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Principal Investigator: JANICE P EVANS
Organization: PURDUE UNIVERSITY
Fiscal Year: 2020
Award: $77,500
Funding agency: Eunice Kennedy Shriver National Institute of Child Health and Human Development
PROJECT SUMMARY
Actin cytoskeleton-based processes play crucial roles in meiosis in mammalian oocytes, including
positioning of the meiotic spindle and asymmetric cell division. These events in oocytes are essential for
reproductive success, as gene knockouts that impair these processes cause female infertility. The overall
goal of the research proposed here is to analyze the roles of key proteins hypothesized to modulate function
of the actin cytoskeleton during female meiosis. The hypotheses to be pursued here are based on our
published data and on new unpublished data presented here. A second goal of this project is a technical
one – to provide valuable proof-of-principle demonstration of the utility of a novel system for post-
translational protein depletion, as an asset to the oocyte biology research community. Standard
approaches for protein depletion that are used in oocytes (i.e., knockout, knockdown) have relatively little
temporal precision for depletion of the target of interest. In contrast, consider the following example, from
study in budding yeast. Chronic depletion of a protein via a null mutation merely produced a slow growth
phenotype, whereas depletion at a specific stage of meiosis provided much sharper insights with a much
more specific and interesting phenotype. This is the inspiration for the project proposed here. This
research will use this same innovative approach – the auxin-inducible degradation (AID) system – to gain
new insights into the mammalian oocyte's progression through meiosis. We show here that we have the
AID system up and running in mouse oocytes. The overall concept for these studies is to express function-
altering variants of proteins of interest (dominant-negative [DN] or constitutively-active [CA]) in wild-type
oocytes to perturb function of a particular pathway. The AID system allows for degradation of this function-
altering protein at different times of meiotic maturation (e.g., M-phase entry [nuclear envelope breakdown],
early M-phase, late M-phase, etc.). In essence, this converts oocytes from a mutant state to wild-type
at a time of our choosing, allowing temporal specificity in assessing protein function. We will use
the AID system to analyze different stages of oocyte meiosis, from entry into M-phase of meiosis I to the
conclusion of meiosis II. Thus, this project will be valuable not only for the discoveries it will provide, but
also for the demonstration of AID system utility at multiple stages of meiosis. Aim 1 focusses on spindle
positioning in meiosis I, and will determine the role of the actin-depolymerization protein cofilin through
studies of when and where cofilin is required for spindle positioning. Aim 2 examines meiosis I, metaphase
II arrest, and completion of meiosis II, and will test the hypothesis that inappropriate activity of the actin-to-
membrane linker protein family known as ERMs impairs spindle function and polar body emission. Taken
together, these studies will provide important insights to advance understanding of these crucial actin-
dependent processes in oocytes, including leading to future work in in vivo models.
Terms: <20S Catalytic Proteasome><20S Core Proteasome><20S Proteasome><20S Proteosome><APF-1><ATP-Dependent Proteolysis Factor 1><Actins><Address><Antimorphic mutation><Auxins><Biology><Breathing><Budding Yeast><Cell Body><Cell Division Process><Cell division><Cells><Cellular Matrix><Chronic><Communities><Complex><Cullin Domain Protein><Cullin Family Gene><Cullin Family Protein><Cullin Proteins><Cullins><Cytoskeletal System><Cytoskeleton><Data><Dominant Negative><Dominant-Negative Mutant><Dominant-Negative Mutation><Embryo Development><Embryogenesis><Embryonic Development><Endomycetales><Event><Experimental Designs><F-Box Domain Protein><F-Box Protein Family><F-Box Proteins><Family member><Female><Female infertility><Fertilized Egg><Fertilized Ovum><Future><Gametes><Generalized Growth><Germ Cells><Germ-Line Cells><Goals><Growth><HMG-20><Haploid><Haploidy><High Mobility Protein 20><Impairment><Investigation><Knock-out><Knockout><Knowledge><Ligase><Ligase Gene><M Phase><Macropain><Macroxyproteinase><Mediating><Meiosis><Membrane><Metabolic Protein Degradation><Metaphase><Methods><Mice><Mice Mammals><Mitosis><Mitosis Stage><Mitotic Metaphase><Molecular><Multicatalytic Proteinase><Murine><Mus><Nuclear Envelope><Nuclear Membrane><Oocytes><Ovocytes><Paper><Pathway interactions><Phenotype><Phytohormones><Plant Growth Regulators><Plant Hormones><Plants><Play><Position><Positioning Attribute><Preparation><Process><Prosome><Proteasome><Proteasome Endopeptidase Complex><Protein Family><Protein Turnover><Proteins><Proteosome><Publishing><Regulatory Protein Degradation><Reproductive Cells><Research><Respiratory Aspiration><Respiratory Inspiration><Role><Running><Saccharomycetales><Sex Cell><Skp1 Protein><Specificity><Sperm><Spermatozoa><Synthetases><System><Testing><Time><Tissue Growth><Ubiquitin><Ubiquitin Ligase Component Gene><Ubiquitin Ligase Gene><Variant><Variation><Work><actin depolymerizing factor><actin depolymerizing proteins><base><cofilin><egg><experiment><experimental research><experimental study><ezrin><genetic approach><genetic strategy><in vivo Model><infertility in women><initial cell><innovate><innovation><innovative><insight><inspiration><interest><intracellular skeleton><knock-down><knockdown><knockout gene><meiotic><membrane structure><membrane-organizing extension spike protein><moesin><multicatalytic endopeptidase complex><mutant><new approaches><novel><novel approaches><novel strategies><novel strategy><null mutation><ontogeny><oocyte maturation><p19 SKP1><p19(SKP1) Protein><pathway><phosphoprotein p81><protein degradation><protein function><public health relevance><radixin><radixin protein><reproductive success><reverse genetics><sexual cell><social role><sperm cell><tool><ubiquitin ligase><unable to bear children><zoosperm><zygote>