Gamete Biology

Donor Centrosome Regulation of Initial Spindle Formation in Mouse Somatic Cell Nuclear Transfer: Roles of Gamma-Tubulin and Nuclear Mitotic Apparatus Protein 1

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. Here we show that, in the mouse, the level of gamma-tubulin in the poles and around the metaphase II spindle declines significantly, whereas only about 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 7-8 µm inner-diameter pipette, which retained the donor cell centrosome. In contrast, removing the donor cell centrosome by a small pipette impaired preimplantation development and prevented full-term development. In conclusion, we found that the initial spindle assembly of a metaphase-like spindle is regulated by the centrosome from the donor cell in the mouse.

Key words: Cumulus cells • Cytokines • Developmental biology • Early development • Ovum

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