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

研究减数分裂 I 期间协调前期到中期转变的保守机制

• 批准号: 10534113
• 负责人: Ian Wolff
• 金额: $ 6.76万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10534113
• 关键词: Acute; Adaptor Signaling Protein; Address; Aneuploidy; Animal Model; Animals; Auxins; Binding Proteins; Biochemical; Biochemistry; Biological Assay; CDC2 gene; CUL1 gene; Caenorhabditis elegans; Cell Cycle; Cell Cycle Progression; Cell Nucleus; Cells; Chromatin; Chromosome Pairing; Chromosome Segregation; Chromosomes; Complex; Conceptions; Coupled; Cullin Proteins; Cyclin B; Cyclins; Data; Defect; Development; Development Plans; Diploidy; Embryo; Ensure; Enzymes; Eukaryota; Event; F Box Domain; Family; Female; Gametogenesis; Genetic; Genetic Crossing Over; Genetic Materials; Germ Cells; Germ Lines; Haploidy; House mice; Human; Individual; Investigation; Knockout Mice; Knowledge; Laboratories; Laboratory mice; Lead; Licensing; Licensing Factor; Life; Lighting; Maturation-Promoting Factor; Mediating; Meiosis; Meiotic Prophase I; Metaphase; Microscopy; Mitosis; Mitotic; Modeling; Molecular; Mus; Mutant Strains Mice; Nematoda; Oogenesis; Organism; Orthologous Gene; Phase Transition; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Play; Positioning Attribute; Pregnancy Complications; Process; Prophase; Proteins; Publishing; RNA Interference; RNA interference screen; Regulation; Reproduction; Reproductive Biology; Research; Resolution; Role; Signal Transduction; Spermatocytes; System; Techniques; Testing; Testis; Training; Ubiquitin-Conjugating Enzymes; Ubiquitination; Universities; Veterinary Medicine; Work; career development; college; competence factor; egg; experimental study; genetic information; inhibitor; male; member; mutant; precursor cell; prevent; sperm cell; three dimensional structure; ubiquitin-protein ligase

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.

性繁殖生物通过减数分裂产生游戏，遗传物质的过程 虽然对所有动物生命至关重要，但减数分裂是惊人的 容易出错的人，估计所有人类概念的5-25％导致非整倍体胚胎，通常是领先的 严重的发育缺陷和妊娠并发症，每年影响数百万人。 因此，研究通过减数分裂到定义进展到的核心分子机制至关重要 进一步了解可能存在哪些检查点以感知错误。该提议调查了 使用两种强大的减数分裂模型的减数分裂进程，Lab Mouse Mus Musculus和线虫 秀丽隐杆线虫。减数分裂我通过配对，突触和跨越（CO）的同源（CO）进行 预言过程中的染色体，只有正确发生这些事件后才可以同源物对齐 适当地在减数分裂纺锤体上，然后在第一个减数分裂部门平均分离。但是，还不清楚 细胞周期机制如何将CO的形成得出如何发展，以将减数分裂发展为M期。适当的公司 形成需要含有蛋白质CNTD1的直系同源细胞周期域，小鼠精子细胞和 Cosa-1，在秀丽隐杆线虫卵子中。有趣的是，CNTD1与cdc34相互作用，cdc34是泛素E2共轭 与E3泛素连接酶的SKP1库蛋白-F-box（SCF）家族作用的酶。 Cdc34的已知目标 有丝分裂系统是细胞周期M期抑制剂WEE1。在CNTD1敲除鼠标精子细胞中，Cdc34为 缺乏核和WEE1一直存在直到减数分裂预言结束，这表明 用于CNTD1促进的WEE1降解中的跨界成熟，并导致成M期。这 导致一个假设，即从减数分裂预言I到M期的进展是通过直接获得许可的 SCFCDC34的WEE1泛素化以跨真核生物保守的交叉依赖性方式。 该提案提出了两个具体的目的：1）是SCFCDC34介导的WEE1的泛素化，这对于进展至关重要 从减数分裂预言i到M相i？ WEE1泛素化和WEE1/CDC34染色质定位将是 直接在野生型和交叉缺陷的突变小鼠中进行测试。将开发一种直接的测定 可视化秀丽隐杆线虫中WEE1直系同源物的种系抽象，WEE-1.3，以测试WEE-1.3在 秀丽隐杆线虫的进展。 2）SCFCDC34介导的WEE1的生化机制是什么 泛素化？ SCFCDC34复合物的分子成分将在小鼠精子细胞中阐明 通过对推定的复合构件的突变分析，以及在秀丽隐杆线虫中通过RNAi屏幕进行分析。这项工作将 可以在康奈尔大学兽医学院的Paula Cohen博士的实验室中进行 对哺乳动物生殖生物学的广泛培训，特别是在早期减数分裂事件中 配子发生。该研究策略以及申请人的培训和职业发展计划将 通过使用完整的模型生物提供独特的减数分裂投资方法。