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Cytoplasmic Asters Are Required for Progression Past the First Cell Cycle in Cloned Mouse Embryos¹

RIKEN Bioresource Center,³ Koyadai, Tsukuba, Ibaraki 305-0074, Japan Graduate School of Life and Environmental Sciences,⁴ University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Meiji University Graduate School,⁵ Kawasaki, Kanagawa 214-8571, Japan

Unlike the oocytes of most other animal species, unfertilized murine oocytes contain cytoplasmic asters, which act as microtubule-organizing centers following fertilization. This study examined the role of asters during the first cell cycle of mouse nuclear transfer (NT) embryos. NT was performed by intracytoplasmic injection of cumulus cells. Cytoplasmic asters were localized by staining with an anti--tubulin antibody. Enucleation of MII oocytes caused no significant change in the number of cytoplasmic asters. The number of asters decreased after transfer of the donor nuclei into these enucleated oocytes, probably because some of the asters participated in the formation of the spindle that anchors the donor chromosomes. The cytoplasmic asters became undetectable within 2 h of oocyte activation, irrespective of the presence or absence of the donor chromosomes. After the standard NT protocol, a spindle-like structure persisted between the pseudopronuclei of these oocytes throughout the pronuclear stage. The asters reappeared shortly before the first mitosis and formed the mitotic spindle. When the donor nucleus was transferred into preactivated oocytes (delayed NT) that were devoid of free asters, the microtubules and microfilaments were distributed irregularly in the ooplasm and formed dense bundles within the cytoplasm. Thereafter, all of the delayed NT oocytes underwent fragmentation and arrested development. Treatment of these delayed NT oocytes with Taxol, which is a microtubule-assembling agent, resulted in the formation of several aster-like structures and reduced fragmentation. Some Taxol-treated oocytes completed the first cell cycle and developed further. This study demonstrates that cytoplasmic asters play a crucial role during the first cell cycle of murine NT embryos. Therefore, in mouse NT, the use of MII oocytes as recipients is essential, not only for chromatin reprogramming as previously reported, but also for normal cytoskeletal organization in reconstructed oocytes.

¹ Supported by grants from MEXT, MHWL, and the Human Foundation, Japan.

² Correspondence: A. Ogura, RIKEN Bioresource Center, 3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan. FAX: 81 29 836 9172; ogura{at}rtc.riken.go.jp

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