Role of Cyclic Nucleotides in Sperm Function

描述(申请人提供)：虽然cAMP在雄配子功能中的作用已被广泛接受，但在精子成熟和受精过程中cAMP的确切产生机制仍很不清楚。我们的实验室使用了生化和遗传学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酰环化酶，一种是膜结合的AC3，另一种是可溶性腺酰环化酶(SAC)，它们与精子细胞和精子中cAMP的产生有关。SAC的性质已被广泛研究，证明该环化酶整合在调节cAMP水平的正、负反馈环路中。切除小鼠的SAC会导致完全的男性不育和严重的精子运动障碍。膜结合的ACs失活也会导致男性生育力下降和精子功能受损。我们建议使用这些体内遗传模型来进一步确定成熟精子中cAMP信号的性质，以及它们在控制精子运动、顶体反应和受精过程中的作用。这项实验计划按照三个具体目标组织。第一个具体目标将致力于进一步表征SAC缺失的表型，以及在没有SAC和AC3的精子中cAMP信号的特性，包括GPCR刺激运动的机制。第二个具体目标将集中在SAC的生化特性和控制其活性的调节反馈上。最后一个具体目标是研究精子中的环化酶和下游靶标之间的关系。我们将研究环化酶、磷酸二酯酶和蛋白激酶的大分子复合体的存在，并确定它们在精子功能中的作用。这些研究将提供对受精必不可少的过程的调节的洞察。它们还将为验证雄性配子的药理操作靶标以及定义雄性因子的新诊断工具提供基础。

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.