Membrane Potential and cAMP Crosstalk in Sperm Capacitation

精子获能过程中的膜电位和 cAMP 串扰

• 批准号: 8121607
• 负责人: Pablo E. Visconti
• 金额: $ 27.17万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8121607
• 关键词: Acrosome Reaction; Agonist; Antibodies; Bicarbonates; Biochemical; Cell membrane; Cells; Cholesterol; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Ejaculation; Epididymis; Event; Experimental Models; Female; Fertilization; Flow Cytometry; Grant; Heterogeneity; Image Analysis; Investigation; Ion Transport; Ions; Membrane Potentials; Methods; Molecular; Mus; Pathway interactions; Pattern; Permeability; Phosphorylation; Play; Population; Process; Proteins; Regulation; Reporting; Rest; Role; Signal Transduction; Sperm Capacitation; System; Time; Tyrosine Phosphorylation; Zona Pellucida; base; cell motility; epithelial Na+ channel; novel; patch clamp; sperm cell; voltage; zygote

DESCRIPTION (provided by applicant): Mammalian sperm are not able to fertilize eggs immediately after ejaculation. They acquire fertilization capacity after residing in the female tract for a finite period of time in a process known as capacitation [24, 25]. Initially, capacitation was defined using fertilization as end-point. However, a variety of evidences suggest that the functional changes occurring in the sperm during capacitation are not one event, but a combination of sequential and concomitant processes. Some of these processes occur as soon as the sperm are released from the epididymis, others are slower and are activated only after sperm incubation for a certain period of time in conditions that support the sperm ability to fertilize the egg. These slow events are associated with changes in the motility pattern (e.g. hyperactivation) and with the acquisition of the sperm capacity to undergo an agonist-stimulated acrosome reaction (AR). Although both, fast and slow events are regulated by HCO3- and by a cAMP-dependent pathway, slower events are limited by the release of cholesterol from the sperm plasma membrane. Using the mouse as an experimental model, we have demonstrated that these last events are associated with a protein kinase A (PKA)-dependent increase in the tyrosine (tyr) phosphorylation of a subset of proteins [23, 26]. Simultaneously with our findings, Zeng et al. (1995) reported that capacitation is accompanied by hyperpolarization of the sperm plasma membrane ootential (Em). It has been hypothesized that hyperpolarization is necessary to drive Low Voltage Activated (LVA) Ca2+ T-channels (Cav3) from an inactive state to a closed state that can be activated by agonists such as the zona pellucida (ZP), triggering the AR. Despite these advances, little is known on how these molecular changes are combined to promote capacitation. As part of the first cycle of this grant, we have shown that HCO3-, Na+ and K+ are involved in the regulation of the sperm Em [21, 27, 28] and that Epithelial Na+ channels (ENaC) are present in sperm and play an important role in the regulation of the sperm resting Em [21]. The objective of this proposal is to understand how changes in cAMP, protein phosphorylation and hyperpolarization integrate to promote capacitation. Investigation of the crosstalk between cAMP and the changes in ion permeability is essential to understand the molecular basis of capacitation and to provide novel targets for pharmacological control of the fertilization process.

描述(申请人提供)：哺乳动物的精子不能在射精后立即使卵子受精。在被称为获能的过程中，她们在女性体内居住一段有限的时间后获得受精能力[24，25]。最初，获能的定义是以受精为终点。然而，各种证据表明，精子在获能过程中发生的功能变化不是一个事件，而是一个连续和伴随的过程的组合。其中一些过程在精子从附睾处释放出来时就会发生，另一些过程则较慢，只有在精子孵化一定时间后才被激活，条件是支持精子使卵子受精。这些缓慢的事件与运动模式的改变(例如，过度激活)和精子获得经历激动剂刺激的顶体反应(AR)的能力有关。尽管快事件和慢事件都受到HCO3和cAMP依赖途径的调节，但慢事件受到精子质膜胆固醇释放的限制。使用小鼠作为实验模型，我们已经证明了这些最后的事件与蛋白激酶A(PKA)依赖的蛋白质子集的酪氨酸(Tyr)磷酸化增加有关[23，26]。与我们的发现同时，曾等人。(1995)报道了精子获能伴随着精子质膜电位(Em)的超极化。据推测，超极化是将低电压激活(LVA)钙离子T通道(Cav3)从非激活状态驱动到闭合状态所必需的，而闭合状态可以被激动剂如透明带(ZP)激活，从而触发AR。尽管有这些进展，但关于这些分子变化是如何结合起来促进获能的，人们知之甚少。作为这项资助的第一个周期的一部分，我们已经证明了HCO3-，Na+和K+参与了对精子Em的调节[21，27，28]，并且上皮性Na+通道(ENaC)存在于精子中，在调节静止的精子Em中起着重要的作用[21]。这项建议的目的是了解cAMP、蛋白质磷酸化和超极化的变化是如何结合起来促进获能的。研究cAMP和离子通透性的变化之间的串扰对于理解获能的分子基础和为受精过程的药物控制提供新的靶点是必不可少的。