alpha-endosulfine in mammalian oocyte meiotic maturation

描述（由申请人提供）：本R03项目将描述1-亚砜（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。从果蝇的令人兴奋的研究来看，内Endos与卵母细胞减数分裂成熟有关，内Endos缺乏的卵母细胞在I前期停滞和I中期纺锤体的组织过程中存在缺陷（Development 135:3697）。此外，在果蝇S2细胞有丝分裂纺锤体组装所需因子的RNAi筛选中鉴定了内切酶（endo） （Science 316:417）。最后，人类Ensa挽救了果蝇enendos缺陷卵母细胞的减数分裂阻滞表型（Development 135:3697），表明enens / Ensa的减数分裂功能在进化上是守恒的。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，验证ENSA /ENSA是控制减数分裂成熟的进化保守途径的一部分这一广泛而全面的假设。这一假设将以两个具体目标进行检验。在Aim 1中，我们将在小鼠卵母细胞减数分裂过程中表征Endos/ENSA的表达和定位。使用抗ENSA抗体的小鼠卵巢切片免疫组化显示卵母细胞和体细胞染色（Development 135:3697），我们在这里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的作用的理解，并为未来的研究奠定基础。此外，对ENSA的表达和定位的研究将通过对ENSA的候选上游调节因子的类似表征进行补充，该激酶被称为Greatwall或MASTL（微管相关丝氨酸/苏氨酸激酶样），我们发现该激酶在小鼠卵中大量表达。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。Endos通过促进polo样激酶和Cdc25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I前期停滞或延迟I前期停滞的进展，并伴有减数分裂纺锤体组装缺陷；(b)小鼠卵母细胞中Ensa的过表达将导致即使在维持这种停滞的培养条件下也会退出I前期停滞，和/或加速减数分裂的进展。我们这项工作的最终目标是建立一个更广泛的、长期的哺乳动物卵母细胞减数分裂成熟的ENSA项目，建立在通过这个试点研究产生的数据的基础上。

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.