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

• 批准号: 8191837
• 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
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-08 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8191837
• 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

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. PUBLIC HEALTH RELEVANCE: Meiosis is a fundamentally important cell division process that generates the haploid gametes (sperm and egg). Errors during female meiosis can be a significant cause of infertility and of chromosomal abnormalities in gametes and embryos; such chromosomal errors are detected in 7-10% of clinically recognized pregnancies. This research project will examine a novel molecule that is a candidate to regulate female meiosis.

描述（由申请人提供）：该 R03 项目将描述 1-endosulfine（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。根据果蝇令人兴奋的研究，Endos 与卵母细胞减数分裂成熟有关，Endos 缺陷的卵母细胞在前期 I 停滞的进展和中期 I 纺锤体的组织中存在缺陷 (Development 135:3697)。此外，Endos 在 RNAi 筛选中被鉴定为果蝇 S2 细胞中有丝分裂纺锤体组装所需的因子 (Science 316:417)。最后，人类 Ensa 挽救了果蝇内壁缺陷卵母细胞中的减数分裂停滞表型 (Development 135:3697)，表明 Endos/ENSA 减数分裂功能的进化保守性。这里提出的研究将采取下一步，直接检查小鼠卵母细胞中的 ENSA 功能，测试 Endos/ENSA 是控制减数分裂成熟的进化保守途径的一部分的广泛的整体假设。该假设将通过两个具体目标进行检验。在目标 1 中，我们将描述减数分裂过程中小鼠卵母细胞中 Endos/ENSA 的表达和定位。使用抗 Endos 抗体对小鼠卵巢切片进行免疫组织化学显示卵母细胞和体细胞染色 (Development 135:3697)，我们在此介绍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哺乳动物雌性减数分裂中 ENSA 功能的理解，并为未来的研究奠定基础。此外，ENSA 的表达和定位这一目标的工作将得到 ENSA 候选上游调节因子的类似表征的补充，该激酶称为 Greatwall 或 MASTL（微管相关丝氨酸/苏氨酸激酶样），我们发现它在小鼠卵中大量表达。在目标 2 中，我们将确定 ENSA 破坏（敲低或过度表达）对小鼠卵母细胞减数分裂成熟的影响。 Endos 通过促进 Polo 样激酶和 Cdc25 的稳定性来影响果蝇减数分裂成熟 (Development 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前期 I 停滞或前期 I 停滞的延迟进展，并伴有减数分裂纺锤体组装缺陷，以及 (b) 小鼠卵母细胞中 Ensa 的过度表达将导致退出前期 I 停滞，甚至应维持这种停滞的培养条件，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这项试点研究产生的数据基础上，针对哺乳动物卵母细胞减数分裂成熟的 ENSA 开发一个更广泛、长期的项目。 公众健康相关性：减数分裂是产生单倍体配子（精子和卵子）的一个极其重要的细胞分裂过程。雌性减数分裂过程中的错误可能是导致不孕以及配子和胚胎染色体异常的重要原因；在临床认可的妊娠中，7-10% 会检测到此类染色体错误。该研究项目将研究一种新分子，该分子是调节雌性减数分裂的候选分子。