Regulation of microtubule organizing centers during mammalian gametogenesis

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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.