ORGANIZATION AND FUNCTIONS OF LIPID RAFTS IN SPERMATOZOA

描述（由申请人提供）：作为“获能”过程的一部分，在细胞外受体将甾醇从精子质膜中去除之前，精子不能与卵子受精。尽管对这种脂质重新排序的要求已经知道了几十年，但仍有几个关键问题没有得到解答。精质膜中的脂质是如何组织的？什么分子介导甾醇外排？固醇外排是如何转化为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性的理解。这些知识也可能对心脏病或糖尿病等细胞正常的固醇代谢受到干扰的疾病有益。

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).