MARCKS, acronym for myristoylated alanine-rich C-kinase substrate, is a major in vivo substrate of protein kinase C (PKC) and interacts with plasma membranes in a phosphorylation-, myristoylation-, and calmodulin-dependent manner. Although MARCKS has been implicated in many cellular processes, its precise function is yet unclear. The failure of MARCKS knockout mice to survive to reproductive age has prevented any assessment of MARCKS function during oocyte development and it is unknown how MARCKS deficiency affects oocyte maturation and fertility. We have reported that MARCKS is expressed in mouse oocytes and that its phosphorylated form, pMARCKS, is a novel centrosome component that also defines a peripheral subdomain of the cortical actin cap in mouse eggs. Furthermore, MARCKS has been associated to cortical granules (CG) exocytosis but its function is unknown. These results suggest that MARCKS might have multiple functions during meiotic oocyte maturation. The long-term objective of this proposal is to determine the function of MARCKS during mouse oocyte maturation and egg activation. First, to identify the role of pMARCKS in meiotic oocyte maturation, mouse oocytes will be microinjected with a specific pMARCKS antibody and the progression of meiosis, particularly spindle assembly, cytoskeleton reorganization, and extrusion of polar bodies will be analyzed by confocal and electron microscopy. Similar experiments will be performed after overexpression of different GFP-pMARCKS mutants. Second, to assess the presence of MARCKS associated to CG membranes, immunoelectron microscopy studies in ultrathin cryosections will be performed. We will also characterize MARCKS function in membrane fusion during CG exocytosis, injecting or delivering into mouse eggs different MARCKS domains. Third, to assess the role of MARCKS in oocyte development and fertility, we will generate MARCKS transgenic knockdown mice in which MARCKS mRNA will be selectively ablated in the oocyte, and their fertility will be analyzed. Ovaries histology will be examined by optical and fluorescence microscopy. Oocyte maturation is essential for meiosis and prepares the oocyte for fertilization. Understanding the molecular mechanism of mammalian oocyte maturation is a necessary prerequisite to the development of new drugs and therapies to improve or prevent fertilization in human beings. ? ? ?
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