Modification of Egg Plasma Membrane

描述（由申请人提供）：利用外部受精的物种（包括模型系统，如海胆、青蛙和鱼）的受精均表现出快速蛋白酪氨酸激酶（PTK）活化，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需的关键步骤。最近的研究表明，哺乳动物卵子与低等动物卵子在控制精子诱导的钙瞬变的机制上存在差异。然而，虽然在小鼠和大鼠的卵母细胞中的研究表明Src家族激酶活性不是钙信号传导所必需的，但有强有力的证据表明这些激酶在Mil恢复和原核聚集中起作用。该提案的主要目标是确定哪些Src家族激酶是MM恢复和原核聚集所需的关键信号通路。第一个具体的目标将测试的假设，个别Src家族的PTKs（Fyn，是，或Src）需要恢复减数分裂和原核大会。实验方法将使用Src家族敲除小鼠和显性阴性构建体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种功能的特定生化途径。在这里，生化分析将确定哪些途径已被每个单独的激酶Fyn，Yes和Src的抑制所中断。将建立每个途径与MM恢复原核大会的关系。第三个具体的目标将测试的假设，Fyn，是的，Src受精后的差异调节，他们的活动增加在特定的点在卵激活，使他们能够控制MM恢复和原核大会。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。这项研究的结果将提供一个了解的生化机制，调节MM恢复和原核大会，并将导致未来的研究，使我们能够人为地调节这些过程。摘要：受精需要多种酶被打开，以使卵子成长为胚胎。这项研究将使用更敏感的现代技术来确定哪些蛋白激酶触发受精卵排出额外的染色体并将雄性和雌性细胞核移到一起。

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.