Phosphorylation Events During Sperm Capacitation

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。它们在雌性生殖道中获得受精能力，这一过程被称为获能。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。虽然快事件和慢事件都受到HCO3-、Adcy10的激活、非典型的可溶腺苷酸环化酶(也称为SAC)以及随后cAMP依赖的途径的激活的调节，但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，我们已经证明这些最后的事件与蛋白激酶A(PKA)依赖的蛋白酪氨酸(Tyr)磷酸化的增加有关。我们还证明了酪氨酸磷酸化的增加以及获能能力的增加受到获能介质中胆固醇受体如牛血清白蛋白(BSA)的调节。在这个建议的第一个周期中，我们发现虽然有必要，但涉及HCO3-/SAC/PKA的经典线性途径不足以诱导下游靶标的磷酸化。在PKA激活的基础上，另一个导致Ser/Thr磷酸酶下调的途径也是必要的。这第二个途径对于调节PKA途径和激活精子获能所需的其他非PKA依赖的磷酸化途径是必不可少的。基于这一假设的假设是，获能是两条途径共同作用的结果，一条是由胆固醇受体(如牛血清白蛋白)调节的，诱导CSRC家族激酶(CSRC Family Kinase)诱导的丝氨酸/苏氨酸磷酸酶(S)下调，另一条是由HCO3-和Ca~(2+)调节并介导SAC和PKA的激活。这项建议的目的是了解这两条途径在获能过程中是如何整合的。

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.