Modification of Egg Plasma Membrane

描述(由申请人提供)：在利用外部受精的物种中受精，包括模型系统，如海胆、青蛙和鱼，都表现出快速的蛋白酪氨酸激酶(PTK)激活，这是精子诱导的钙瞬变、退出减数分裂II、原核聚集和有丝分裂所必需的关键步骤。最近的研究表明，哺乳动物的卵子在控制精子诱导的钙瞬变的机制上与低等物种不同。然而，尽管对小鼠和大鼠卵母细胞的研究表明，钙信号不需要Src家族的激酶活性，但有强有力的证据表明，这些激酶在MIL恢复和原核聚集中发挥作用。该方案的主要目的是确定哪些Src家族的蛋白激酶是多发性骨髓瘤恢复和原核融合的关键信号通路。第一个特定的目的将检验这样的假设，即单个的Src家族PTK(Fyn、Yes或Src)是恢复减数分裂和原核融合所必需的。实验方法将使用src家族基因敲除小鼠和显性-负性结构的组合来定义每个激酶在这些过程中的作用。第二个目标将确定这些激酶参与完成每项功能的特定生化途径。在这里，生化分析将确定哪些通路被每个单独的激酶Fyn、Yes和Src的抑制所中断。将建立每条途径与MM恢复原核聚集的关系。第三个特定目标将检验这样的假设，即Fyn、Yes和Src在受精后受到不同的调控，并且它们的活性在卵子激活期间的特定时间点增加，从而使它们能够控制MM的恢复和原核聚集。这一目标还将确定这些激酶在受精卵中被激活的调节机制。本研究的结果将有助于理解MM恢复和原核聚集的生化机制，并将为我们未来的研究提供人工调控这些过程的基础。受精：受精需要多种酶的激活才能使卵子发育成胚胎。这项研究将使用更敏感、更现代的技术来确定哪些蛋白激酶能触发受精卵排出额外的染色体，并将男性和女性的细胞核一起移动。

DESCRIPTION (provided by applicant): Fertilization in species that utilize external fertilization, including model systems such as sea urchin, frog, and fish, all exhibit rapid protein tyrosine kinase (PTK) activation as a critical step required for the sperm- induced calcium transient, exit from meiosis II, pronuclear congression, and mitosis. Recent studies have demonstrated that mammalian eggs differ from lower species as to the mechanism controlling the sperm- induced calcium transient. However, while studies in oocytes of mice and rats indicate that Src-family kinase activity is not required for calcium signaling, there is strong evidence that these kinases play a role in Mil resumption and pronuclear congression. The main goals of this proposal are to establish which Src-family kinases are required signaling pathways critical to MM resumption and pronuclear congression. The first specific aim will test the hypothesis that individual Src-family PTKs (Fyn, Yes, or Src) are required for resumption of meiosis and pronuclear congression. The experimental approach will use a combination of Src-family knockout mice and dominant-negative constructs to define the role of each kinase in these processes. The Second aim will identify the specific biochemical pathways in which these kinases participate to accomplish each function. Here biochemical analyses will establish which pathways have been interrupted by suppression of each individual kinase Fyn, Yes, and Src. The relationship of each pathway to MM resumption of pronuclear congression will be established. The third specific aim will test the hypothesis that Fyn, Yes, and Src are differentially regulated after fertilization and that their activity increases at specific points during egg activation that allow them to control MM resumption and pronuclear congression. This aim will also identify the regulatory mechanisms by which these kinases are activated in the fertilized egg. The results of this study will provide an understanding of the biochemical mechanisms regulating MM resumption and pronuclear congression and will lead to future studies enabling us to modulate these processes artificially. Lay Summary: Fertilization requires that multiple enzymes become turned on in order for the egg to grow into an embryo. This study will use more sensitive, modern techniques to establish which protein kinases trigger the fertilized egg to eject extra chromosomes and move the male and female nuclei together.