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）：哺乳动物的精子缩质体是一种膜有限的细胞器，其中包含各种水解酶，可用于穿透鸡蛋投资。精子血浆和粘膜膜被划分为不同分子组成的结构域，这些结构域执行哺乳动物施肥所需的特定功能。我们对外脊柱膜 - 膜 - 矩阵复合物（OMC）多肽的组装和功能的理解受到限制。该提案的远距离目标是确定与外脊髓膜外膜（OAM）结构域组装有关的分子和结构机制，及其粘附的Arosomal基质，并定义其组成型多肽在膜融合中的作用，以及在水质反应过程中释放的水解酶。高度纯化的OMC级分由三个主要（54、50和45KDA）和几个小（38-19KDA）多肽组成。最近，我们通过连续洗脱SDS-PAGE发表了OMC高pH不溶性部分的纯化45KDA多肽（OMC45）。然而，OMC45多肽的分子表征尚不清楚。三个目标将解决这些目标。 AIM＃1-实验＃1A：OMC45多肽的生化和蛋白质组学鉴定：蛋白质组学分析得出的OMC45多肽序列数据将用于合成与OMC45的N-和C-termini相对应的20-MER肽。这些将用于实验＃1B中的Adrosom反应竞争抑制测定法。实验＃1B：使用透化的精子模型来定义OMC32（从高pH溶可溶部分和OMC45多肽中分离出的32KDA多肽的功能），以分析ACROSOMES反应：本研究将确定OMC32和OMC45多粒细胞在钙化型杂种型杂种型杂种中的作用。实验＃1C：通过交联研究确定OMC复合物的已知成员的相互作用：该实验将揭示OMC复合物的已知成员的相互作用。目标＃2-鉴定蛋白质，这些蛋白质介导OAM和AROSOMAL基质之间的粘附。成熟精子的粘体基质和成熟精子的粘膜膜被隔离为独特的结构，组成和功能的域。我们发现，仓鼠精子教素基质的不同元素是精确局部的，特别与OAM的熔融结构域相关联，该结构域暗示了OAM和基质之间的特定粘合剂相互作用。我们假设，除了其在Acrosom反应中的熔融过程函数外，顶体膜和基质多肽在受体配体类型的相互作用中相互作用，以建立斜视外膜膜膜复合物并调节独特的蛇耳元素的组装和分布。亲和力色谱和免疫沉淀将用于鉴定负责膜 - 矩阵粘附的蛋白质。目标＃3-确定附生传输过程中科素体基质多肽的分子内翻译后修饰：我们的假设是，在成熟过程中，在成熟过程中发生的脊柱质体基质蛋白的转化后修饰可能与这些生物化学修饰有关。多肽的处理将通过2-D页和免疫印迹分析确定。这些研究的完成将为哺乳动物施肥期间的杂色段及其功能的组装提供新的见解。这项工作带来的潜在实际利益可能包括制定维持储存的精子中的劣质完整性的策略，促进了缩小体反应，并提高了功能受损的精子的受精能力。 公共卫生相关性：精子血浆的不同领域和耕种膜在受精过程中执行特定功能。我们对外顶膜 - 马trix复合物（OMC）的蛋白质成分的组装和功能的理解都受到限制。该提案的目标是确定在质体体内结合和调节粘膜体基质组件的分布并定义OMC多肽在膜融合中的作用以及在Acrosoms反应期间水解酶释放的作用。这项研究不仅将洞悉精子外瘤膜在哺乳动物施肥中的作用，而且还将为本科生和研究生提供精子生物学研究机会。这项研究将在北卡罗来纳州费耶特维尔的历史悠久的黑人学院和大学在费耶特维尔州立大学进行，为少数族裔本科/研究生提供了学习科学伦理的绝佳机会，并获得了生物化学现代技术的经验。