Role of Cyclic Nucleotides in Sperm Function

描述（由申请人提供）：尽管cAMP在雄性配子功能中的作用被广泛接受，但在精子成熟和受精过程中cAMP产生的确切机制在很大程度上是未知的。我们的实验室使用生化和遗传方法来研究这一途径的组成部分，以及它在精子运动中的作用。我们确定至少有两种腺苷酸环化酶，一种是膜结合的AC3酶，另一种是可溶性腺苷酸环化酶（sAC），它们参与精子和精子中cAMP的产生。sAC的特性已被广泛研究，表明该环化酶整合在调节cAMP水平的正负反馈回路中。在小鼠中消融sAC会导致完全的雄性不育和精子活力的严重破坏。膜结合ACS的失活也会导致男性生育能力下降并损害精子功能。我们建议使用这些遗传体内模型来进一步确定成熟精子中cAMP信号的特性，以及它们在运动、顶体反应和受精过程中的控制作用。实验计划是按照三个具体目标组织的。第一个特异性目标将致力于进一步表征sAC无表型，以及缺乏sAC和AC3的精子中cAMP信号的特性，包括GPCR刺激运动的机制。第二个特定目标将侧重于sAC的生化特性和控制其活性的调节反馈。最后一个专题将探讨环化酶和精子下游靶标之间的关系。组织环化酶、磷酸二酯酶和蛋白激酶的大分子复合物的存在将被研究，它们在精子功能中的作用将被确定。这些研究将对受精过程的调控提供深入的见解。他们还将为验证男性配子的药理学操作靶标提供基础，并为定义男性因素提供新的诊断工具。

DESCRIPTION (provided by applicant): Although a role of cAMP in the function of the male gamete is widely accepted, the exact mechanisms of cAMP generation during the maturation of the spermatozoon and at fertilization are largely unknown. Our laboratory has used both biochemical and genetic approaches to investigate the components of this pathway, as well as its role in sperm motility. We determined that at least two adenylyl cyclases, a membrane-bound AC3 and soluble adenylyl cyclases (sAC), contribute to cAMP production in spermatids and in spermatozoa. The properties of sAC have been extensively investigated demonstrating that this cyclase is integrated in positive and negative feedback loops regulating cAMP levels. Ablation of sAC in mice produces complete male infertility and major disruption in sperm motility. Inactivation of the membrane-bound ACS also causes a decrease in male fertility and impairs sperm functions. We propose to use these genetic in vivo models to further define the properties of cAMP signaling in the maturing spermatozoon, and their role in the control of motility, acrosome reaction, and during fertilization. The experimental plan is organized along three Specific Aims. The first Specific Aim will be devoted to further characterization of the sAC null phenotype, and the properties of cAMP signaling in spermatozoa devoid of sAC and AC3, including the mechanism of GPCR stimulation of motility. The second Specific Aim will focus on the biochemical properties of sAC and on the regulatory feedback controlling its activity. The last Specific Aim will investigate the relationship between cyclases and downstream targets in the spermatozoon. The existence of macromolecular complexes organizing cyclases, phosphodiesterases, and protein kinases will be investigated and their role in sperm function will be determined. These studies will offer insight into the regulation of processes essential for fertilization. They will also provide the groundwork for validation of targets for pharmacological manipulation of the male gamete, and new diagnostic tools useful to define the male factor.