alpha-endosulfine in mammalian oocyte meiotic maturation

描述（由申请人提供）：本R 03项目将描述1-硫丹（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。从果蝇中令人兴奋的研究中发现，Endos与卵母细胞减数分裂成熟有关，Endos缺陷的卵母细胞在从前期I停滞的进展和中期I纺锤体的组织中具有缺陷（Development 135：3697）。此外，在果蝇S2细胞中有丝分裂纺锤体组装所需的因子的RNAi筛选中鉴定了Endos（Science 316：417）。最后，人Ensa挽救了果蝇endos缺陷型卵母细胞中的减数分裂停滞表型（Development 135：3697），表明Endos/ENSA的减数分裂功能的进化保守性.本文提出的研究将采取下一步措施，直接检查ENSA在小鼠卵母细胞中的功能，测试广泛的总体假设，即Endos/ENSA是控制减数分裂成熟的进化保守途径的一部分。我们将以两个具体目标来检验这一假设。在目的1中，我们将表征Endos/ENSA在小鼠卵母细胞减数分裂过程中的表达和定位。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），我们在此提出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的表达和定位的工作将通过ENSA的候选上游调节子的类似表征来补充，该候选上游调节子是称为Greatwall或MASTL（微管相关丝氨酸/苏氨酸激酶样）的激酶，我们发现其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Cdc 25的稳定性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，与减数分裂纺锤体组装缺陷相结合，和（B）小鼠卵母细胞中Ensa的过表达将导致退出前期I停滞，即使培养条件应该维持这种停滞，和/或加速减数分裂的进展。我们这项工作的最终目标是开发一个更广泛的，长期的项目ENSA在哺乳动物卵母细胞减数分裂成熟，通过这项试点研究产生的数据的基础上建立。 公共卫生相关性：减数分裂是产生单倍体配子（精子和卵子）的一个非常重要的细胞分裂过程。女性减数分裂过程中的错误可能是不育和配子和胚胎染色体异常的重要原因;在临床确认的妊娠中检测到7-10%的染色体错误。这项研究项目将研究一种新的分子，这是一个候选人，以调节女性减数分裂。

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.