Regulation of microtubule organizing centers during mammalian gametogenesis

PROJECT SUMMARY Regulation of microtubule organizing centers during mammalian gametogenesis Establishing bipolar spindles during meiotic divisions ensures accurate chromosome segregation. Characterizing microtubule organizing center (MTOC) dynamics will help understand the 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 1A of our proposal is focused on how two rounds of centriole duplication are controlled and coordinated with the meiotic division during mammalian spermatogenesis. We will test the hypothesis that specific protein phosphatase 2 (PP2A) regulators control the function of Polo-like kinase 4 (PLK4), which we have confirmed to be the “master regulator” of centriole duplication during spermatogenesis. Beyond meiosis and centriole duplication, we have discovered that PLK4 has post-meiotic functions during spermiogenesis that ensure spermatids undergo key morphological changes to form functional spermatozoa. In Aim 1B, we delve into the mechanistic relationship between PLK4 and newly discovered interaction partners that mediate the formation of unique structures in the sperm, including the sperm head, manchette, perinuclear ring, basal body, and flagella. It is known that oocytes mediate chromosome segregation with microtubule organizing center (MTOC) that is acentriolar (aMTOC). For Aim 2A, we will use a new mouse model that combines the auxin-inducible degron (AID) system with protein epitope tagging for live cell imaging and protein interactome analysis. We will discover key regulatory processes that drive aMTOC formation in oocytes and assess the properties required for bipolar spindle formation and maintenance. From our studies on PLK4 and centriole duplication, we determined that spermatocytes are capable of undergoing meiosis in the absence of centrioles and centrosome components. In Aim 2B, we will elucidate the non-centrosomal MTOC processes critical for mediating chromosome segregation during spermatogenesis. By defining the novel processes required for MTOC biogenesis during mammalian meiosis, we will develop new concepts of how meiotic chromosome dynamics and segregation are regulated. Furthermore, the importance of MTOCs in post-meiotic spermatocytes is under-explored, and our work in this area will help fill this knowledge gap. Our research will contribute to diagnosing causes of gamete aneuploidy and help efforts to reduce events that cause birth defects, affect physical and mental development, and increase the risk of infertility.