ORGANIZATION AND FUNCTIONS OF LIPID RAFTS IN SPERMATOZOA

描述(申请人提供)：精子不能使卵子受精，直到精子质膜上的类固醇被细胞外受体从精子质膜中移除，这是“获能”过程的一部分。尽管这种脂类重新排序的要求已经知道几十年了，但仍有几个关键问题没有得到回答。精子质膜中的脂质是如何组织起来的？哪些分子参与了类固醇的外流？类固醇外流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对男性不育的诊断，改善精子的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脂微环境如何影响单个蛋白质和信号通路的活性的理解。这些知识还可能有助于心脏病或糖尿病等细胞的正常固醇代谢受到干扰的疾病。 PI最近发现，精子质膜被组织成被称为“脂筏”的子域，这一发现表明，类固醇外流与精子功能变化之间存在令人兴奋的联系。脂筏是细胞膜上富含甾醇和鞘磷脂(如神经节苷脂)的亚域，在体细胞中既组织和调节信号通路。1)精子至少具有两种亚型的脂筏，一种富含甾醇，一种富含神经节苷脂，GM1，2)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)获能过程中排出的固醇导致富含甾醇的脂筏的溶解和富含GM1的脂筏的重新分布，以及4)脂质微环境的这些变化引起膜蛋白活性的变化，使精子能够受精。 该模型将通过表征脂筏中的脂类和蛋白质含量进行检验(目标1)。靶向(例如免疫印迹)和蛋白质组学(例如双向凝胶电泳法和蛋白质测序法)的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)。研究不同的甾醇受体的能力和动力学将有助于推断(S)介导外排的蛋白质的性质(目标3)。将改变异源表达系统的膜脂含量以及精子的膜脂含量，以测试甾醇和神经节苷脂对钾通道活性和精子经历顶体胞吐的能力的影响(目标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).