Regulation of microtubule organizing centers during mammalian gametogenesis

项目摘要 哺乳动物配子发生过程中微管组织中心的调控 在减数分裂期间双极纺锤体的建立确保了染色体的精确分离。 微管组织中心（MTOC）动力学的表征将有助于了解配子的原因 非整倍性。形成双极MTOC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MTOC介导的，中心粒在精子发生前复制一次。 减数分裂I和减数分裂II之前再次。相反，卵母细胞形成多个无中心MTOC片段， 结合在一起形成双极纺锤体。 我们已经开发了新的研究工具和采用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。我们建议的目标1侧重于确定 中心粒复制的关键调控因子Polo样激酶4（PLK4）是中心粒的“主调节因子 有丝分裂细胞中的复制。此外，SAS 4是中心粒的关键组成部分。然而， 关于配子发生过程中中心粒复制的控制在Aim 1A中，我们使用条件敲除小鼠， 模型，以帮助确定在精母细胞中心粒复制过程中PLK4和SAS 4的需求。 此外，我们将使用这些模型来解决它们在卵子发生过程中是否有功能。在目标1B中， 将发现新的PLK4相互作用伙伴和磷酸化靶点，这些靶点对时间依赖性至关重要。 精子发生过程中中心粒复制的调节。 减数分裂过程中中心体成熟和分离是形成双极纺锤体所必需的过程 大多是不确定的。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激酶都被证明在有丝分裂细胞中调节中心体成熟 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， 尚待阐明。在Aim 2A中，我们将使用条件性基因敲除小鼠模型来发现 PLK1和Aurora A激酶在配子发生中的作用，重点关注MTOC过程和染色体 种族隔离在Aim 2B中，我们将确定PLK1如何调节Aurora A激酶活性以避免中心粒 过度重复我们还将发现新的生殖细胞特异性中心体成分， 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中心体和无中心粒MTOC生物发生所需的新过程， 减数分裂我们将发展减数分裂染色体动力学和分离是如何调节的新概念。 我们提出的研究将有助于诊断配子非整倍体的原因，并有助于减少 这些事件会导致出生缺陷，影响身心发育，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.