An Izumo-Like Protein in C. elegans and its Role during Fertilization

线虫中的出云样蛋白及其在受精过程中的作用

• 批准号: 8064601
• 负责人: STEVEN W. L'HERNAULT
• 金额: $ 23.25万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8064601
• 关键词: Animals; Antibodies; Binding; Biological Assay; Biological Models; Caenorhabditis elegans; Cell fusion; Cell membrane; Cells; Child; Chimera organism; Complex; Couples; Data; Development; Evolution; Fertility; Fertilization; Fertilization in Vitro; Foundations; Genes; Germ Cells; Goals; Homologous Gene; Human; In Vitro; Infertility; Integral Membrane Protein; Invertebrates; Knowledge; Link; Location; Mammals; Mediating; Membrane Fusion; Membrane Proteins; Modeling; Molecular; Mus; Nematoda; Oocytes; Orthologous Gene; Ovum; Play; Process; Proteins; Public Health; Publishing; RNA Interference; Recovery; Reproductive Biology; Role; Sperm-Ovum Interactions; Surface; System; Techniques; Tertiary Protein Structure; Testing; Transgenic Organisms; Translational Research; Work; base; contraceptive target; cost; egg; extracellular; high throughput screening; in vivo; meetings; mutant; prevent; receptor; research study; small molecule; sperm cell; tool; zygote

DESCRIPTION (provided by applicant): Fertilization requires that two dissimilar cells fuse their plasma membranes to create a zygote. This process is poorly understood and there has been no evidence it is well-conserved. We recently discovered C. elegans Izumo-like (CEIZUMO), which is the first case of a homologous membrane protein required for fertilization in both mammals and nematodes. Under specific aim 1, we will identify the location and role of CeIzumo during fertilization in vivo. Our studies will first use immunological approaches to characterize and visualize C. elegans CEIZUMO. This is will determine where CEIZUMO is localized to facilitate cell-cell fusion and reveal other features it might share with other well-characterized C. elegans and mouse fertilization mutants. We will use a transgenic approach to determine which parts of CeIzumo and worm/mouse gene chimeras restore self-fertility to the CeIzumo mutant; we already know that the mouse Ig domain can be swapped into CeIzumo without compromising its function. Under specific aim 2, we will determine if CeIzumo is a C. elegans sperm fusogen in vitro. We will develop an in vitro fertilization system to allow determination of the precise point where CEIZUMO acts. We will also establish whether an extracellular fragment of CEIZUMO can bind to unfertilized eggs. If successful, this will be followed by an RNAi screen for egg surface CEIZUMO receptors. The emerging picture is that worm and mammalian Izumo homologs are orthologous and could define a "core" cell-cell fusion mechanism that has been conserved during fertilization in animals. Antibodies to Izumo are known to block human sperm-egg fusion, suggesting that this protein is an outstanding contraceptive target. The C. elegans experiments described in this proposal already suggest that a high throughput assay for small molecules that block human Izumo function might be possible. PUBLIC HEALTH RELEVANCE: Fertilization is a process with significant public health implications because ~15% of couples desiring children are infertile and many cases are due to defective sperm. We have discovered that cell-cell-fusion during fertilization in nematodes may be similar to that which occurs in humans. We will exploit the significant experimental advantages offered by nematodes for analyzing this important problem in reproductive biology.

描述(申请人提供)：受精需要两个不同的细胞融合它们的质膜来产生受精卵。人们对这一过程知之甚少，也没有证据表明它得到了很好的保护。我们最近发现了类秀丽线虫(Celegans Izumo-like，CEIZUMO)，这是哺乳动物和线虫受精所需的第一例同源膜蛋白。在特定目标1下，我们将确定CeIzumo在体内受精过程中的位置和作用。我们的研究将首先使用免疫学方法来表征和可视化线虫CEIZUMO。这将确定CEIZUMO的本地化位置，以促进细胞-细胞融合，并揭示它可能与其他特征良好的线虫和小鼠受精突变体共享的其他特征。我们将使用转基因方法来确定CeIzumo和虫/鼠基因嵌合体的哪些部分可以恢复CeIzumo突变体的自我育性；我们已经知道，小鼠的Ig结构域可以被交换到CeIzumo中，而不会影响其功能。在特定目标2下，我们将在体外确定CeIzumo是否是线虫精子FusoGen。我们将开发一种体外受精系统，以确定CEIZUMO作用的精确点。我们还将确定CEIZUMO的胞外片段是否能与未受精卵结合。如果成功，接下来将进行鸡蛋表面CEIZUMO受体的RNAi筛选。出现的情况是，蠕虫和哺乳动物的出云同源物是同源的，可能定义了一种在动物受精过程中被保守的“核心”细胞-细胞融合机制。众所周知，出云的抗体可以阻止人类精子和卵子的融合，这表明这种蛋白质是一个出色的避孕靶点。这项提案中描述的线虫实验已经表明，对阻断人类出云功能的小分子进行高通量检测是可能的。 公共卫生相关性：受精是一个对公共健康有重大影响的过程，因为大约15%的夫妇想要孩子是不育的，许多病例是由于精子缺陷。我们已经发现，线虫受精过程中的细胞-细胞融合可能与人类相似。我们将利用线虫提供的重要实验优势来分析生殖生物学中的这个重要问题。