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.

该项目的长期目标是阐明相互作用 已知的精子获能介质之间（精子的改变） 精子在交配和受精之间必须发生的情况） 描述建立细胞内的元调节机制 介体浓度和分布。 理解更全面 控制精子关键细胞功能的机制获得 这些研究将允许更有选择性和有效的干预 临床不孕症所需的姑息治疗意义，以及 针对精子的抗生育措施所需的预防意识。 这 该项目的具体目标是： 描绘出以下机制： 维持精子静息细胞内 [Ca2+] 和 pH 的功能（ 特别注意 Na+/H+ 交换活性）；以确定 细胞内 pH 和游离 [Ca2+] 作为介质的调节作用 精子活力的启动和过度激活；定义角色 磷脂代谢改变膜对 Ca2+ 的通透性 这似乎是运动和精子顶体过度激活的基础 反应。 一些强大的新工具可用于实现这些目标 目标。 荧光探针，羧基荧光素，quin-2 和 fura-2， 提供连续、灵敏和具体的测定方法和 分别监测细胞质 p​​H 值和游离 [Ca2+] 由渗透酯前体原位生成。 这些探测器将 与一价和二价阳离子离子载体一起使用， 系统地建立细胞内 pH 和游离 [Ca2+] 的新平衡 同时通过视频显微镜检查对精子活力的影响。 这些相同的探针，以及离子的选择性激动剂和拮抗剂 运输，也将允许检查和表征机制 细胞 pH 值和 [Ca2+] 稳态和元调节控制 - 在 系统地但短暂地改变细胞内的实验 介体浓度。 磷脂代谢的作用是 通过改进溶血磷脂产生的测定来定义 使用和不使用已确定的药理学诱导剂的精子获能。 磷脂酶 A2 将通过新的亲和层析进行纯化 技术并以已知介体的控制为特征。 将检查精子膜的磷脂酶 C 是否可能 GTP 和 Ca2+ 与表面受体的元调节耦合。