(13) CEP192 depletion during murine gametogenesis using the auxin-inducible degron (AID) system

Marnie W. Skinner^1,2,3, Philip W. Jordan^1,2,4

¹ Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA

² Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA

³ The Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA

⁴ School of Biomedicine, The University of Adelaide, Adelaide, SA, 5000, Australia

Abstract Text:

Microtubule organizing centers (MTOCs) are a key component for successful chromosome segregation during cell division. During gametogenesis, MTOCs mediate two rounds of chromosome segregation; first when homologous chromosomes are segregated during meiosis I and again when sister chromatids are segregated during meiosis II. During spermatogenesis, the centrosome is the canonical MTOC and consists of two centrioles and surrounding proteins known as the pericentriolar matrix (PCM). Oocytes, however, rely on an acentriolar MTOC (aMTOC) for bipolar spindle assembly. While this aMTOC lacks centrioles, it harbors many of the same proteins found in the PCM of centrosomes. Centrosomal protein 192 (CEP192) is one protein that is present in centrosomes and aMTOCs. CEP192 aids in recruiting other PCM components to the MTOC during mitosis and is important for MTOC organization, spindle assembly, and centriole duplication. However, the role of CEP192 in meiotically dividing cells has not been previously assessed. To assess CEP192 depletion in a meiotic-specific context, we have utilized the auxin-inducible degron 2 (AID2) system in a mouse model. In this system, an optimized Oryza sativa F-box transport inhibitor response 1 (OsTIR1(F74G)) transgene was incorporated into the Rosa26 locus, and CEP192 was C-terminally tagged with a 47-amino-acid miniAID (mAID) tag and a moxNeonGreen reporter. The TIR1 protein binds with the SCF E3 complex, and administration of auxin results in the polyubiquitination and proteasomal degradation of CEP192. The nature of auxin-induced protein degradation allows for both rapid and reversible CEP192 degradation. Reproductive assessments of this model system, including histological and cell-based immunofluorescent (IF) analyses, have demonstrated the successful depletion of CEP192 in gametes. Additional assessments of the effect of CEP192 depletion on meiotic progression are currently underway. In spermatocytes, tubule squash approaches coupled with IF assessment of centrosome and meiotic markers are being implemented to assess the effect of CEP192 depletion. Additionally, fixed and live cell IF techniques to observe similar markers are being used to assess the role of CEP192 in oocytes. Initial observations suggest that CEP192 depletion perturbs meiotic progression. Based on the previously reported mitotic role of CEP192 coordinating spindle assembly, we anticipate further assessments of this model will likely identify errors in MTOC organization and/or chromosome segregation during meiosis. Couples worldwide continue to struggle with infertility, and errors during meiosis that lead to chromosome segregation are a major cause. Furthering our knowledge of the mechanisms behind successful MTOC biogenesis and spindle assembly in gametes will contribute to our understanding of the causes of gamete aneuploidy and infertility.