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

描述(申请人提供)：哺乳动物精子顶体是一个有膜结合的细胞器，它含有多种水解酶，用于穿透卵子包被物。精子浆和顶体膜被分成不同的分子组成区域，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-基质复合体(OMC)多肽的组装和功能的了解有限。这项建议的长期目标是确定参与组装顶体外膜(OAM)结构域及其附着的顶体基质的分子和结构机制，并确定它们的组成多肽在膜融合和顶体反应过程中水解酶的释放中的作用。高纯度的OMC组分由三个主要多肽(54、50和45 kDa)和几个次要多肽(38-19 kDa)组成。最近，我们报道了用连续洗脱的方法从OMC的高pH不溶性部分中分离纯化出一种45 kDa的多肽(OMC45)。然而，OMC45多肽的分子特征尚不清楚。三个目标将解决这些目标。目的#1-实验#1a：OMC45多肽的生化和蛋白质组学鉴定：通过蛋白质组学分析得到的OMC45多肽序列数据将用于合成与OMC45的N-末端和C-末端相对应的20聚体多肽。这些将用于实验1b中的顶体反应竞争抑制分析。实验1b：利用分析顶体反应的渗透性精子模型确定OMC32(从OMC的高pH可溶部分中分离出的32 kDa多肽)和OMC45多肽的功能：本研究将确定OMC32和OMC45多肽在顶体胞吐作用的钙依赖膜融合事件中的作用。实验#1c：通过交联研究确定OMC复合体的已知成员之间的相互作用：本实验将揭示OMC复合体的已知成员的相互作用。目的#2-鉴定介导OAM和顶体基质之间黏附的蛋白质。成熟精子的顶体基质和顶体膜被分成具有独特结构、组成和功能的区域。我们发现仓鼠精子顶体基质的不同成分被精确定位，并与OAM的未来生成结构域特异相关，提示OAM与基质之间存在特异性的黏附作用。我们推测，除了顶体反应中的未来过程功能外，顶体外膜和基质多肽以受体-配体类型的相互作用建立顶体外膜-基质复合体，并调节不同顶体基质元素的组装和分布。亲和层析和免疫沉淀将被用来鉴定负责膜-基质黏附的蛋白质(S)。目的#3-鉴定附睾期顶体基质多肽的翻译后修饰：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，这些生化修饰可能参与了顶体水解酶与基质多肽的相互作用。多肽的加工将通过2D-PAGE和免疫印迹分析进行鉴定。这些研究的完成将为哺乳动物受精过程中顶体片段的组装及其功能提供新的见解。这项工作的潜在实际好处可能包括开发保持保存精子顶体完整性的策略，促进顶体反应，以及提高功能受损精子的受精能力。 与公共卫生相关：精子浆和顶体膜的不同区域在受精过程中发挥特定的功能。我们对顶体外膜-基质复合体(OMC)蛋白质组分的组装和功能的了解是有限的。该方案的目的是鉴定顶体外膜(OAM)中与顶体基质组装结合并调节顶体内分布的蛋白质，并确定OMC多肽在顶体反应中膜融合和水解酶释放中的作用。这项研究不仅将深入了解精子顶体外膜在哺乳动物受精中的作用，还将为本科生和研究生提供精子生物学方面的研究机会。这项研究将在费耶特维尔州立大学进行，这是一所位于北卡罗来纳州费耶特维尔的历史上一直是黑人的学院和大学，为少数族裔本科生/研究生提供了学习科学伦理和获得现代生物化学技术实践的绝佳机会。

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. PUBLIC HEALTH RELEVANCE: Different domains of the spermatozoan plasma and acrosomal membranes perform specific functions during fertilization. Our understanding of both the assembly and functions of protein constituents of the outer acrosomal membrane-matrix complex (OMC) is limited. The goals of this proposal are to identify the proteins of the outer acrosomal membrane (OAM) which bind to and regulate the distribution of acrosomal matrix assemblies within the acrosome and to define the role of OMC polypeptides in the membrane fusion and the release of hydrolases during the acrosome reaction. This study will not only provide insight into the role of sperm outer acrosomal membrane in mammalian fertilization, but will also provide undergraduate and graduate students with research opportunities in sperm biology. This study will be performed at Fayetteville State University, a historically black college and university in Fayetteville, North Carolina that provides an excellent opportunity for minority undergraduate/graduate students to learn the ethics of science and to receive hands on experience with modern techniques in biochemistry.