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alpha-endosulfine in mammalian oocyte meiotic maturation

α-硫辛在哺乳动物卵母细胞减数分裂成熟中的作用

基本信息

批准号：
8299422
负责人：
JANICE P EVANS
金额：
$ 8.2万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-08 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8299422
关键词：
Address Affect Antibodies Cell Division Process Cells Chromosome abnormality Complement Complex Cues Cyclic AMP-Dependent Protein Kinases Cytoplasm Data Defect Development Drosophila genus Embryo Female Foundations Future Germ Cells Goals Growth Haploidy Hormonal Human Immunoblotting Immunofluorescence Immunologic Immunohistochemistry Infertility Lead Mammals Meiosis Meiotic Prophase I Metaphase Microtubules Mitotic spindle Molecular Mus Nomenclature Oocytes Organism Ovarian PLK1 gene Pathway interactions Phenotype Phosphotransferases Pilot Projects Pregnancy Process Prophase Protein-Serine-Threonine Kinases Proteins RNA Interference Regulation Research Research Project Grants Science Somatic Cell Staging Staining method Stains Testing Vesicle Work alpha-endosulfine anaphase-promoting complex base cross reactivity egg endosulfine human PLK1 protein insight mutant novel overexpression sperm cell

项目摘要

DESCRIPTION (provided by applicant): This R03 project will characterize the functions in female meiosis of 1-endosulfine (known as Endos in Drosophila and ENSA in mammals). Endos is implicated in oocyte meiotic maturation from exciting studies in Drosophila, with endos-deficient oocytes having defects in progression from prophase I arrest and in organization of the metaphase I spindle (Development 135:3697). Additionally, Endos was identified in an RNAi screen for factors required for mitotic spindle assembly in Drosophila S2 cells (Science 316:417). Finally, human Ensa rescues the meiotic arrest phenotype in Drosophila endos- deficient oocytes (Development 135:3697), suggesting evolutionary conservation of a meiotic function for Endos/ENSA. The research proposed here will take the next step and directly examine ENSA functions in mouse oocytes, testing the broad, overall hypothesis that Endos/ENSA is part of an evolutionarily conserved pathway controlling meiotic maturation. This hypothesis will be examined with two Specific Aims. In Aim 1, we will characterize the expression and localization of Endos/ENSA in mouse oocytes during progression through meiosis. Immunohistochemistry of mouse ovarian sections using an anti-Endos antibody shows staining of oocytes and somatic cells (Development 135:3697), and our preliminary studies presented here show that mouse oocytes express ENSA. More detailed data will advance our understanding of ENSA function in mammalian female meiosis, and will set the stage for future studies. Additionally, the work in this aim on the expression and localization of ENSA will be complemented by similar characterization of a candidate upstream regulator of ENSA, the kinase known as Greatwall or MASTL (Microtubule-Associated Serine/Threonine kinase-Like), which we find is abundantly expressed in mouse eggs. In Aim 2, we will determine the effects of ENSA disruption (knockdown or overexpression) on meiotic maturation of mouse oocytes. Endos affects Drosophila meiotic maturation by promoting the stability of Polo-like kinase and Cdc25 (Development 135:3697). Based on these results, we hypothesize that (a) knockdown of Ensa in mouse oocytes will result in persistent prophase I arrest or delayed progression out of prophase I arrest, combined with meiotic spindle assembly defects, and (b) overexpression of Ensa in mouse oocytes will result in exit from prophase I arrest even culture conditions that should maintain this arrest, and/or accelerated progression through meiosis. Our ultimate goal for this work is to develop a broader, long-term project on ENSA in mammalian oocyte meiotic maturation, built on the foundation of data generated through this pilot study.

描述(申请人提供)：这个R03项目将描述1-内硫醚(在果蝇中称为endos，在哺乳动物中称为ensa)在雌性减数分裂中的功能。ENDOS与果蝇卵母细胞的减数分裂成熟有关，在果蝇中，ENTOS缺陷的卵母细胞从前期I停滞和中期I纺锤体的组织开始都有缺陷(Development 135：3697)。此外，在RNAi筛选中，在果蝇S2细胞中鉴定了有丝分裂纺锤体组装所需的因子(Science 316：417)。最后，人类Ensa挽救了果蝇内源性缺陷卵母细胞的减数分裂停滞表型(Development 135：3697)，表明Endos/Ensa的减数分裂功能在进化上是保守的。本文提出的研究将迈出下一步，直接检测ENSA在小鼠卵母细胞中的功能，检验广泛的、全面的假设，即Endos/ENSA是控制减数分裂成熟的进化保守途径的一部分。这一假设将以两个具体目标进行检验。在目标1中，我们将研究Endos/ENSA在小鼠卵母细胞减数分裂过程中的表达和定位。用抗Endos抗体对小鼠卵巢切片的免疫组织化学染色显示卵母细胞和体细胞(Development 135：3697)，我们的初步研究表明小鼠卵母细胞表达ENSA。更详细的数据将促进我们对ENSA在哺乳动物雌性减数分裂中的功能的理解，并为未来的研究奠定基础。此外，在ENSA的表达和定位方面的工作将得到ENSA的一个候选上游调节因子的类似表征的补充，该激酶被称为Greatwall或Mastl(微管相关丝氨酸/苏氨酸激酶样蛋白)，我们发现它在小鼠的卵子中大量表达。在目标2中，我们将确定ENSA干扰(敲除或过度表达)对小鼠卵母细胞减数分裂成熟的影响。Endos通过促进Polo-like激酶和CDC25的稳定性影响果蝇的减数分裂成熟(Development 135：3697)。基于这些结果，我们假设：(A)在小鼠卵母细胞中敲除Ensa将导致持续性的前期I期停滞或延缓前期I期停滞的进程，并伴随着减数分裂纺锤体组装缺陷，以及(B)Ensa在小鼠卵母细胞中的过表达将导致退出早期I期停滞，甚至在应该维持这种停滞的培养条件下，和/或通过减数分裂加速进展。我们这项工作的最终目标是在通过这项初步研究产生的数据的基础上，开发一个关于哺乳动物卵母细胞减数分裂成熟中ENSA的更广泛、更长期的项目。