Maintaining genomic integrity in mammalian early embryos, which are deficient in DNA damage repair, is critical for normal preimplantation and subsequent development. Abnormalities in DNA damage repair in preimplantation embryos can cause not only developmental arrest, but also diseases such as congenital disorders and cancers. Histone H4 lysine 20 monomethylation (H4K20me1) is involved in DNA damage repair and regulation of gene expression. However, little is known about the role of H4K20me1 during mouse preimplantation development. In this study, we revealed that H4K20me1 mediated by SETD8 is involved in maintaining genomic integrity. H4K20me1 was present throughout preimplantation development. In addition, reduction in the level of H4K20me1 by inhibition of SETD8 activity or a dominant-negative mutant of histone H4 resulted in developmental arrest at the S/G2 phase and excessive accumulation of DNA double-strand breaks. Together, our results suggest that H4K20me1, a type of epigenetic modification, is associated with the maintenance of genomic integrity and is essential for preimplantation development. A better understanding of the mechanisms involved in maintaining genome integrity during preimplantation development could contribute to advances in reproductive medicine and technology.

Maternal RNA and proteins accumulate in mouse oocytes and regulate initial developmental stages. Sperm DNA combines with protamine, which is exchanged after fertilization with maternal histones, including H3.3; however, the effect of H3.3 on development post-fertilization remains unclear.Herein, we established an electroporation method to introduce H3.3 siRNA into germinal vesicle (GV)stage oocytes without removing cumulus cells. Oocyte-attached cumulus cells need to be removed during the traditional microinjection method; however, we confirmed that artificially removing cumulus cells from oocytes reduced fertilization rates, and oocytes originally free of cumulus cells had reduced developmental competence. On introducing H3.3 siRNA at the GV stage, H3.3 was maintained in the maternal pronucleus and second polar body but not in the paternal pronucleus, resulting in embryonic lethality after fertilization. These findings indicate that H3.3 protein was not incorporated into the paternal pronucleus, as it was repeatedly translated and degraded over a relatively short period.Conversely, H3.3 protein incorporated into the maternal genome in the GV stage escaped degradation and remained in the maternal pronucleus after fertilization. This new method of electroporation into GVstage oocytes without cumulus cell removal is not skill-intensive and is essential for the accurate analysis of maternal effect genes.

Maternal RNAs and proteins accumulate in mouse oocytes and control the initial stages of development. The DNA in sperm combines with protamine, which is exchanged after fertilization with maternal histones, including H3.3, but the effect of H3.3 on post-fertilization development has been unclear. In this study, we established an electroporation method to introduce H3.3 siRNA into germinal vesicle (GV)-stage oocytes without removing cumulus cells. In the traditional microinjection method, cumulus cells attached to oocytes must be removed; however, we confirmed that artificially removing cumulus cells from oocytes reduced fertilization rates, and oocytes originally free of cumulus cells had reduced developmental competence. When H3.3 siRNA was introduced at the GV stage, H3.3 was still present in the maternal pronucleus and second polar body, but not in the paternal pronucleus, resulting in embryonic lethality after fertilization. This indicates that the H3.3 protein was not incorporated into the paternal pronucleus because it was repeatedly translated and degraded in a relatively short time. On the other hand, H3.3 protein incorporated into the maternal genome in the GV stage escaped degradation and remained in the maternal pronucleus after fertilization. This new method of electroporation into GV-stage oocytes without removing cumulus cells is not skill intensive and is essential for the accurate analysis of maternal effect genes.

In mouse preimplantation development, zygotic genome activation (ZGA), which synthesizes new transcripts from the embryos, begins in the S phase of the one-cell stage, with major ZGA occurring especially at the late two-cell stage. Myc is a transcription factor expressed in parallel with ZGA, but its direct association with the major ZGA has not been clarified. In this study, we found that developmental arrest occurs at the two-cell stage when mouse embryos were treated with antisense oligos targeting Myc or inhibitors specific for MYC from the one-cell stage. In order to identify when MYC inhibition affected development, we applied time-limited inhibitor treatment, and found that inhibition of MYC at the two-cell, four-cell, and morula stages had no effect on preimplantation development, whereas treatment with the inhibitor at the early two-cell stage arrested development at the two-cell stage. Furthermore, transcriptome analysis revealed that when MYC function was inhibited, genes expressed in the major ZGA phase were suppressed. These results suggest that Myc is essential for the induction of major ZGA and its subsequent development. Revealing the function of Myc in preimplantation development is expected to contribute to advances in assisted reproductive technology.