Cyclic AMP and the Control of the Meiotic Cell Cycle

描述（由申请人提供）：本提案侧重于雌性配子减数分裂细胞周期中G2/M转变的调节机制。这一过程发生在由体细胞创造的一个明确定义的“生态位”中，可以作为细胞-细胞相互作用的范例。了解所涉及的信号传导将为cAMP如何在多细胞结构中调节细胞周期的进入和退出提供一个模型，从而揭示细胞复制调节的旁分泌机制。在过去的五年中，我们已经确定了cAMP信号级联中的关键成分，这些成分控制小鼠卵母细胞中MPF （cyclinB/cdc2复合物）的活性。我们在体内灭活这些成分的遗传模型表明，卵母细胞中cAMP信号的破坏与女性不育的减少或完全以及G2/M转换的阻断有关。我们建议使用这些模型以及我们已经生成的生化工具来了解cAMP如何作为再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。第一个特定目标将致力于了解cAMP合成如何在卵母细胞中受到调节。我们将分析gpcr缺陷小鼠以及卵母细胞中表达的PDEs的减数分裂恢复情况，以及与细胞内cAMP水平和细胞周期调节因子活性相关的细胞周期状态。此外，还将对体细胞产生的gpcr及其配体的特性进行研究。第二个特定目标将致力于了解PDE3A是如何通过磷酸化调节的。PDE3A是小鼠卵母细胞中鉴定出的主要PDE，在减数分裂成熟过程中不可或缺。最后一个具体目标将集中在PKA下游的cAMP级联步骤。我们将分析PKA对双磷酸酶cdc25B和新发现的激酶wee1b的磷酸化作用及其在卵母细胞中的定位。这两种酶的活性和定位对MPF激活状态的影响也将被探讨。所提出的研究将进一步加深我们对减数分裂细胞特化周期调控的认识

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