Gamete Interactions in Caenorhabditis elegans

说明(申请人提供)：受精是一种生物过程，具有重要的医学、社会和经济意义。通过广泛的研究，人们对受精事件有了更详细的了解。然而，这些事件的分子基础通常仍然难以捉摸。以前关于受精的大多数工作都依赖于生化和免疫学方法。我们的工作在将经典基因分析应用于这一重要研究领域方面具有开创性。我的实验室一直在帮助率先使用线虫来研究精子和卵子相互作用的机制。为线虫开发的许多遗传和分子工具在传统上用于研究受精的其他生物中是不可用的或非常困难的。线虫最显著的优势之一是我们能够分离和保持影响精子或卵子的突变株，而不影响其他细胞。以前，通过对不育突变体的研究，我们已经鉴定出线虫中产生配子相互作用所需的一些第一批精子分子。这些精子分子包括SPE-9和SPE-38。SPE-9是一种精子表面跨膜分子，其胞外结构域包含10个表皮生长因子(EGF)样重复序列。我们假设SPE-9作为卵子表面精子受体的配体发挥作用。SPE-38基因编码一种新的四遍跨膜分子。基于蛋白质定位和遗传相互作用研究，我们假设SPE-38调节钙离子通道(SPE-41/Trp-3)和其他精子分子的定位和/或活性。我们最近发现了线虫受精所需的第一个卵子分子。鸡蛋-1和鸡蛋-2基因编码鸡蛋表面低密度脂蛋白受体重复序列蛋白。我们推测，在受精过程中，卵子-1和卵子-2半冗余地作为精子的卵面受体发挥作用。这项建议的目的是通过进行以下实验目标来加深我们对线虫受精的理解：1)通过开发试剂来测试它们与其他分子之间潜在的配体-受体相互作用，进一步表征SPE-9和Eg-1/Eg-2在受精中的作用。2)通过蛋白质相互作用和诱变研究SPE-38和SPE-41/Trp-3在受精过程中的作用，探讨SPE-38和SPE-41/Trp-3在受精过程中的作用。3)克隆精子功能基因spe-36，以确定其分子性质及其在受精中的作用。Spe-36基因是由与spe-9、spe-38和spe-41/trp-3突变体相同的不育突变表型定义的。这项工作将补充其他生物的生育研究，并为细胞-细胞相互作用的机制和生殖策略的多样性提供见解。与公共卫生相关：尽管对许多物种的受精事件有相当好的描述，但这一过程的分子基础还没有被很好地理解。我们正在模型系统秀丽线虫中鉴定受精的分子和分子机制。这项工作将补充对其他物种受精的研究，并最终可能导致更好地了解不孕不育的原因、生殖策略的多样性和新避孕药的设计。

DESCRIPTION (provided by applicant): Fertilization is biological process with important medical, social and economic implications. From extensive study, the events of fertilization are known in some detail. However, the molecular underpinnings of these events generally remain elusive. Most previous work on fertilization has relied on biochemical and immunological approaches. Our work is groundbreaking in the application of classic genetic analysis to this vital area of research. My lab has been helping to pioneer the use of C. elegans for addressing the mechanisms of sperm-egg interactions. Many of the genetic and molecular tools developed for C. elegans are not available or are very difficult to utilize in other organisms traditionally used for studying fertilization. One of the most significant advantages of C. elegans is our ability to isolate and maintain mutants that affect sperm or eggs and no other cells. Previously, through the study of sterile mutants, we have identified some of the first sperm molecules required for productive gamete interactions in C. elegans. These sperm molecules include SPE-9 and SPE-38. SPE-9 is a sperm surface transmembrane molecule with an extracellular domain that contains ten epidermal growth factor (EGF)-like repeats. We hypothesize that SPE-9 functions as a ligand for an egg surface sperm receptor. The spe-38 gene encodes a novel four pass transmembrane molecule. Based on protein localization and genetic interaction studies, we hypothesize that SPE-38 regulates the localization and/or activity of a calcuim channel (SPE-41/TRP-3) and other sperm molecules. We recently identified the first egg molecules required for fertilization in C. elegans. The egg-1 and egg-2 genes encode egg surface Low Density Lipoprotein (LDL) receptor repeat-containing proteins. We hypothesize that EGG-1 and EGG-2 function semi-redundantly as egg surface receptors for sperm during fertilization. The goal of this proposal is to further our understanding of fertilization in C. elegans by conducting the following experimental aims: 1) to further characterize the roles of SPE-9 and EGG-1/EGG-2 in fertilization by developing reagents to test for potential ligand-receptor interactions between them and/or other molecules. 2) to gain a better understanding SPE-38 and SPE-41/TRP-3 function in fertilization by investigating the connection between them through protein interaction studies and mutagenesis. 3) to clone the sperm function gene spe-36 in order to determine its molecular nature and role in fertilization. The spe-36 gene is defined by a sterile mutant phenotype that is identical to spe-9, spe-38 and spe-41/trp-3 mutants. This work will complement fertility studies in other organisms as well as provide insights into the mechanisms of cell-cell interactions and the diversity of reproductive strategies. PUBLIC HEALTH RELEVANCE: Although the events of fertilization are fairly well described for a number of species, the molecular underpinnings of the process are not well understood. We are identifying the molecules and molecular mechanisms of fertilization in the model system Caenorhabditis elegans. This work will complement studies of fertilization in other species and could eventually lead to a better understanding of the causes of infertility, the diversity of reproductive strategies and the design of new contraceptives.