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
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