Role of MARCKS during mouse oocyte maturation and egg activation

MARCKS 在小鼠卵母细胞成熟和卵子激活过程中的作用

• 批准号: 7290566
• 负责人: MARCELA ALEJANDRA MICHAUT
• 金额: $ 5.08万
• 依托单位: NATIONAL UNIVERSITY OF CUYO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7290566
• 关键词: Achyrocline; Acrosome Reaction; Actins; Affect; Age; Antibodies; Biological; Calcium; Calmodulin; Cell membrane; Cell physiology; Centrosome; Complex; Cryoultramicrotomy; Cytoplasmic Granules; Cytoskeletal Proteins; Cytoskeleton; DNA Sequence Rearrangement; Development; Diploidy; Electron Microscopy; Exocytosis; Failure; Female; Fertility; Fertilization; Fluorescence Microscopy; Germ Cells; Haploidy; Histology; Human; Immunoelectron Microscopy; Infertility; Investigation; Ionophores; Knockout Mice; Meiosis; Meiotic Prophase I; Membrane; Membrane Fusion; Messenger RNA; Metaphase; Modification; Molecular; Mus; Nuclear; Nuclear Envelope; Oocytes; Optics; Ovary; Peripheral; Pharmacotherapy; Phorbol Esters; Phosphorylation; Play; Process; Protein Kinase C; Protein Overexpression; Proteins; RNA Interference; Rattus; Reaction; Reporting; Reproduction; Research Personnel; Role; Signal Transduction Pathway; Staging; Structure; Substrate Domain; Technology; Testing; Time; Transgenic Organisms; atypical protein kinase C; base; cell type; citrate carrier; egg; improved; in vivo; male; mutant; myristoylated alanine-rich C kinase substrate; myristoylation; novel; oocyte maturation; prevent; programs; reproductive; research study; secretion process; success; zygote

DESCRIPTION (provided by applicant): 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.

描述(申请人提供)：MARCKS，富含肉豆蔻酰化丙氨酸的C-激酶底物的首字母缩写，是体内蛋白激酶C(PKC)的主要底物，以磷酸化、肉豆蔻酸化和钙调素依赖的方式与质膜相互作用。尽管Marcks参与了许多细胞过程，但其确切功能尚不清楚。Marcks基因敲除小鼠未能存活到生殖年龄，阻碍了对Marcks在卵母细胞发育过程中功能的评估，目前尚不清楚Marcks缺陷是如何影响卵母细胞成熟和生育的。我们已报道Marcks在小鼠卵母细胞中表达，其磷酸化形式pMARCKS是一种新的中心体成分，它还定义了小鼠卵中皮质肌动蛋白帽的外周亚域。此外，Marcks与皮质颗粒(CG)胞吐有关，但其功能尚不清楚。这些结果表明，Marcks在卵母细胞减数分裂成熟过程中可能具有多种功能。这项建议的长期目标是确定Marcks在小鼠卵母细胞成熟和卵子激活过程中的功能。首先，为了确定pMARCKS在减数分裂卵母细胞成熟中的作用，将一种特定的pMARCKS抗体显微注射到小鼠卵母细胞中，并用共聚焦显微镜和电子显微镜分析减数分裂的进展，特别是纺锤体组装、细胞骨架重组和极体排出。在不同的GFP-pMARCKS突变体过表达后，将进行类似的实验。其次，为了评估与CG膜相关的MARCKS的存在，将在超薄冰冻切片中进行免疫电子显微镜研究。我们还将研究Marcks在CG胞吐过程中膜融合中的功能，将不同的Marcks结构域注入或运送到小鼠卵子中。第三，为了评估Marcks在卵母细胞发育和生育中的作用，我们将产生Marcks转基因敲除小鼠，在其中Marcks mRNA将在卵母细胞中被选择性地去除，并对它们的生育能力进行分析。卵巢组织学检查将通过光学显微镜和荧光显微镜进行。卵母细胞成熟是减数分裂的关键，为受精做好准备。了解哺乳动物卵母细胞成熟的分子机制是开发新的药物和治疗方法以改善或防止人类受精的必要前提。