ORGANIZATION AND FUNCTIONS OF LIPID RAFTS IN SPERMATOZOA

描述（由申请方提供）：精子不能与卵子受精，除非作为“获能”过程的一部分，细胞外受体从精子质膜上除去甾醇。“虽然几十年来人们已经知道这种脂质重新排序的要求，但几个关键问题仍然没有答案。精子质膜中的脂质是如何组织的？什么分子介导固醇流出？固醇流出是如何转化为使精子受精的功能变化的？回答这些问题的健康相关性包括改善男性不育症的诊断，改善精子冷冻保存和体外处理，以及帮助设计男性避孕药。这些研究将促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动的理解。这些知识也可能有益于心脏病或糖尿病等疾病，这些疾病中细胞的正常固醇代谢受到干扰。 PI最近发现精子质膜被组织成称为“脂筏”的子域，这一发现提出了甾醇流出和精子功能变化之间令人兴奋的联系。脂筏是富含固醇和鞘脂（如神经节苷脂）的膜的亚结构域，其在体细胞中组织和调节信号通路。本提案中提供了来自我实验室的新数据，这些数据导致创建了以下模型：1）精子具有至少两种亚型的脂筏，一种富含甾醇，一种富含神经节苷脂GM 1，2）在非获能精子中，这些脂筏通过与潜在的细胞骨架蛋白的相互作用而严格分离，3）获能过程中的甾醇流出导致富含甾醇的筏的溶解和富含GM1的筏的重新分布，以及4）脂质微环境中的这些改变导致膜蛋白活性的变化，使得精子能够受精。 将通过表征脂筏的脂质和蛋白质含量来测试该模型（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（目的2）。研究不同甾醇受体的能力和动力学将有助于推断蛋白质介导外排的性质（目的3）。将改变异源表达系统以及精子的膜脂质含量，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的能力的影响（目的4）。

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).