Phosphorylation Events During 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。它们在雌性生殖道中获得受精能力，这一过程被称为获能。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。尽管快速和缓慢的事件都受到HCO3-、Adcy10的激活、非典型可溶性腺苷酸环化酶（也称为sAC）以及随后camp依赖性途径的激活的调节，但较慢的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）依赖性蛋白酪氨酸（tyr）磷酸化的增加有关。我们还证明，酪氨酸磷酸化的增加以及容化是由容化介质中胆固醇受体如牛血清白蛋白（BSA）的存在所调节的。在本提案的第一个周期中，我们发现，尽管有必要，但涉及HCO3-/SAC/PKA的经典线性途径不足以引发下游靶点的磷酸化。在PKA激活的基础上，另一条导致丝氨酸/苏氨酸磷酸酶下调的途径也是必要的。第二种途径对于调节PKA途径和激活精子获能所需的其他不依赖于PKA的磷酸化途径至关重要。这一提议的基础假设是两种途径的共同作用，一种是由胆固醇受体（如BSA）诱导证监会家族激酶（SFK）诱导丝氨酸/瑟氨酸磷酸酶下调，另一种是由HCO3-和Ca2+调节，由sAC和PKA的激活介导。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。

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.