Regulation of microtubule organizing centers during mammalian gametogenesis

项目总结 哺乳动物配子发生过程中微管组织中心的调控 减数分裂过程中两极纺锤体的建立确保了染色体的准确分离。 微管组织中心(MTOC)动力学特征将有助于理解配子的原因 非整倍体。形成两极MTOCs所需的过程是性二态的。染色体 精子发生过程中的分离是由含有中心粒的MTOCs介导的，中心粒在精子发生之前复制一次 减数分裂I和减数分裂II之前。相反，卵母细胞形成多个无着丝粒MTOC片段， 结合在一起形成两极心轴。 我们开发了新的研究工具并采用了新的技术来定义和比较MTOC 哺乳动物精子发生和卵子发生之间的过程。我们提案的目标1侧重于确定 中心粒复制的关键调控因子。Polo-like kinase4(Plk4)被认为是中心粒的“主调节器” 有丝分裂细胞中的复制。此外，SAS4是中心粒的关键成分。然而，人们对此知之甚少。 关于配子发生过程中中心粒复制的控制。在目标1A中，我们使用条件基因敲除小鼠 有助于确定精母细胞中心粒复制过程中对Plk4和SAS4的需求的模型。 此外，我们将使用这些模型来研究它们在卵子发生过程中是否具有功能。在AIM 1B中，我们 将发现新的Plk4相互作用伙伴和磷酸化靶点，它们对时间 精子发生过程中中心粒复制的调控。 减数分裂中两极纺锤体形成所需的中心体成熟和分离过程 大部分都是未定义的。在目标2中，我们将评估精子发生过程中中心体生物发生的关键阶段。 PLK1和Aurora A激酶都被证明调节有丝分裂细胞的中心体成熟 以重叠的方式。然而，它们在减数分裂过程中的作用，特别是与中心体生物发生有关的作用， 目前尚不清楚。在目标2A中，我们将使用条件性基因敲除小鼠模型来发现 配子发生过程中的PLK1和Aurora A激酶，重点是MTOC过程和染色体 种族隔离。在目标2B中，我们将确定PLK1如何调节Aurora A激酶活性以避免中心粒 过度复制。我们还将发现新的生殖细胞特有的中心体成分，这些成分是由 PLK1和Aurora A激酶，以确保熟练的中心体生物发生。 通过定义哺乳动物中心体和无着丝点MTOC生物发生所需的新过程 减数分裂，我们将提出减数分裂染色体动力学和分离如何被调节的新概念。 我们提出的研究将有助于诊断配子非整倍体的原因，并有助于努力减少 这些事件会导致出生缺陷，影响身体和精神发育，并增加不孕的风险。

PROJECT SUMMARY Regulation of microtubule organizing centers during mammalian gametogenesis Establishment of bipolar spindles during meiotic divisions ensures accurate chromosome segregation. Characterization of microtubule organizing center (MTOC) dynamics will help understand causes of gamete aneuploidy. The processes required for the formation of bipolar MTOCs are sexually dimorphic. Chromosome segregation during spermatogenesis is mediated by MTOCs containing centrioles that duplicate once prior to meiosis I and again prior to meiosis II. In contrast, oocytes form multiple acentriolar MTOC fragments that coalesce together to form bipolar spindles. We have developed new research tools and adapted novel techniques to define and compare MTOC processes between mammalian spermatogenesis and oogenesis. Aim 1 of our proposal focuses on determining key regulators of centriole duplication. Polo-like kinase 4 (PLK4) is known as the “master regulator” of centriole duplication in mitotic cells. In addition, SAS4 is a key component of the centriole. However, very little is known about the control of centriole duplication during gametogenesis. In Aim 1A, we use conditional knockout mouse models to help determine the requirements for PLK4 and SAS4 during centriole duplication in spermatocytes. Furthermore, we will use these models to address whether they have functions during oogenesis. In Aim 1B we will discover the novel PLK4 interaction partners and phosphorylation targets that are critical for temporal regulation of centriole duplication during spermatogenesis. The processes of centrosome maturation and separation required for bipolar spindle formation during meiosis are mostly undefined. In Aim 2 we will assess key stages of centrosome biogenesis during spermatogenesis. PLK1 and Aurora A kinases have both been shown to regulate centrosome maturation in mitotically dividing cells in an overlapping manner. However, their roles during meiosis, particularly relating to centrosome biogenesis, are yet to be elucidated. In Aim 2A, we will use conditional knockout mouse models to discover the functions of PLK1 and Aurora A kinases during gametogenesis, with focus on MTOC processes and chromosome segregation. In Aim 2B we will determine how PLK1 regulates Aurora A kinase activity to avoid centriole overduplication. We will also discover novel germ cell specific centrosomal components that are targeted by PLK1 and Aurora A kinases to ensure proficient centrosome biogenesis. By defining the novel processes required for centrosome and acentriolar MTOC biogenesis during mammalian meiosis we will develop new concepts of how meiotic chromosome dynamics and segregation are regulated. Our proposed research will contribute to diagnosing causes of gamete aneuploidy and help with efforts to reduce these events that cause birth defects, affect physical and mental development, and increase the risk of infertility.