Administrative Supplement request for GM117155

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PROJECT SUMMARY The establishment of bipolar spindles during meiotic divisions ensures accurate chromosome segregation. Characterization of 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. In Aim 1, we will assess meiotic progression in the absence of key microtubule organizing center (MTOC) regulators. In Aim 2, we aim to discover and characterize novel components of MTOCs during oogenesis and spermatogenesis. In order to achieve our objectives, we have developed a method to increase the magnification of spermatocytes and oocytes by combining cryosectioning with tissue expansion and super-resolution microscopy to enable us to view fine structural features of MTOCs. In addition, with donated, deidentified human gonad samples that we receive via approved protocols with organ procurement organizations, we are performing short-term culture experiments and chemical modulation using specific small molecule inhibitors against cell cycle kinases. Our aims can only be addressed with the appropriate equipment. Unfortunately, our cryostat recently broke down without an option for repair (discontinued model). Therefore, we are requesting a new, improved cryostat system to alleviate this issue. The Epredia Cryostar NX70 we propose to obtain with equipment supplement funding is optimal for our needs, a superior product, and will solve our current cryosectioning issue. 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.