Cyclic AMP and the Control of the Meiotic Cell Cycle

描述（由申请人提供）：本提案重点关注雌性配子特化减数分裂细胞周期中G2/M转换的调节机制。这是一个发生在由体细胞创建的明确定义的“小生境”中的过程，可以用作细胞-细胞相互作用的范例。了解所涉及的信号转导将提供一个模型，cAMP如何调节进入和退出细胞周期的多细胞结构，从而阐明旁分泌机制的调节细胞复制。在过去的五年中，我们已经确定了cAMP信号级联的关键组成部分，控制MPF（cyclinB/cdc 2复合物）在小鼠卵母细胞的活动。我们的遗传模型，其中这些组件已在体内失活表明，在卵母细胞中的cAMP信号的中断与减少或完全女性不育和G2/M转换的封锁。我们建议使用这些模型以及我们已经产生的生化工具来了解cAMP如何作为重新进入减数分裂细胞周期的信号以及体细胞如何控制生殖细胞中的这一途径。实验计划分为三个具体目标。第一个具体目标将致力于了解cAMP合成是如何调节卵母细胞。将分析GPCR以及卵母细胞中表达的PDE缺陷的小鼠的减数分裂恢复以及与细胞内cAMP水平和细胞周期调节剂活性相关的细胞周期状态。此外，将研究体细胞产生的GPCR及其配体的性质。第二个具体目标将致力于了解PDE 3A是如何通过磷酸化调节的。PDE 3A是小鼠卵母细胞中鉴定的主要PDE，其对于减数分裂成熟是必不可少的。最后一个具体目标将集中在PKA下游的cAMP级联反应中的步骤。将分析双重磷酸酶cdc 25 B和新发现的激酶Wee 1 B的PKA磷酸化作用以及它们在卵母细胞中的定位。还将探讨这两种酶的活性和定位对MPF激活状态的影响。这些研究将进一步加深我们对特化减数分裂细胞周期的调控以及

DESCRIPTION (provided by applicant): The present proposal focuses on the mechanisms of regulation of the G2/M transition in the specialized meiotic cell cycle of the female gamete. This is a process that occurs in a well defined "niche" created by somatic cells that can be used as a paradigm of cell-cell interaction. Understanding the signaling involved will provide a model for how cAMP regulates entry and exit from the cell cycle in a multicellular structure thereby shedding light on paracrine mechanisms of regulation of cell replication. Over the past five years, we have identified key components in the cAMP signaling cascade that control the activity of MPF (cyclinB/cdc2 complex) in mouse oocytes. Our genetic models where these components have been inactivated in vivo show that disruption in cAMP signaling in the oocytes is associated with reduced or complete female infertility and blockade in G2/M transition. We propose to use these models as well as biochemical tools that we have generated to understand how cAMP functions as a signal for reentry into the meiotic cell cycle and how somatic cells control this pathway in germ cells. The experimental plan is organized into three Specific Aims. The first Specific Aim will be devoted to understand how cAMP synthesis is regulated in oocytes. Mice defective in GPCRs as well PDEs expressed in oocytes will be analyzed for meiotic resumption and the state of the cell cycle related to intracellular cAMP levels and activity of cell cycle regulators. In addition, the properties of the GPCRs and their ligands produced by somatic cells will be investigated. The second Specific Aim will be devoted to understanding how PDE3A is regulated by phosphorylation. PDE3A is the major PDE identified in mouse oocytes, which is indispensable for meiotic maturation. The last Specific Aim will focus on the steps in the cAMP cascade downstream of PKA. The effect of PKA phosphorylation of the dual phosphatase cdc25B and the newly discovered kinase Wee1 B as well as their localization in the oocytes will be analyzed. The impact of the activity and localization of these two enzymes on the MPF state of activation also will be explored. The studies proposed will further our understanding of the regulation of the specialized meiotic cell cycle as well as the