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

牛精子顶体膜基质复合物在受精中的作用

• 批准号: 8075184
• 负责人: Subir K. Nagdas
• 金额: $ 9.49万
• 依托单位: FAYETTEVILLE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8075184
• 关键词: Acrosome; Acrosome Reaction; Address; Adhesions; Adhesives; Affinity Chromatography; Binding; Biochemical; Biochemistry; Biological Assay; Biology; Calcium; Cattle; Complex; Data; Development; Elements; Ethics; Event; Exocytosis; Fertilization; Goals; Hamsters; Hand; Historically Black Colleges and Universities; Hydrolase; Immunoblotting; Immunoprecipitation; Investments; Learning; Ligands; Mediating; Membrane; Membrane Fusion; Minor; Minority; Modeling; Modification; Molecular; North Carolina; Organelles; Peptides; Plasma; Post-Translational Protein Processing; Process; Proteins; Proteomics; Publishing; Research; Role; Science; Structure; Techniques; Universities; Work; crosslink; egg; experience; graduate student; improved; insight; member; polypeptide; protein function; receptor; research study; sperm cell

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.

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投资的水解酶。精子质和顶体膜被分成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。我们对外部顶体膜基质复合物（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顶体外膜（OAM）结构域及其粘附顶体基质组装所涉及的分子和结构机制，并确定其组成多肽在膜融合中的作用，以及顶体反应过程中水解酶的释放。高度纯化的 OMC 级分由三种主要（54、50 和 45kDa）和几种次要（38-19kDa）多肽组成。最近，我们发表了通过连续洗脱 SDS-PAGE 从 OMC 的高 pH 不溶部分中纯化 45kDa 多肽（OMC45）。然而，OMC45多肽的分子特征尚不清楚。三个目标将实现这些目标。目标#1-实验#1a：OMC45 多肽的生化和蛋白质组学鉴定：源自蛋白质组学分析的OMC45 多肽序列数据将用于合成对应于OMC45 N 端和C 端的20 聚体肽。这些将用于实验#1b 中的顶体反应竞争性抑制测定。实验#1b：使用透化精子模型分析顶体反应来定义 OMC32（从 OMC 的高 pH 可溶性级分中分离出的 32kDa 多肽）和 OMC45 多肽的功能：本研究将确定 OMC32 和 OMC45 多肽在顶体反应中的作用。顶体胞吐作用的钙依赖性膜融合事件。实验#1c：通过交联研究识别 OMC 复合物已知成员的相互作用：该实验将揭示 OMC 复合物已知成员的相互作用。目标#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为具有独特结构、组成和功能的区域。我们发现仓鼠精子顶体基质的不同元素精确定位并与 OAM 的融合域特异性相关，表明 OAM 和基质之间存在特定的粘附相互作用。我们假设除了顶体反应中的融合过程功能外，顶体外膜和基质多肽以受体-配体类型的相互作用相互作用，以建立顶体外膜-基质复合物并调节不同顶体的组装和分布。矩阵元素。将利用亲和层析和免疫沉淀来鉴定负责膜-基质粘附的蛋白质。目标#3-鉴定顶体基质多肽在附睾转运过程中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顶体基质蛋白的相互作用。基质多肽。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顶体节段的组装及其在哺乳动物受精过程中的功能提供新的见解。这项工作的潜在实际效益可能包括制定维持储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。 公共卫生相关性：精子质和顶体膜的不同区域在受精过程中发挥特定的功能。我们对顶体膜-基质复合物（OMC）外层蛋白质成分的组装和功能的了解是有限的。该提案的目标是鉴定顶体外膜 (OAM) 的蛋白质，这些蛋白质与顶体基质组件结合并调节顶体内的分布，并确定 OMC 多肽在膜融合和水解酶释放过程中的作用。顶体反应。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悠久的黑人学院和大学，为少数族裔本科生/研究生提供了学习科学伦理并获得现代生物化学技术实践经验的绝佳机会。