Phosphorylation Events During Sperm Capacitation

描述（由申请人提供）：哺乳动物精子在射精后不能立即使卵子受精。它们通过一个被称为获能的过程在雌性生殖道中获得受精能力。最初，获能被定义为使用受精作为终点。然而，各种证据表明，精子获能过程中发生的功能变化不是一个事件，而是一个顺序和伴随过程的组合。其中一些过程发生在精子从附睾中释放出来时，另一些则较慢，只有在精子孵育一定时间后才能在支持精子使卵子受精的条件下激活。这些缓慢的事件与运动模式的变化（例如超活化）和精子获得进行激动剂刺激的顶体反应（AR）的能力有关。尽管快速和缓慢事件都受到HCO 3-、Adcy 10激活、非典型可溶性腺苷酸环化酶（也称为sAC）和随后cAMP依赖性途径激活的调节，但较慢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，我们已经表明，这些最后的事件与蛋白酪氨酸（tyr）磷酸化的蛋白激酶A（PKA）依赖性增加。我们还证明，酪氨酸磷酸化以及获能的增加受到胆固醇受体如牛血清白蛋白（BSA）在获能介质中的存在的调节。在该提议的第一个周期中，我们发现，尽管有必要，但涉及HCO 3-/SAC/PKA的经典线性途径不足以引起下游靶点的磷酸化。除了PKA激活，另一条导致Ser/Thr磷酸酶下调的途径也是必要的。第二条途径对调节PKA途径和激活精子获能所需的其他PKA非依赖性磷酸化途径至关重要。这一提议的基础假设是，获能是由两种途径的联合作用引起的，一种途径由胆固醇受体（例如BSA）调节，诱导cSrc家族激酶（SFK）诱导的ser/thr磷酸酶下调，另一种途径由HCO 3-和Ca 2+调节，并由sAC和PKA的活化介导。本提案的目的是了解这两种途径在获能过程中是如何整合的。 公共卫生相关性：早期哺乳动物卵子体外受精的困难主要是由于缺乏对精子生理学的理解。本研究旨在了解精子获能的分子基础，重点是精子蛋白磷酸化信号通路的调控。这些目标的实现将导致新的见解在男性不育症的临床诊断，并提供评估药物避孕目标的工具。

DESCRIPTION (provided by applicant): Mammalian sperm are not able to fertilize eggs immediately after ejaculation. They acquire fertilization capacity in the female tract in a process known as capacitation. 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- , activation of Adcy10, the atypical soluble adenylyl cyclase (also known as sAC) and the subsequent activation of 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 shown that these last events are associated with a protein kinase A (PKA)-dependent increase in protein tyrosine (tyr) phosphorylation. We have also demonstrated that the increase in tyr phosphorylation as well as capacitation was regulated by the presence of cholesterol acceptors such as Bovine Serum Albumin (BSA) in the capacitation media. During the first cycle of this proposal we have discovered that although necessary, the classical linear pathway involving HCO3-/SAC/PKA is not sufficient to elicit phosphorylation of downstream targets. On top of PKA activation, another pathway leading to downregulation of ser/thr phosphatases is also necessary. This second pathway is essential to modulate the PKA pathway and for the activation of other PKA- independent phosphorylation pathways needed for sperm capacitation. The hypotheses underlying this proposal postulate that capacitation results from the combined action of two pathways, one regulated by cholesterol acceptors (e.g. BSA) inducing a cSrc family kinase (SFK)-induced downregulation of ser/thr phosphatase(s) and the other regulated by HCO3- and Ca2+ and mediated by the activation of sAC and PKA. The objective of this proposal is to understand how these two pathways are integrated during capacitation. PUBLIC HEALTH RELEVANCE: Difficulties in earlier efforts to fertilize mammalian eggs in vitro were due mainly to a lack of comprehension of sperm physiology. This proposal is aimed to understand the molecular basis of sperm capacitation with emphasis in the regulation of signaling pathways involving protein phosphorylation in sperm. Accomplishment of these goals will lead to novel insights in clinical diagnosis of male infertility and provide tools for evaluating pharmacological contraceptive targets.