Spindle Formation and Function in Oocytes and Early Embryos

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DESCRIPTION (provided by applicant): Correct segregation of chromosomes during meiosis and mitosis is essential for embryonic development and perpetuation of the species. Nuclear transfer (cloning) provides a powerful means for studying spindle formation and function during both processes. We find that removal of the spindle-chromosome complex (SCC) from the oocyte during the first step in cloning by adult somatic cell nuclear transfer (SCNT) depletes the oocyte of a number of proteins, but these proteins become replenished within a few hours. We also find that spindles that form in clones made by SCNT are deficient in calmodulin, whereas the spindles that form in clones made with embryonic nuclei (ECNT) acquire calmodulin appropriately. This correlates with better chromosome congression, reduced incidence of tetraploidy and enhanced development in ECNT clones. Because calmodulin remains abundant in the ooplasm, these observations indicate that the association of calmodulin, and possibly other proteins, with the spindle is controlled by factors associated with the nuclei, and that these factors differ between embryonic and somatic nuclei. It is our hypothesis that the factors that recruit the appropriate proteins to the SCC constitute a novel form of non-genetic, nuclear inheritance present in the oocyte and expressed in cells of the early embryo, but not in somatic cells. These factors are required for correct SCC formation specifically within the context of the ooplasm, and are thus dispensable in somatic cells. This inheritance is removed during cloning, and thus lacking in SCNT embryos, but is present in ECNT embryos. The deficiency in SCNT embryos likely contributes to the poor success of cloning. Identifying the factors that direct SCC formation SCC in the oocyte and early embryo will provide an important new advancement in our basic understanding of meiosis and mitosis in early embryos, and should provide an important key to improving cloning for applied and therapeutic purposes. We will pursue these objectives using a combination of state-of-the-art proteomics approaches and manipulation of gene expression in oocytes and cloned embryos: We will (1) Identify the protein(s) that recruit calmodulin to the meiotic SCC, (2) Undertake a proteomics analysis focused on SCC proteins to identify proteins that are depleted from oocytes and lacking specifically in the SCNT SCC, and (3) Restore or augment expression of these proteins in SCNT embryos and donor cells, and test for enhanced viability of SCNT embryos.