Regulation of Centrosome Biogenesis During Mammalian Spermatogenesis

不孕症影响着全世界约15%的育龄夫妇。与不孕症有关的问题往往 由于减数分裂过程中的错误而出现，减数分裂是一种特殊的细胞分裂，其中单个二倍体细胞产生 四个单倍体配子。减数分裂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），然后姐妹染色单体的分离（减数分裂II）。 在染色体分离的过程中，细胞组织双极纺锤体以均匀地分配遗传物质。 材料像大多数有丝分裂细胞一样，精母细胞依赖中心体作为微管 负责形成两极纺锤体的组织中心。中心体由两个中心粒组成 和相关的蛋白质称为中心粒周围物质（PCM）。中心粒是一种圆柱形细胞器 成对的，一个中心粒垂直于另一个。围绕它们的PCM由有序的， 动态蛋白质是细胞功能所必需的，如蛋白质降解，细胞器运输， 主轴组件PCM还通过集中起作用的γ-微管蛋白而充当纺锤体组织中心 作为组装双极纺锤体形成和染色体所必需的微管的成核位点 种族隔离在细胞分裂过程中，中心粒复制发生。Polo样激酶4（PLK4）负责 有丝分裂中的复制过程。PLK4通过自身磷酸化和蛋白酶体降解调节 以确保中心粒复制在细胞周期中仅发生一次。然而，在减数分裂中，细胞必须经历 中心粒复制两次，中心粒复制不再与DNA合成偶联，而是 发生在前期I和间期。此外，PLK4蛋白水平在精子发生期间保持高水平 其活性可能受一种新机制的调节。由于这种增加的复杂性，PLK4在 与减数分裂期间中心粒复制的关系尚不清楚。为了确定PLK4在 减数分裂、条件性敲除（cKO）和过表达（OE）小鼠模型，其允许消耗或 在早期前期精母细胞中PLK4的上调。在Plk4 cKO模型中，由于缺乏 功能性PLK4，中心粒不能复制，精母细胞以单个中心体进入减数分裂I。在 相反，中心粒在Plk4 OE模型中被过度复制。我们的目标是利用这些模型进一步 描述PLK4的作用，并阐明其他调控机制和所需的组件， 哺乳动物精子发生过程中的中心体生物发生。由于中心体的生物发生对于 精子发生过程中染色体分离，精子鞭毛的形成需要中心粒， 研究将有助于了解不育和配子非整倍性的原因。我们将解决以下问题 本提案的目标： 目的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 目的2：探讨哺乳动物精子发生过程中中心粒复制的调控机制

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