ORGANIZATION AND FUNCTIONS OF LIPID RAFTS IN SPERMATOZOA

精子中脂筏的组织和功能

• 批准号: 6986836
• 负责人: ALEXANDER J TRAVIS
• 金额: $ 26.19万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-16 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6986836
• 关键词: SDS polyacrylamide gel electrophoresis; acrosome; cell membrane; cytoskeletal proteins; exocytosis; gas chromatography; intermolecular interaction; laboratory mouse; lipids; matrix assisted laser desorption ionization; potassium channel; proteins; sperm; sterols; thin layer chromatography

DESCRIPTION (provided by applicant): Sperm cannot fertilize an egg until sterols have been removed from the sperm plasma membrane by extracellular acceptors as part of the process of "capacitation." Although the requirement for this re-ordering of lipids has been known for decades, several critical questions remain unanswered. How are lipids organized in the sperm plasma membrane? What molecules mediate sterol efflux? How is sterol efflux transduced into the functional changes that enable the sperm to fertilize? The health relevance of answering these questions include improving diagnostics for male infertility, improving sperm cryopreservation and handling in vitro, and helping design male contraceptives. These studies will advance our understanding of how a membrane's lipid microenvironment affects the activities of single proteins and signaling pathways. Such knowledge might also benefit conditions such as cardiac disease or diabetes in which the normal sterol metabolism of cells is perturbed. The recent discovery by the PI that the sperm plasma membrane is organized into sub-domains known as "lipid rafts" presents an exciting link between sterol efflux and changes in sperm function. Lipid rafts are sub-domains of membrane enriched in sterols and sphingolipids (such as gangliosides), which both organize and regulate signaling pathways in somatic cells. New data from my laboratory are presented in this proposal which have led to the creation of the following model: 1) sperm possess at least two sub-types of lipid raft, one enriched in sterols and one enriched in the ganglioside, GM1, 2) in non-capacitated sperm, these rafts are strictly segregated by interactions with underlying cytoskeletal proteins, 3) sterol efflux during capacitation causes dissolution of sterol-enriched rafts and re-distribution of rafts enriched in GM1, and 4) these alterations in the lipid microenvironment cause changes in the activities of membrane proteins, enabling the sperm to fertilize. The model will be tested by characterizing the lipid and protein content of the lipid rafts (Aim 1). Both targeted (e.g. immunoblots) and proteomic (e.g. two dimensional gel electrophoresis and protein sequencing) experimental approaches will determine which cytoskeletal proteins interact with lipid rafts, and how they regulate dynamic movements of rafts (Aim 2). Studying the ability and kinetics of different sterol acceptors will help deduce the nature of the protein(s) mediating efflux (Aim 3). The membrane lipid content of a heterologous expression system as well as of sperm will be altered to test the effect of sterols and gangliosides on the activity of a potassium channel and a sperm's ability to undergo acrosomal exocytosis (Aim 4).

描述（由申请人提供）：在细胞外受体将甾醇从精子质膜上除去（作为“获能”过程的一部分）之前，精子无法使卵子受精。尽管人们几十年来就知道这种脂质重新排序的要求，但仍有几个关键问题尚未得到解答。精子质膜中的脂质是如何组织的？什么分子介导甾醇流出？甾醇外流如何转化为使精子受精的功能变化？回答这些问题与健康的相关性包括改进男性不育症的诊断、改进精子冷冻保存和体外处理，以及帮助设计男性避孕药具。这些研究将增进我们对膜的脂质微环境如何影响单个蛋白质和信号通路的活性的理解。这些知识也可能有益于心脏病或糖尿病等细胞正常甾醇代谢受到干扰的疾病。 PI 最近发现，精子质膜被组织成称为“脂筏”的子域，这表明甾醇流出与精子功能变化之间存在令人兴奋的联系。脂筏是富含甾醇和鞘脂（例如神经节苷脂）的膜的子结构域，它们组织和调节体细胞中的信号传导途径。本提案中提出了我实验室的新数据，从而创建了以下模型：1) 精子至少拥有两种脂筏子类型，一种富含甾醇，另一种富含神经节苷脂 GM1，2)在非获能精子中，这些筏通过与底层细胞骨架蛋白的相互作用被严格隔离，3) 获能过程中甾醇流出导致富含甾醇的溶解筏和富含 GM1 的筏的重新分布，4) 脂质微环境的这些变化导致膜蛋白活性的变化，使精子能够受精。 该模型将通过表征脂筏的脂质和蛋白质含量进行测试（目标 1）。靶向（例如免疫印迹）和蛋白质组学（例如二维凝胶电泳和蛋白质测序）实验方法将确定哪些细胞骨架蛋白与脂筏相互作用，以及它们如何调节脂筏的动态运动（目标 2）。研究不同甾醇受体的能力和动力学将有助于推断介导外流的蛋白质的性质（目标 3）。将改变异源表达系统和精子的膜脂含量，以测试甾醇和神经节苷脂对钾通道活性和精子进行顶体胞吐作用的能力的影响（目标 4）。