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（由申请人提供）：该R03项目将表征1-硫磺胺女性减数分裂的功能（果蝇中被称为胚胎和哺乳动物中的ensa）。恩德斯与果蝇激动人心的研究有关的卵母细胞减少成熟，而内斯缺陷型卵母细胞在预言I停滞和中期I纺锤体中的进展中具有缺陷（开发135：3697）。此外，在RNAi筛选中鉴定出了内核，该果皮S2细胞中有丝分裂纺锤体组装所需的因素（科学316：417）。最后，人类Ensa营救了果蝇内部缺陷的卵母细胞中的减数分裂停滞表型（开发135：3697），表明对内生物/ENSA的减数分裂功能的进化保守。这里提出的研究将采取下一步，并直接检查小鼠卵母细胞中的ENSA功能，检验了广泛的总体假设，即endos/ensa是控制减数分裂成熟的进化保守途径的一部分。该假设将以两个具体的目的进行审查。在AIM 1中，我们将表征通过减数分裂进展过程中小鼠卵母细胞中内生物/ENSA的表达和定位。小鼠卵巢切片的免疫组织化学使用抗ENDOS抗体显示出卵母细胞和体细胞的染色（发育135：3697），此处介绍的我们的初步研究表明，小鼠卵母细胞表达ENSA。更详细的数据将提高我们对哺乳动物女性减数分裂中ENSA功能的理解，并为将来的研究奠定了基础。此外，此目的的工作将通过类似的ENSA候选调节剂（称为Greatwall或MASTL）或MASTL（微管相关/苏氨酸激酶类似）的激酶的候选表征来补充ENSA的表达和定位，我们发现我们发现在小鼠蛋中表达了丰富的表达。在AIM 2中，我们将确定ENSA破坏（敲低或过表达）对小鼠卵母细胞减数分裂成熟的影响。恩迪斯通过促进类似polo样激酶和cdc25的稳定性影响果蝇减数分裂成熟（开发135：3697）。基于这些结果，我们假设（a）小鼠卵母细胞中ENSA的敲低将导致持续的预言I逮捕或延迟从预言I逮捕中，与减数分裂的纺锤体组件缺陷相结合，（b）小鼠卵母细胞中ENSA的过度表达将导致暂停的捕获，甚至可以使我的疗法暂停，甚至可以维持这种疗法，甚至可以维持这种情况。这项工作的最终目标是在哺乳动物卵母细胞减数分裂成熟中开发一个更广泛的长期项目，这是基于通过这项试验研究产生的数据的基础。