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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

基本信息

批准号：
8098169
负责人：
MARCELA ALEJANDRA MICHAUT
金额：
$ 5.03万
依托单位：
NATIONAL UNIVERSITY OF CUYO
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-01 至 2013-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8098169
关键词：
Achyrocline Acrosome Reaction Actins Affect Age Antibodies Biological Calcium Calmodulin Cell membrane Cell physiology Centrosome Complex Confocal Microscopy 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 Proteins RNA Interference Rattus Reaction Reporting Reproduction Research Personnel Role Signal Transduction Pathway Staging Structure Substrate Domain Technology Testing Time Transgenic Organisms acronyms atypical protein kinase C base cell type egg improved in vivo male mutant myristoylated alanine-rich C kinase substrate myristoylation novel oocyte maturation overexpression prevent programs reproductive research study secretion process success zygote

项目摘要

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（由申请人提供）：MARCKS，豆蔻酰化富含丙氨酸的C-激酶底物的首字母缩写，是蛋白激酶C（PKC）的主要体内底物，并以磷酸化、豆蔻酰化和钙调蛋白依赖性方式与质膜相互作用。虽然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育年龄，这阻碍了对MARCKS在卵母细胞发育过程中的功能进行任何评估，目前还不清楚MARCKS缺陷如何影响卵母细胞成熟和生育力。我们已经报道，MARCKS在小鼠卵母细胞中表达，其磷酸化形式，pMARCKS，是一种新的中心体组件，也定义了小鼠卵皮质肌动蛋白帽的外围子域。此外，MARCKS与皮质颗粒（CG）胞吐作用有关，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。本研究的长期目标是确定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，将小鼠卵母细胞用特异性pMARCKS抗体显微注射，并通过共聚焦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挤出。在过表达不同的GFP-pMARCKS突变体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，将在冷冻切片中进行免疫电镜研究。我们还将表征MARCKS在CG胞吐过程中的膜融合功能，将不同的MARCKS结构域注射或递送到小鼠卵中。第三，为了评估MARCKS在卵母细胞发育和生育力中的作用，我们将产生MARCKS转基因敲除小鼠，其中MARCKS mRNA将在卵母细胞中被选择性地去除，并将分析它们的生育力。将通过光学和荧光显微镜检查卵巢组织学。卵母细胞成熟是减数分裂所必需的，并为受精做好准备。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阻止人类受精的必要前提。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

RIM, Munc13, and Rab3A interplay in acrosomal exocytosis.

DOI：
10.1016/j.yexcr.2012.01.002
发表时间：
2012-03-10
期刊：
EXPERIMENTAL CELL RESEARCH
影响因子：
3.7
作者：
Bello, Oscar D.;Natalia Zanetti, M.;Mayorga, Luis S.;Michaut, Marcela A.
通讯作者：
Michaut, Marcela A.

Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs.

DOI：
10.1016/j.yexcr.2016.07.005
发表时间：
2016-09-10
期刊：
Experimental cell research
影响因子：
3.7
作者：
Bello OD;Cappa AI;de Paola M;Zanetti MN;Fukuda M;Fissore RA;Mayorga LS;Michaut MA
通讯作者：
Michaut MA

α-SNAP is expressed in mouse ovarian granulosa cells and plays a key role in folliculogenesis and female fertility.

DOI：
10.1038/s41598-017-12292-9
发表时间：
2017-09-18
期刊：
Scientific reports
影响因子：
4.6
作者：
Arcos A;de Paola M;Gianetti D;Acuña D;Velásquez ZD;Miró MP;Toro G;Hinrichsen B;Muñoz RI;Lin Y;Mardones GA;Ehrenfeld P;Rivera FJ;Michaut MA;Batiz LF
通讯作者：
Batiz LF

Live imaging of cortical granule exocytosis reveals that in vitro matured mouse oocytes are not fully competent to secrete their content.