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Principal Investigator: Marnie Skinner
Organization: JOHNS HOPKINS UNIVERSITY
Fiscal Year: 2024
Award: $48,974
Funding agency: Eunice Kennedy Shriver National Institute of Child Health and Human Development
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
Terms: <Active Follow-up><Affect><Age><Aneuploid><Aneuploidy><Biogenesis><Biologic Models><Biological Models><Cannot achieve a pregnancy><Cell Body><Cell Cycle><Cell Division Cycle><Cell Function><Cell Physiology><Cell Process><Cell division><Cells><Cellular Function><Cellular Physiology><Cellular Process><Centrioles><Centrosome><Chromosome Segregation><Chromosomes><Coupled><Couples><Cytoplasmic Organelle><DNA Replication><DNA Synthesis><DNA biosynthesis><Data><Defect><Difficulty conceiving><Diploid Cells><Ensure><Event><Failure><Flagella><Gametes><Gametogenesis><Genetic Materials><Germ Cells><Germ-Line Cells><Goals><Haploid><Haploidy><Immune Precipitation><Immunoblotting><Immunofluorescence Microscopy><Immunohistochemistry><Immunohistochemistry Cell/Tissue><Immunohistochemistry Staining Method><Immunoprecipitation><Infertility><Investigation><KO mice><Knock-out Mice><Knockout Mice><Lead><Link><Literature><M Phase><MTOC><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Meiosis><Meiotic Prophase I><Metabolic Protein Degradation><Micro-tubule><Microtubule Polymerization><Microtubule-Organizing Center><Microtubules><Mitosis><Mitosis Stage><Mitotic><Model System><Modeling><Null Mouse><Organelles><Origin of Life><PLK Gene><PLK1><PLK1 gene><Pathway interactions><Pb element><Phase Transition><Phosphorylation><Play><Polo-Like Kinase><Process><Process Assessment><Prophase><Prophase I><Protein Dynamics><Protein Phosphorylation><Protein Turnover><Proteins><Regulation><Regulatory Protein Degradation><Reporting><Reproductive Cells><Role><S Period><S phase><STPK13><Serine/Threonine Protein Kinase 13><Sex Cell><Sister Chromatid><Site><Sperm><Spermatocytes><Spermatogenesis><Spermatozoa><Spermiocytes><Subcellular Process><Synthesis Period><Synthesis Phase><Techniques><Testing><Up-Regulation><Upregulation><Western Blotting><Western Immunoblotting><active followup><ages><chromosomal missegregation><chromosome division><chromosome missegregation><conditional knock-out><conditional knockout><experiment><experimental research><experimental study><experiments><fertility cessation><fertility loss><follow up><follow-up><followed up><followup><gamma Tubulin><heavy metal Pb><heavy metal lead><in silico><infertile><initial cell><male germ cells><meiotic><mouse model><murine model><mutant mouse model><novel><overexpress><overexpression><pathway><polo-like kinase 1><protein blotting><protein degradation><reproductive><sexual cell><social role><sperm cell><trafficking><zoosperm><γ-Tubulin>