Regulation of Centrosome Biogenesis During Mammalian Spermatogenesis

哺乳动物精子发生过程中中心体生物发生的调控

• 批准号: 10749144
• 负责人: Marnie Skinner
• 金额: $ 4.77万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-17 至 2025-10-16
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10749144
• 关键词: Affect; Age; Aneuploidy; Biogenesis; Biological Models; Cell Cycle; Cell division; Cell physiology; Cells; Centrioles; Centrosome; Chromosome Segregation; Chromosomes; Coupled; Couples; Cytoplasmic Organelle; DNA biosynthesis; Data; Defect; Diploid Cells; Ensure; Event; Failure; Flagella; Gametogenesis; Genetic Materials; Germ Cells; Goals; Haploidy; Immunofluorescence Microscopy; Immunohistochemistry; Immunoprecipitation; Infertility; Investigation; Knockout Mice; Lead; Link; Literature; Mass Spectrum Analysis; Meiosis; Meiotic Prophase I; Microtubule Polymerization; Microtubule-Organizing Center; Microtubules; Mitosis; Mitotic; Modeling; Organelles; PLK1 gene; Pathway interactions; Phase Transition; Phosphorylation; Play; Process; Process Assessment; Prophase; Protein Dynamics; Proteins; Regulation; Reporting; Role; S phase; Sister Chromatid; Site; Spermatocytes; Spermatogenesis; Techniques; Testing; Up-Regulation; Western Blotting; conditional knockout; experimental study; follow-up; gamma Tubulin; in silico; mouse model; mutant mouse model; novel; overexpression; protein degradation; reproductive; segregation; sperm cell; trafficking

Infertility effects around 15% of couples that are of reproductive age worldwide. Issues relating to infertility often arise due to errors in the process of meiosis, the specialized cell division where a single diploid cell gives rise to four haploid gametes. The general sequence of events in meiosis includes one round of DNA replication, the separation of homologous chromosomes (meiosis I), and then the separation of sister chromatids (meiosis II). During the process of chromosome segregation, the cell organizes bipolar spindles to evenly divide the genetic material. Like most mitotically dividing cells, spermatocytes rely on the centrosome to be the microtubule organizing center responsible for the formation of bipolar spindles. The centrosome consists of two centrioles and associated proteins known as pericentriolar material (PCM). Centrioles are cylindrical organelles that exist in pairs, one centriole oriented perpendicular to the other. The PCM that surrounds them consists of ordered, dynamic proteins that are necessary for cellular functions such as protein degradation, organelle trafficking, and spindle assembly. The PCM also acts as the spindle organizing center by concentrating the γ-tubulin that serves as nucleation sites for assembling the microtubules necessary for bipolar spindle formation and chromosome segregation. During cellular division, centriole duplication occurs. Polo-like kinase 4 (PLK4) is responsible for this duplication event in mitosis. PLK4 is regulated through autophosphorylation and proteasomal degradation to ensure centriole duplication only occurs once during the cell cycle. However, in meiosis the cell must undergo centriole duplication twice and centriole duplication is no longer coupled with DNA synthesis, but rather takes place during prophase I and interkinesis. Furthermore, PLK4 protein levels remain high during spermatogenesis and its activity is likely regulated by a novel mechanism. Because of this added complexity, the role of PLK4 in relationship to centriole duplication during meiosis is not yet understood. To determine the role of PLK4 in meiosis, conditional knockout (cKO) and overexpression (OE) mouse models, which allow for depletion or upregulation of PLK4 in early prophase spermatocytes, are utilized. In the Plk4 cKO model, due to the lack of functional PLK4, centrioles fail to duplicate, and the spermatocytes enter meiosis I with a single centrosome. In contrast, centrioles are over duplicated in the Plk4 OE model. It is our goal to utilize these models to further characterize the role of PLK4 as well as elucidate other regulatory mechanisms and components required for centrosome biogenesis during mammalian spermatogenesis. As centrosome biogenesis is critical for chromosome segregation during spermatogenesis, and centrioles are required for sperm flagella formation, our studies will contribute to understanding causes of infertility and gamete aneuploidy. We will address the following aims in this proposal: Aim 1: Determine novel aspects of bipolar spindle formation during mammalian spermatogenesis Aim 2: Explore the regulatory mechanisms required for centriole duplication during mammalian spermatogenesis

全球生殖年龄的夫妇中，不育会影响大约15％的夫妇。经常与不育有关的问题 由于减数分裂过程中的错误而引起 四个单倍游戏。减数分裂中事件的一般顺序包括一轮DNA复制， 同源染色体的分离（减数分裂I），然后分离姐妹染色质酸（减数分裂II）。 在染色体分离的过程中，细胞组织双极纺锤体均匀分割遗传 材料。像大多数有丝分裂的细胞一样，精子细胞依赖于中心体是微管 组织中心负责形成双极纺锤体。中心体由两个centriles组成 和相关蛋白质称为丁香三元素材料（PCM）。中心是存在的圆柱体细胞器 成对，一个垂直于另一个中心的中心。它们周围环境的PCM由有序组成， 动态蛋白质是细胞功能所必需的，例如蛋白质降解，细胞器运输和 主轴组件。 PCM还通过集中使用的γ-微管蛋白作为主轴组织中心 作为组装双极纺锤体形成和染色体所需的微管的核位点 隔离。在细胞分裂期间，发生中心重复。 polo样激酶4（PLK4）负责 有丝分裂的重复事件。 PLK4通过自磷酸化和蛋白酶体降解来调节 为了确保中心复制仅在细胞周期中发生一次。但是，在减数分裂中，细胞必须经历 Centriole重复两次，Centriole重复不再与DNA合成相结合，而是采用 在预言I和Interkinesis期间放置。此外，PLK4蛋白水平在精子发生过程中保持较高 它的活性很可能受到新机制的调节。由于这种增加的复杂性，PLK4在 在减数分裂过程中与中心重复的关系尚未理解。确定PLK4在 减数分裂，有条件敲除（CKO）和过表达（OE）小鼠模型，这允许耗尽或 使用早期丙酶精子细胞中PLK4的上调。在PLK4 CKO模型中，由于缺乏 功能性PLK4，中心元素无法复制，精子细胞以单个中心体进入减数分裂I。在 对比度，Centrioles在PLK4 OE模型中重复过多。我们的目标是利用这些模型进一步 表征PLK4的作用以及阐明其他调节机制和所需的组件 哺乳动物精子发生过程中的中心体生物发生。由于中心体生物发生对 精子发生过程中的染色体分离，精子形成需要中心元素，我们 研究将有助于理解不育症和配子型的原因。我们将解决以下内容 目的是在此提案中： 目标1：确定哺乳动物精子发生过程中双极纺锤体形成的新方面 目标2：探索哺乳动物精子发生过程中中心重复所需的调节机制