot1L mRNA level was low at the two-cell stage. In the analysis by the immunocytochemistry, DOT1L protein was not observed in the nuclei at this stage. Microinjection of Flag-tagged Dot1L cRNA revealed that the DOT1L protein was localized in the nucleus of the embryos at the one-cell and four-cell stages but not at the two-cell stage. However, C-terminus-truncated DOT1L was localized in the nucleus of two-cell-stage embryos. Expression of the truncated DOT1L caused hypermethylation on H3K79 and the formation of chromocenter-like structures at the two-cell stage. Intriguingly, the expression of catalytically inactive truncated DOT1L also caused the formation of chromocenter-like structures without an increase in H3K79 methylation. Most embryos expressing the truncated DOT1L or its inactive form were arrested at the two-cell stage. These results suggest that the absence of DOT1L, which is involved in the formation of a specific configuration of heterochromatin at the two-cell stage, is essential for early preimplantation development.