The Role of Bovine Sperm Acrosomal Membrane-Matrix Complex in Fertilization

描述（由申请人提供）：哺乳动物精子顶体是一种膜结合的细胞器，含有多种用于穿透卵子包埋物的水解酶。精子质膜和顶体膜被划分为不同分子组成的区域，这些区域执行哺乳动物受精所需的特定功能。我们对顶体外膜-基质复合物（OMC）多肽的组装和功能的理解是有限的。该提案的长期目标是确定参与顶体外膜（OAM）结构域及其粘附顶体基质组装的分子和结构机制，并确定其组成多肽在膜融合中的作用，以及顶体反应期间水解酶的释放。高度纯化的OMC组分由三种主要（54、50和45 kDa）和几种次要（38- 19 kDa）多肽组成。最近，我们用连续洗脱SDS-PAGE从OMC的高pH不溶性组分中纯化了一个45 kDa的多肽（OMC 45）。然而，OMC 45多肽的分子表征尚不清楚。三个目标将实现这些目标。目的#1-实验#1a：OMC 45多肽的生物化学和蛋白质组学鉴定：将来自蛋白质组学分析的OMC 45多肽序列数据用于合成对应于OMC 45的N-和C-末端的20-mer肽。这些将用于实验1b中的顶体反应竞争性抑制试验。实验1b：使用用于分析顶体反应的透化精子模型来确定OMC 32（从OMC的高pH可溶性级分分离的32 kDa多肽）和OMC 45多肽的功能：本研究将确定OMC 32和OMC 45多肽在顶体胞吐的钙依赖性膜融合事件中的作用。实验1c：通过交联研究鉴定OMC复合物已知成员的相互作用：本实验将揭示OMC复合物已知成员的相互作用。目的#2-鉴定介导OAM和顶体基质之间粘附的蛋白质。成熟精子的顶体基质和顶体膜各自分离成具有独特结构、组成和功能的域。我们发现，仓鼠精子顶体基质的不同元素被精确地定位，并具体地与OAM的融合域，这表明OAM和基质之间的特定粘附相互作用。我们推测，除了其融合过程中的顶体反应的功能，外顶体膜和基质多肽相互作用的受体-配体类型的相互作用，以建立外顶体膜-基质复合物，并调节不同的顶体基质元素的组装和分布。亲和层析和免疫沉淀将用于鉴定负责膜-基质粘附的蛋白质。目的#3-鉴定附睾转运过程中顶体基质多肽的分子内翻译后修饰：我们的假设是顶体基质蛋白的翻译后修饰发生在成熟过程中，这些生化修饰可能涉及顶体水解酶与基质多肽的相互作用。多肽的加工将通过2-D PAGE和免疫印迹分析来鉴定。这些研究的完成将为哺乳动物受精过程中顶体节的组装及其功能提供新的见解。从这项工作中获得的潜在的实际利益可能包括发展战略，以保持顶体的完整性储存的精子，促进顶体反应，并提高功能受损的精子的再生能力。

DESCRIPTION (provided by applicant): The mammalian sperm acrosome is a membrane bounded organelle which contains a variety of hydrolases that are utilized to penetrate the egg investments. The spermatozoan plasma and acrosomal membranes are partitioned into domains of distinct molecular composition which perform specific functions required for mammalian fertilization. Our understanding of both the assembly and functions of the outer acrosomal membrane-matrix complex (OMC) polypeptides is limited. The long range goals of this proposal are to identify the molecular and structural mechanisms involved in the assembly of the outer acrosomal membrane (OAM) domain, and its adherent acrosomal matrix, and to define the role of their constituent polypeptides in the membrane fusion, and the release of hydrolases during the acrosome reaction. A highly purified OMC fraction is comprised of three major (54, 50, and 45kDa) and several minor (38-19kDa) polypeptides. Recently, we published the purification a 45kDa polypeptide (OMC45) from the high pH insoluble fraction of OMC by continuous elution SDS-PAGE. However, the molecular characterization of OMC45 polypeptide is not known. Three aims will address these goals. Aim#1- Experiment #1a: Biochemical and Proteomic Identification of OMC45 Polypeptide: The OMC45 polypeptide sequence data derived from proteomic analyses will be used for synthesis of 20-mer peptides corresponding to the N- and C-termini of OMC45. These will be used for the acrosome reaction competitive inhibition assay in experiment #1b. Experiment #1b: To Define the Function of OMC32 (32kDa polypeptide isolated from high pH soluble fraction of OMC) and OMC45 Polypeptides Using a Permeabilized Sperm Model for Analysis of the Acrosome Reaction: This study will determine the role of OMC32 and OMC45 polypetides in the calcium-dependent membrane fusion events of acrosomal exocytosis. Experiment #1c: To identify the Interaction of the known Members of the OMC Complex by Cross-Linking Studies: This experiment will reveal the interaction of the known members of the OMC complex. Aim#2- Identification of Proteins which Mediate the Adhesion Between the OAM and the Acrosomal Matrix. The acrosomal matrix and the acrosomal membrane of mature spermatozoa are each segregated into domains of unique structure, composition and function. We found that different elements of hamster sperm acrosomal matrix are precisely localized and specifically associated with the fusigenic domains of OAM suggesting specific adhesive interactions between OAM and matrix. We hypothesize that in addition to its fusigenic process function in the acrosome reaction, the outer acrosomal membrane and matrix polypeptides interact in a receptor-ligand type of interaction to establish the outer acrosomal membrane-matrix complex and to regulate the assembly and distribution of distinct acrosomal matrix elements. Affinity chromatography and immunoprecipitation will be utilized to identify protein(s) responsible for membrane-matrix adhesion. Aim#3- To Identify the Intramolecular Post-translational Modifications of Acrosomal Matrix Polypeptides during Epididymal Transit: Our hypothesis is that the post- translational modifications of acrosomal matrix proteins occur during maturation and these biochemical modifications may be involved in the interaction of acrosomal hydrolases with matrix polypeptides. Processing of the polypeptides will be identified by 2-D PAGE and immunoblot analyses. Completion of these studies will provide new insights into the assembly of the acrosomal segment and of its functions during mammalian fertilization. Potential practical benefits derived from this work may include the development of strategies for maintaining acrosomal integrity in stored spermatozoa, promoting the acrosome reaction, and improving the fertilizing capacity of functionally impaired spermatozoa.