Spindle Formation and Function in Oocytes and Early Embryos

描述(申请人提供)：在减数分裂和有丝分裂过程中正确的染色体分离对于胚胎发育和物种的永久存在是必不可少的。核移植(克隆)为研究这两个过程中纺锤体的形成和功能提供了有力的手段。我们发现，在成熟体细胞核移植(SCNT)克隆的第一步中，从卵母细胞中去除纺锤体-染色体复合体(SCC)会耗尽卵母细胞中的一些蛋白质，但这些蛋白质在几个小时内就会被补充。我们还发现，在SCNT克隆中形成的纺锤体缺乏钙调蛋白，而在胚胎核克隆(ECNT)中形成的纺锤体适当地获取钙调蛋白。这与更好的染色体聚集、减少四倍体的发生率和促进ECNT克隆的发育有关。由于钙调蛋白在卵质中仍然丰富，这些观察表明，钙调蛋白与纺锤体的联系是由与核相关的因素控制的，这些因素在胚胎核和体细胞核中不同。我们的假设是，将适当的蛋白质招募到SCC的因子构成了一种新的非遗传核遗传形式，存在于卵母细胞中，在早期胚胎的细胞中表达，但不在体细胞中表达。这些因子是正确的鳞癌形成所必需的，特别是在卵质的背景下，因此在体细胞中是必不可少的。这种遗传在克隆过程中被移除，因此在SCNT胚胎中缺失，但在ECNT胚胎中存在。SCNT胚胎的缺陷可能是导致克隆不成功的原因之一。识别在卵母细胞和早期胚胎中引导SCC形成的因素将为我们对早期胚胎减数分裂和有丝分裂的基本理解提供重要的新进展，并将为提高克隆的应用和治疗目的提供重要的关键。我们将结合最先进的蛋白质组学方法和对卵母细胞和克隆胚胎中基因表达的调控来实现这些目标：(1)确定将钙调蛋白招募到减数分裂SCC的蛋白质(S)，(2)进行聚焦于SCC蛋白的蛋白质组学分析，以确定从卵母细胞中耗尽的、在SCNT SCC中特异缺失的蛋白质，以及(3)恢复或增强这些蛋白质在SCNT胚胎和供体细胞中的表达，并测试SCNT胚胎的活力是否提高。

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.