Meiosis is the fundamentally important cell division process that generates the haploid gametes. If the oocyte fails to undergo meiosis correctly, embryonic development is doomed to fail. Regulation of M-phase in meiosis I and II in the oocyte - from entry to maintenance to exit - is crucial for reproductive success. The kinase MASTL (Microtubule Associated Serine/Threonine kinase-Like; also known as Greatwall) is a newly appreciated major player in the cell cycle. However, MASTL functions in the mammalian oocyte are unclear, with conflicting data from different depletion models about whether MASTL functions in the G2?M transition (i.e., exit from prophase I arrest) and/or in progression out of meiosis I and to meiosis II. The work here overcomes the challenges presented by these disparate results. What would be ideal is a precise, specific, and temporally controllable way to eliminate protein function - which is exactly the approach we propose here. We will use the auxin-inducible degron (AID) system for protein degradation, which can induce proteosome-mediated destruction of an AID-tagged protein in 20-60 minutes. The workhorse of the AID system is the auxin-dependent F-box protein TIR1, and work to date has used cell lines transfected to express TIR1. Treatment of TIR1-expressing cells with the plant hormone auxin promotes the interaction of AID-tagged proteins with TIR1. Degradation of AID-tagged proteins occurs as a result of TIR1 forming a functional Skp1-Cullin-F-Box (SCF) ubiquitin E3 ligase with endogenous proteins. The AID system has been used to deplete a variety of proteins in numerous systems, from yeast to Plasmodium to vertebrate cells, and has uncovered exciting phenotypes that were missed in null mutants. Use of the AID system in mammalian oocytes will involve the development of genetically engineered mouse models.
In Aim 1, we will use CRISPR-Cas9-mediated genome editing to make mouse models to express TIR1 and MASTL tagged with the AID. With these mouse models, our work will have the advantage of using small molecule pharmacological inhibitors (precisely-timed, short-term perturbation of the target protein) with virtually no worry of off-target effects. Thus, the approach here combines desirable aspects of genetic approaches and drug-based approaches.
In Aim 2, we will address specific hypotheses related to MASTL function in progression of mammalian oocytes through meiosis. Oocytes from these mice will be treated in vitro with auxin to induce degradation of MASTL at specific times to test the hypotheses that MASTL functions in entry to meiosis I, transition through meiosis I-to-II, and in maintaining metaphase II arrest. En toto, this project will advance understanding of meiotic maturation in mammalian oocytes, and of the functions of MASTL in the cell cycle. This work also will lead to development of a widely applicable and highly valuable research resource for conditional depletion of any protein of interest.
Meiosis is a fundamentally important cell division process that generates the haploid gametes (sperm and egg). Errors during female meiosis can be a significant cause of infertility and of chromosomal abnormalities in gametes and embryos; such chromosomal errors are detected in 7-10% of clinically recognized pregnancies. This research project will analyze the molecular mechanisms that regulate the correct progression of the mammalian oocyte through meiosis.