A more accurate analysis of maternal effect genes by siRNA electroporation into mouse oocytes

Abstract

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.

Improved mRNA electroporation into immature oocytes to examine protein dynamics during development

“…As a result, we found that a relatively higher impedance value of 590 Ω allows for the lowest invasiveness and effective introduction of mRNAs into the GV oocytes enclosed with multiple layers of cumulus cells than the standard impedance value in the TAKE method and found that the MII oocyte is not suitable for EP with these settings. Yamamoto et al recently reported a successful suppression of the H3f3a/b gene expression by introducing siRNA (-23 bp) into mouse GV oocytes using electroporation in a manner similar to our study (Yamamoto et al, 2023). In this study, we introduced a much longer size of mRNAs of approximately 1.2 kbp and succeeded in the efficient expression of fluorescent proteins in the oocytes and embryos.…”