Modification of the Egg Extracellular Matrix at Fertilization

受精时的成功既需要精子-卵子的融合，也需要阻止过多的精子融合或多精受精。在大多数动物中，多精受精是致命的，卵母细胞已经进化出了将其发生率降至最低的机制。在整个系统发育过程中，最保守的机制涉及受精后通过分泌皮质颗粒快速重塑卵子细胞外基质，皮质颗粒是卵子和卵母细胞特有的依赖刺激的分泌囊泡。皮质颗粒内容物可改变新生卵细胞外基质，形成对多精受精的永久阻碍，并为早期胚胎发育提供保护。在这一应用中解决的一般问题是：鸡蛋如何如此快速有效地构建其新的细胞外基质？我们将在这个项目中使用海胆卵，因为：1)我们可以获得平均每个雌性106个卵，这使得大规模的生化方法成为可能；2)这一过程的每个组成部分-皮质颗粒、受精膜和卵黄层-可以以功能形式集体分离；3)已经分离出代表这一过程中每个主要功能蛋白的cdna克隆；以及4)海胆卵在受精时经历了最极端的修改之一。他们投入6%的蛋白质质量和近20%的mRNA，在60秒内将新生的细胞外卵黄层转化为不渗透的受精包膜。这个项目的更广泛的影响是，本科生和研究生将接受不同的当代细胞、分子和发育生物学方法的培训，受精和细胞外基质生物学的一般原理将被整合到一个经典描述的、易于处理的教育系统中。此外，我们实验室网站的教育资源页面将不断更新K-16的项目结果和其他教育材料，(http://www.brown.edu/Research/Wessel_Lab/).Three提出了具体的研究目标：1.确定受精包膜构建所必需的卵黄层蛋白质。2.确定皮质颗粒蛋白中构建细胞外基质所需的结构域，并在受精时阻止多精受精。3.确定包膜交联所必需的过氧化氢产生机制。

0315657WesselSuccess at fertilization requires both sperm-egg fusion and blockage of excess sperm fusions, or polyspermy. Polyspermy is lethal in most animals, and oocytes have evolved mechanisms to minimize its occurrence. The most conserved mechanism throughout phylogeny involves a rapid remodeling of the egg extracellular matrix following fertilization by secretion of cortical granules, which are stimulus-dependent secretory vesicles unique to eggs and oocytes. Cortical granule contents modify the nascent egg extracellular matrix to form both a permanent block to polyspermy and to provide protection for early embryonic development. The general question addressed in this application is: How do eggs so rapidly and efficiently construct their new extracellular matrix? We will use sea urchin eggs for this project because: 1) we can obtain, on average, 106 eggs per female, enabling large-scale biochemical approaches; 2) each of the constituents of this process - the cortical granules, the fertilization envelope, and the vitelline layer, can be isolated en masse in a functional form; 3) cDNA clones have been isolated that represent each major functional protein of this process; and 4) sea urchin eggs undergo one of the most extreme modifications at fertilization. They invest 6% of their protein mass and nearly 20% of their mRNA to this transformation of the nascent extracellular, vitelline layer into an impervious fertilization envelope within 60 seconds.The broader implications of this project are that undergraduate and graduate students will be trained in diverse contemporary methods of cell, molecular, and developmental biology, and that general principles of fertilization and extracellular matrix biology will be integrated in a classically described, tractable system for educational purposes. In addition, project results and other educational material for K-16 will be continually updated on the Educational Resources page of our lab web site (http://www.brown.edu/Research/Wessel_Lab/).Three Specific Research Aims are proposed:1. Identify the vitelline layer proteins essential for fertilization envelope construction. 2. Determine the domains in cortical granule proteins required for construction of the extracellular matrix and block to polyspermy at fertilization. 3. Identify the peroxide-generating mechanism essential for envelope cross-linking.