Genetics and Proteomics of Mouse Egg Activation

小鼠卵子激活的遗传学和蛋白质组学

• 批准号: 10209649
• 负责人: John C Schimenti
• 金额: $ 19.45万
• 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: 2021-03-15 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10209649
• 关键词: Affect; Animals; Assisted Reproductive Technology; Biochemical; Biological Markers; Biological Models; Calcium; Calcium Signaling; Cell Cycle; Cell Differentiation process; Cell Nucleus; Cells; Chemicals; Data; Deposition; Detection; Development; Diagnosis; Diagnostic; Disease; Drosophila genus; Embryo; Embryonic Development; Enzymes; Event; Failure; Fertility; Fertilization; Fertilization in Vitro; Future; Gene Expression; Gene Proteins; Genetic; Genetic Transcription; Genetic study; Genome; Human; Infertility; Invertebrates; Ionophores; Mammals; Maternal Messenger RNA; Mediating; Meiosis; Membrane; Metaphase; Methodology; Modeling; Modification; Molecular; Molecular Genetics; Monitor; Mus; Nature; Nucleic Acids; Oocytes; Oogenesis; Organism; Pathway interactions; Phosphorylation; Phosphotransferases; Placenta; Post-Translational Protein Processing; Procedures; Process; Production; Protein Isoforms; Proteins; Proteome; Proteomics; Rana; Reporting; Role; Sea Urchins; Sterility; Strontium; Testing; Totipotent; Translations; analytical tool; base; calmodulin-dependent protein kinase II; egg; female fertility; macromolecule; men; phosphoproteomics; protein expression; sperm cell; tool; transcriptome; translation factor; zygote

PROJECT SUMMARY/ABSTRACT Ovulated human and mouse oocytes are stalled in meiosis II. They are transcriptionally quiescent but have a maternally-loaded transcriptome and proteome. Fertilization triggers “egg activation”, in which a rise(s) of calcium in the oocyte induces several key events that allow transition to embryonic development, namely, meiotic resumption and completion, changes to the egg’s proteome, and genome activation after the first zygotic division. Therefore, egg activation is required for the oocyte to become a totipotent zygote. Despite the essential nature of this process for female fertility, the molecular events of egg activation are not well understood, primarily for technical reasons. Egg activation occurs without new transcription; thus nucleic acids- based ‘omics comparisons are uninformative. The macromolecules that transduce the calcium signal to effect downstream cellular events are not known in humans or any other mammal. Recent studies in MW’s lab exploited technical advantages of the Drosophila model system to show that there is large phospho-modulation of the maternally-provided proteome during egg activation (this also occurs in frogs and sea urchins). We hypothesized, and our genetic data supported, that this posttranslational modification regulated the activity of stored proteins to permit transition of an arrested mature oocyte to a cell that can undertake embryogenesis. We then showed that a calcium-regulated phospho-regulatory enzyme mediates these phospho-changes in the cell cycle machinery, translation factors and other proteins needed to transition the egg to an embryo. In this R21 we propose to test this model for mammalian oocytes, using mouse as a model. Following procedures analogous to those used for Drosophila, we will determine whether there are phosphoproteome changes during mouse egg activation, and which proteins undergo these changes. We will then test the role of CamKII, a calcium-regulated kinase that has been shown to be required for egg activation in mouse, in making these phospho-changes. The results of our studies will lay the groundwork for the field in several ways, including developing phosphoproteomics for mouse oocytes and determining proteins that are phospho-regulated during egg activation. The results will provide information essential for future studies into the roles of the regulated proteins that we identify here, and the effects of specific phosphomodulations during this critical developmental transition. Such fundamental studies will be important for identifying the molecular and genetic bases of human infertilities associated with defective egg activation, providing biomarkers to monitor this process, and potentially for optimizing conditions for assisted reproductive technologies.

项目总结/摘要 排卵的人类和小鼠卵母细胞在减数分裂II中停滞。它们在转录上是静止的， 一个母体携带的转录组和蛋白质组受精触发了“卵子激活”，在这种情况下， 卵母细胞中的钙会诱导几个关键事件，从而允许过渡到胚胎发育，即， 减数分裂的恢复和完成，卵子蛋白质组的变化，以及第一次减数分裂后的基因组激活 合子分裂因此，卵母细胞成为全能合子需要卵激活。尽管 这一过程的本质是女性生育力，卵子激活的分子事件并不好 这主要是出于技术原因。卵细胞的激活没有新的转录，因此核酸- 基于“组学”的比较是没有信息的。抑制钙信号发挥作用的大分子 人类或任何其他哺乳动物的下游细胞事件尚不清楚。 MW实验室最近的研究利用了果蝇模型系统的技术优势，表明 在卵激活期间母体提供的蛋白质组存在大的磷酸化调节（这也发生在 青蛙和海胆）。我们假设，我们的遗传数据支持，这种翻译后 修饰调节储存蛋白质的活性，以允许停滞的成熟卵母细胞过渡到细胞 可以进行胚胎发育的细胞然后，我们发现，钙调节磷酸调节酶， 介导细胞周期机制、翻译因子和其他蛋白质中的磷酸化变化， 将卵子转化为胚胎。 在此R21中，我们建议使用小鼠作为模型来测试哺乳动物卵母细胞的该模型。以下 类似于果蝇的程序，我们将确定是否存在磷酸化蛋白质组， 小鼠卵子激活过程中的变化，以及哪些蛋白质经历这些变化。然后我们将测试 CamKII是一种钙调节激酶，已被证明是小鼠卵子激活所必需的， 这些磷的变化。 我们的研究结果将在几个方面为该领域奠定基础，包括开发 小鼠卵母细胞的磷酸化蛋白质组学和确定卵母细胞中磷酸化调节的蛋白质 activation.这些结果将为未来研究受监管的 我们在这里确定的蛋白质，以及在这个关键的发育过程中特定磷酸调节的影响， 过渡这些基础性研究对于确定人类免疫缺陷病毒的分子和遗传基础具有重要意义。 与卵子激活缺陷相关的不孕症，提供生物标志物来监测这一过程， 可能用于优化辅助生殖技术的条件。