Membrane Potential and cAMP Crosstalk in Sperm Capacitation

描述（由申请人提供）：哺乳动物的精子不能在射精后立即使卵子受精。它们在雌性生殖道停留一段有限的时间后获得受精能力，这一过程被称为获能[24,25]。最初，能化被定义为以受精为终点。然而，各种证据表明，在获能过程中发生的精子功能变化不是一个事件，而是一系列连续和伴随过程的组合。其中一些过程在精子从附睾中释放出来时立即发生，另一些则较慢，并且只有在精子在支持精子与卵子受精能力的条件下孵育一段时间后才被激活。这些缓慢的事件与运动模式的改变（如过度激活）和获得精子进行激动剂刺激顶体反应（AR）的能力有关。尽管快速和慢速事件都受HCO3-和camp依赖性途径的调节，但慢速事件受到精子质膜释放胆固醇的限制。使用小鼠作为实验模型，我们已经证明，这些最后的事件与蛋白激酶a （PKA）依赖性的酪氨酸（tyr）磷酸化的增加有关[23,26]。与我们的发现同时，Zeng等人（1995）报道了获能伴随着精子质膜卵电位（Em）的超极化。据推测，超极化是驱动低电压激活（LVA） Ca2+ t通道（Cav3）从无活性状态到封闭状态所必需的，这种状态可以被透明带（ZP）等激动剂激活，从而触发AR。尽管取得了这些进展，但人们对这些分子变化如何结合起来促进容能知之甚少。作为第一期资助的一部分，我们已经证明HCO3-、Na+和K+参与精子Em的调节[21,27,28]，并且上皮Na+通道（Epithelial Na+ channels, ENaC）存在于精子中，并在精子静息Em bb0的调节中发挥重要作用。本研究的目的是了解cAMP、蛋白磷酸化和超极化的变化是如何共同促进能化的。研究cAMP与离子通透性变化之间的串扰关系，对于理解细胞能化的分子基础和为受精过程的药理控制提供新的靶点具有重要意义。

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.