Reconstruction of artificial nuclei with nuclear import activity in living mouse oocytes

• Published in: Genes to cells : devoted to molecular & cellular mechanisms 2024-10, Vol.29 (10), p.820-837
• Main Authors: Yonezawa, Nao, Shindo, Tomoko, Oda, Haruka, Kimura, Hiroshi, Hiraoka, Yasushi, Haraguchi, Tokuko, Yamagata, Kazuo
• Format: Article
• Language: English
• Subjects: Active Transport, Cell Nucleus; Animals; artificial nucleus; Cell Nucleus - metabolism; Deoxyribonucleic acid; DNA; Female; Fluorescence microscopy; long‐strand DNA; Metaphase; Mice; Microinjection; Microinjections - methods; mouse oocyte; nuclear import activity; Nuclear pores; Nuclear transport; Nucleotide sequence; Oocytes; Oocytes - metabolism; pronucleus; Somatic cells; Strontium
• ISSN: 1356-9597; 1365-2443; 1365-2443
• DOI: 10.1111/gtc.13149

In eukaryotes, DNA is housed within the cell nucleus. Molecules required for the formation of a nucleus have been identified using in vitro systems with frog egg extracts and in vivo imaging of somatic cells. However, little is known about the physicochemical factors and conditions required for nuclear formation in mouse oocytes. In this study, using a reconstitution approach with purified DNA, we aimed to determine factors, such as the amount and timing of DNA introduction, required for the formation of nuclei with nuclear transport activity in mouse oocytes. T4 phage DNA (~166 kbp) was microinjected into strontium‐activated oocytes to evaluate the conditions appropriate for nuclear formation. Microinjection of 100–500 ng/μL of T4 DNA, but not 20 ng/μL, was sufficient for the formation of nucleus‐like structures. Furthermore, microinjection of DNA during metaphase II to telophase II, but not during interphase, was sufficient. Electron and fluorescence microscopy showed that T4 DNA‐induced nucleus‐like structures had nuclear lamina and nuclear pore complex structures similar to those of natural nuclei, as well as nuclear import activity. These results suggest that exogenous DNA can form artificial nuclei with nuclear transport functions in mouse oocytes, regardless of the sequence or source of the DNA. We succeeded in the reconstruction of an artificial nucleus in mouse oocytes using purified long‐length DNA.