Regulation of Centrosome Biogenesis During Mammalian Spermatogenesis

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Infertility effects around 15% of couples that are of reproductive age worldwide. Issues relating to infertility often arise due to errors in the process of meiosis, the specialized cell division where a single diploid cell gives rise to four haploid gametes. The general sequence of events in meiosis includes one round of DNA replication, the separation of homologous chromosomes (meiosis I), and then the separation of sister chromatids (meiosis II). During the process of chromosome segregation, the cell organizes bipolar spindles to evenly divide the genetic material. Like most mitotically dividing cells, spermatocytes rely on the centrosome to be the microtubule organizing center responsible for the formation of bipolar spindles. The centrosome consists of two centrioles and associated proteins known as pericentriolar material (PCM). Centrioles are cylindrical organelles that exist in pairs, one centriole oriented perpendicular to the other. The PCM that surrounds them consists of ordered, dynamic proteins that are necessary for cellular functions such as protein degradation, organelle trafficking, and spindle assembly. The PCM also acts as the spindle organizing center by concentrating the γ-tubulin that serves as nucleation sites for assembling the microtubules necessary for bipolar spindle formation and chromosome segregation. During cellular division, centriole duplication occurs. Polo-like kinase 4 (PLK4) is responsible for this duplication event in mitosis. PLK4 is regulated through autophosphorylation and proteasomal degradation to ensure centriole duplication only occurs once during the cell cycle. However, in meiosis the cell must undergo centriole duplication twice and centriole duplication is no longer coupled with DNA synthesis, but rather takes place during prophase I and interkinesis. Furthermore, PLK4 protein levels remain high during spermatogenesis and its activity is likely regulated by a novel mechanism. Because of this added complexity, the role of PLK4 in relationship to centriole duplication during meiosis is not yet understood. To determine the role of PLK4 in meiosis, conditional knockout (cKO) and overexpression (OE) mouse models, which allow for depletion or upregulation of PLK4 in early prophase spermatocytes, are utilized. In the Plk4 cKO model, due to the lack of functional PLK4, centrioles fail to duplicate, and the spermatocytes enter meiosis I with a single centrosome. In contrast, centrioles are over duplicated in the Plk4 OE model. It is our goal to utilize these models to further characterize the role of PLK4 as well as elucidate other regulatory mechanisms and components required for centrosome biogenesis during mammalian spermatogenesis. As centrosome biogenesis is critical for chromosome segregation during spermatogenesis, and centrioles are required for sperm flagella formation, our studies will contribute to understanding causes of infertility and gamete aneuploidy. We will address the following aims in this proposal: Aim 1: Determine novel aspects of bipolar spindle formation during mammalian spermatogenesis Aim 2: Explore the regulatory mechanisms required for centriole duplication during mammalian spermatogenesis