CHARACTERIZATION OF SPERM MEMBRANES

描述（改编自申请人的摘要）：长期 目的是表征公羊精子的结构-功能关系 受精所必需的表面膜结构域。 研究者已 表明脂质组成和膜结构域性质的变化 发生在附睾成熟过程中，并且：（a）独特的胆固醇 男性存在转运蛋白（CTP）和脂蛋白颗粒（LPP 道液;（B）脂质修饰改变精子功能（诱导 顶体反应（AR）和运动性）;和（c）来自两个 结构域（质膜（PM）覆盖顶体和PM 覆盖尾部）可以被隔离。解决两个问题。“怎么样？ 开发和维护独特的PM域？以及“细胞膜层是如何 (e.g., PM和内膜的“洋葱状”排列）在 发展？“一种假说将公羊精子PM 脂质通过管腔/内部脂质转移蛋白和管腔LPP， 获取膜功能。实验目标和问题是： 目标1（确定顶体上PM的脂质不对称性） 回答了这个问题：成熟与不成熟的PM地形是否不同 精子？两个实验表征了磷脂脂肪酶活性， 孤立膜囊泡的脂质不对称性。 目标2（消除贫穷的后果 顶体上PM的脂质不对称性）回答了这个问题： 胆固醇（chol）的跨双层运动是否允许两个 表面（IPM和外顶体膜）独特地富含 不饱和磷脂三个实验评估脂质的程度 不对称的PM的未成熟精子，测试模型系统，以确定 阻断未成熟精子的AR，并检查跨双分子层的作用。 膜不稳定中的胆固醇运动。 目标3（确定血脂 组成/不对称的PM覆盖的尾巴）回答了这个问题： 覆盖尾部的PM的脂质组成和不对称性 在附睾运输过程中发生了变化 两个实验确定脂质 尾膜结构域的组成，并评估这些脂质不对称性 膜囊泡 目标4（确定脂质代谢的机制） 在PM中改变）回答了这个问题：鉴于精子细胞 存在于LPP和CTP的海洋中，如何使单个表面域 在附睾运输过程中被选择性地改变？ 四个实验测试 CTP的膜结构域特异性结合，确定CTP是否可以转移 chol进入特定的膜结构域，将PC转移蛋白定位在细胞膜上。 顶体区，并测试附睾液中的PE转移蛋白。

DESCRIPTION (Adapted from the applicant's abstract): The long term objective is to characterize structure-function relationships of ram sperm surface membrane domains essential to fertilization. The investigator has shown that changes in lipid composition and properties of membrane domains occur during epididymal maturation and that: (a) unique cholesterol transfer protein(s) (CTP) and lipoprotein particles (LPP) exist in male tract fluids; (b) lipid modifications alter sperm function (induction of the acrosome reaction (AR) and motility); and (c) membranes from two domains (the plasma membrane (PM) overlying the acrosome and the PM overlying the tail) can be isolated.Two questions are addressed. "How are unique PM domains developed and maintained?"and "How do layers of membranes (e.g., "onion-like" arrangements of PM and inner membranes) interact during development?" A hypothesis links progressive modifications of ram sperm PM lipids by luminal/internal lipid transfer proteins and luminal LPP to acquisition of membrane function. Experimental goals and questions are: Goal 1 (determination of lipid asymmetry of PM overlying the acrosome) answers the question: Is PM topography distinct in mature vs immature sperm?Two experiments characterize phospholipid flipase activities and lipid asymmetry of isolated membrane vesicles. Goal 2 (consequences of lipid asymmetry of the PM overlying the acrosome) answers the question: Does transbilayer movement of cholesterol (chol) permit fusion between two surfaces (IPM and outer acrosomal membrane) uniquely enriched in unsaturated phospholipid? Three experiments assess the extent of lipid asymmetry in the PM of immature sperm, test a model system to determine the block to the AR in immature sperm, and examine the role of transbilayer chol movement in membrane destabilization. Goal 3 (determination of lipid composition/asymmetry of PM overlying the tail) answers the question: Has the lipid composition and asymmetry of the PM overlying the tail been altered during epididymal transit? Two experiments determine the lipid composition of the tail membrane domain and assess lipid asymmetry of these membrane vesicles. Goal 4 (determination of the mechanism by which lipids are altered in the PM) answers the question: Given that the sperm cell exists in a sea of LPPs and CTPs, how are individual surface domains selectively modified during epididymal transit? Four experiments test for membrane domain specific binding of CTP)(s), determine if CTP can transfer chol into specific membrane domains, localize a PC transfer protein in the acrosomal region, and test for a PE transfer protein in epididymal fluids.