IONIC MEDIATORS & METAREGULATIONS IN SPERM CAPACITATION

离子介体

• 批准号: 3322957
• 负责人: Donner F Babcock
• 金额: $ 9.92万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-08-01 至 1989-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3322957
• 关键词: acid base balance; acrosome; affinity chromatography; calcium metabolism; cyclic AMP; cytoplasm; fluorescent dye /probe; guanosine triphosphate; ion transport; ionophores; lipid metabolism; membrane permeability; microscopy; phospholipase A2; phospholipase C; phospholipids; sperm capacitation; sperm motility

The long range goals of this project are to elucidate the interactions between the known mediators of sperm capacitation (the alterations to sperm that occur obligatorily to sperm between mating and fertilization) and to describe the metaregulatory mechanisms that establish intracellular mediator concentrations and distributions. The more complete understanding of the mechanisms that control crucial cellular functions in sperm gained from these studies will allow more selective and effective intervention in the palliative sense required for so clinical infertility, and in the preventative sense required for sperm-directed antifertility measures. The specific aims of this project are: to delineate the mechanisms that function in maintaining resting intracellular [Ca2+] and pH in sperm (with particular attention to Na+/H+ exchange activity); to determine the regulatory roles of intracellular pH and free [Ca2+] as mediators of the initiation and hyperactivation of sperm motility; to define the role of phospholipid metabolism in the alteration of membrane permeability to Ca2+ that appears basic to hyperactivation of motility and the sperm acrosome reaction. Some powerful new tools are available for achievement of these goals. The fluorescent probes, carboxyfluorescein, quin-2 and fura-2, provide continuous, sensitive and specific means for determination and monitoring of cytosolic pH and free [Ca2+], respectively, after their generation in situ from permeant ester precursors. These probes will be used, together with monovalent and divalent cation ionophores, to systematically establish new equilibria of intracellular pH and free [Ca2+] while examining the consequences to sperm motility by video microscopy. These same probes, together with selective agonists and antagonists of ion transport, will also allow examination and characterization of mechanisms of cellular pH and [Ca2+] homeostasis and metaregulatory control - in experiments that systematically, but transiently, alter intracellular mediator concentrations. The role of phospholipid metabolism will be defined by an improved determination of lysophospholipid production during sperm capacitation with and without established pharmacological inducers. Phospholipase A2 will be purified with a new affinity chromatography technique and characterized with respect to control by known mediators. Phospholipase C of sperm membranes will be examined for possible metaregulatory coupling by GTP and Ca2+ to surface receptors.

该项目的远距离目标是阐明互动 在已知的精子电容介质之间（精子的改变 这是在交配和受精之间的精子中强制发生的）和 描述建立细胞内的元调节机制 介体的浓度和分布。 更完整的理解 控制精子中至关重要的细胞功能的机制 从这些研究中将允许对 如此临床不育所需的姑息性意义，在 精子指导的抗毒性措施所需的预防性意义。 这 该项目的具体目的是：描述的机制 在精子中维持静止的细胞内[Ca2+]和pH的功能（带有 特别注意Na+/H+交换活动）；确定 细胞内pH和游离[Ca2+]作为介体的调节作用 精子运动的起始和过度激活；定义角色 磷脂代谢改变了膜渗透率向Ca2+ 这似乎是运动性过度激活和精子缩小型体的基础 反应。 一些有力的新工具可用于实现这些 目标。 荧光探针，羧基氟菌素，Quin-2和Fura-2， 提供持续，敏感和特定的确定手段 分别监测胞质pH和自由[Ca2+] 从渗透酯前体的原位产生。 这些探针将是 与单价和二价阳离子离子载体一起使用 系统地建立新的细胞内pH和游离[CA2+]的平衡 同时通过视频显微镜检查对精子运动的后果。 这些相同的探针，以及离子的选择性激动剂和拮抗剂 运输，还将允许检查和表征机制 细胞pH和[Ca2+]稳态和元调节控制 - 在 系统但瞬时会改变细胞内的实验 介体浓度。 磷脂代谢的作用将是 通过改善溶物磷脂产生的确定定义 具有和没有既定药理诱导剂的精子电容。 磷脂酶A2将用新的亲和力色谱纯化 技术和对控制的特征是已知的介体。 将检查精子膜的磷脂酶C，以便可能 GTP和Ca2+与表面受体的元调节耦合。