CHARACTERIZATION OF SPERM MEMBRANES

描述(改编自申请人的摘要)：长期 目的是研究公羊精子的结构与功能的关系 受精所必需的表面膜结构域。调查员已经 表明脂质组成和膜结构域性质的变化 发生在附睾成熟期间，并且：(A)独特的胆固醇 男性体内存在转运蛋白(S)和脂蛋白颗粒(LPP)。 体液；(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(覆盖在顶体和质膜上的质膜 覆盖尾巴)可以隔离。解决了两个问题。“你好吗？ 开发和维护独特的PM结构域？“和”膜层如何 (例如，PM和内膜的“洋葱状”排列)在 发育？“一种假说将公羊精子PM的渐进性修改联系起来 通过腔/内脂转移蛋白和腔内LPP来调节血脂 膜功能的获得。实验目标和问题包括： 目标1(测定覆盖在顶体上的PM的脂质不对称性) 回答问题：PM地形成熟与不成熟有区别吗？ 精子？两个实验表征了磷脂脂肪酶的活性和 离体膜囊泡的脂质不对称。目标2( 覆盖在顶体上的PM的脂质不对称)回答了这个问题： 胆固醇的跨双分子层移动(Chol)允许两个 表面(IPM和顶体外膜)独特地富含 不饱和磷脂？三个实验评估了脂质的程度 未成熟精子的PM不对称，测试模型系统以确定 阻断未成熟精子中的AR，并检测跨双层的作用 膜失稳中的胆碱迁移。目标3(脂质的测定 尾巴上的PM的组成/不对称)回答了问题：有 尾部PM的脂质组成和不对称性 在附睾期改变了吗？两项实验测定了血脂 尾膜结构域的组成和评估这些结构域的脂质不对称性 膜泡。目标4(确定脂质的作用机制 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，单个表面域是如何 在附睾运输过程中有选择地改变？四个实验测试 膜结构域特异性结合CTP)(S)，确定CTP是否可以转移 Chol进入特定的膜区，将PC转运蛋白定位在 并检测附睾液中的PE转运蛋白。

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.