Investigating conserved mechanisms that orchestrate the prophase to metaphase transition during meiosis I

有性繁殖的生物通过减数分裂产生配子， 细胞分裂成两半，形成一个单倍体精子或卵子。尽管减数分裂对所有动物生命都是必不可少的， 容易出错，估计有5 - 25%的人类受孕导致非整倍体胚胎，通常导致 严重的发育缺陷和妊娠并发症，每年影响数百万人。 因此，关键是要研究核心的分子机制，确定通过减数分裂的进展， 进一步了解可能存在哪些检查点来检测错误。本建议探讨控制 减数分裂进程使用两个强大的减数分裂模型，实验室小鼠小家鼠和线虫 秀丽隐杆线虫减数分裂I通过同源染色体的配对、联会和交换（CO）进行。 只有当这些事件正确发生时，同源染色体才能对齐 适当地位于减数分裂纺锤体上，然后在第一次减数分裂时均等分离。但目前尚不清楚 如何成功的CO形成被细胞周期机制感知，从而将减数分裂推进到M期。适当CO 在小鼠精母细胞中，其形成需要含有蛋白质CNTD1的正向细胞周期蛋白样结构域， COSA-1，在C.线虫卵子发生有趣的是，CNTD1与CDC34相互作用，CDC34是一种泛素E2缀合物， 与E3泛素连接酶的Skp1-Cullin-F-Box（SCF）家族作用的酶。CDC34的一个已知靶点， 有丝分裂系统是细胞周期M期抑制剂，WEE1。在CNTD1基因敲除小鼠精母细胞中，CDC34表达上调。 WEE1在减数分裂前期一直持续到结束，表明WEE1在减数分裂前期起着重要作用。 对于CNTD1促进的WEE 1降解中的交叉成熟和导致的进入M期的进展。这 导致了这样的假设，即从减数分裂前期I到M期I的进展是通过直接的 WEE 1泛素化由SCFCDC 34以交叉依赖的方式，在真核生物中保守。 该提议提出了两个具体的目标：1）SCFCDC 34介导的WEE 1泛素化是否对进展至关重要 从减数分裂前期I到M期I WEE1泛素化和WEE1/CDC 34染色质定位将在 在野生型和交叉缺陷突变小鼠中直接测试。将开发一种检测方法， 可视化C中WEE 1直系同源物的生殖系丰度。elegans，WEE-1.3，以测试WEE-1.3在 C.秀丽隐杆线虫减数分裂进程2)SCFCDC 34介导WEE 1的生化机制是什么 泛素化？将在小鼠精母细胞中阐明SCFCDC 34复合物的分子组分 通过对假定复合体成员的突变体分析，并在C. elegans通过RNAi筛选。这项工作将 在康奈尔大学兽医学院Paula Cohen博士的实验室进行，提供 在哺乳动物生殖生物学方面的广泛培训，特别是在早期减数分裂事件中， 配子发生该研究策略与申请人的培训和职业发展计划相结合， 提供了一个独特的方法，通过使用互补的模式生物减数分裂的调查。

Sexually reproducing organisms generate gametes through meiosis, the process by which the genetic material of the cell is halved to form a haploid sperm or egg. Although essential for all animal life, meiosis is strikingly error-prone, with an estimated 5-25% of all human conceptions resulting in an aneuploid embryo, often leading to severe developmental defects and pregnancy complications that effect millions of individuals every year. Therefore, it is critical to investigate the core molecular mechanisms that define progression through meiosis to further understand what checkpoints may exist to sense errors. This proposal investigates the control of meiotic progression using two powerful meiotic models, the lab mouse Mus musculus and the nematode Caenorhabditis elegans. Meiosis I proceeds through pairing, synapsis, and crossing over (CO) of homologous chromosomes during prophase, and only once these events have occurred correctly can homologs align appropriately on the meiotic spindle and then segregate equally at the first meiotic division. However, it is unclear how successful CO formation is sensed by cell cycle machinery to progress meiosis into M-phase. Proper CO formation requires orthologous cyclin-like domain containing proteins CNTD1, in mouse spermatocytes, and COSA-1, in C. elegans oogenesis. Interestingly, CNTD1 interacts with CDC34, a ubiquitin E2 conjugating enzyme that acts with the Skp1-Cullin-F-Box (SCF) family of E3 ubiquitin ligases. A known target of CDC34 in mitotic systems is the cell cycle M-phase inhibitor, WEE1. In CNTD1 knockout mouse spermatocytes, CDC34 is absent from nuclei and WEE1 persists aberrantly through until the end of meiotic prophase, suggesting a role for CNTD1-promoted crossover maturation in WEE1 degradation and resulting progression into M-phase. This leads to the hypothesis that progression from meiotic prophase I to M-phase I is licensed through direct WEE1 ubiquitination by SCFCDC34 in a crossover-dependent manner that is conserved across eukaryotes. This proposal presents two specific aims: 1) Is SCFCDC34-mediated ubiquitination of WEE1 critical for progression from meiotic prophase I to M-phase I? WEE1 ubiquitination and WEE1/CDC34 chromatin localization will be directly tested in both wild type and crossover deficient mutant mice. An assay will be developed to directly visualize the germ line abundance of the WEE1 ortholog in C. elegans, WEE-1.3, to test the role of WEE-1.3 in C. elegans meiotic progression. 2) What is the biochemical mechanism of SCFCDC34 mediated WEE1 ubiquitination? The molecular components of the SCFCDC34 complex will be elucidated in mouse spermatocytes through mutant analysis of putative complex members, and in C. elegans through a RNAi screen. This work will be performed in the lab of Dr. Paula Cohen in the Cornell University College of Veterinary Medicine, providing extensive training in mammalian reproductive biology, specifically in the early meiotic events during gametogenesis. This research strategy coupled with the applicant’s training and career development plans will provide a unique approach towards investigation of meiosis through using complementary model organisms.