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.