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Research Article |
RIKEN Kobe Institute,3 Center for Developmental Biology, Laboratory for Genomic Reprogramming, Chuo-ku, Kobe City, Hyogo 650-0047, Japan
Department of Life Science,4 Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan
ABSTRACT
During the process of spindle-chromosome complex depletion in the oocyte, it is unclear whether both gamma-tubulin and nuclear mitotic apparatus protein 1 (NUMA1), which are required for mitotic organization and spindle assembly, are removed. The role of the donor cell centrosome and donor nuclear NUMA1 in the initial spindle morphogenesis and chromosome remodeling also remains unclear. In the present study, we show that in the mouse, the level of gamma-tubulin in the poles and around the metaphase II spindle declines significantly, whereas only approximately 10% of NUMA1 is removed during spindle-chromosome complex depletion in the recipient oocyte. This process does not impede initial spindle morphogenesis and is regulated by the centrosome of the donor cumulus cell. Retaining the donor cell centrosome establishes a monopolar spindle, whereas prior removal of the centrosome by a narrow-bore micropipette leads to bipolar spindle formation. Our data show that the centrosome of the donor cell regulates initial spindle morphogenesis and that the donor cumulus cell NUMA1 compensates for the deficiency in recipient NUMA1 during the formation of metaphase-like structures after nuclear transfer. Full-term offspring of cloned mice were obtained after injection of donor cells only with a pipette having an inner diameter of 7–8 µm, which retained the donor cell centrosome. In contrast, removing the donor cell centrosome with a small pipette impaired preimplantation development and prevented full-term development. In conclusion, the initial spindle assembly of a metaphase-like spindle is regulated by the centrosome from the donor cell in the mouse.
cloned embryos, cumulus cells, cytokines, developmental biology, donor cell centrosome, early development, gamma-tubulin, nuclear transfer, NUMA1, ovum, spindle morphogenesis
1 Supported by grants-in-aid for Creative Scientific Research (13GS0008) and for Young Scientists (A) (15681014) to T. W. from MEXT, Japan.
2 Correspondence: Nguyen Van Thuan, RIKEN Kobe Institute, Center for Developmental Biology, Laboratory for Genomic Reprogramming, 2–2–3 Minatojima-minamimachi, Chuo-ku, Kobe City, Hyogo 650-0047, Japan. FAX: 81 78 306 3095; nvthuan{at}cdb.riken.jp
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