In both mitosis and meiosis, metaphase to anaphase transition requires the activity of a ubiquitin ligase known as anaphase promoting complex/cyclosome (APC/C). The activation of APC/C in metaphase is under the control of the checkpoint mechanism, called the spindle assembly checkpoint (SAC), which monitors the correct attachment of all kinetochores to the spindle. It has been shown previously in somatic cells that exposure to a small molecule inhibitor, prodrug tosyl-l-arginine methyl ester (proTAME), resulted in cell cycle arrest in metaphase, with low APC/C activity. Interestingly, some reports have also suggested that the activity of SAC is required for this arrest. We focused on the characterization of proTAME inhibition of cell cycle progression in mammalian oocytes and embryos. Our results show that mammalian oocytes and early cleavage embryos show dose-dependent metaphase arrest after exposure to proTAME. However, in comparison to the somatic cells, we show here that the proTAME-induced arrest in these cells does not require SAC activity. Our results revealed important differences between mammalian oocytes and early embryos and somatic cells in their requirements of SAC for APC/C inhibition. In comparison to the somatic cells, oocytes and embryos show much higher frequency of aneuploidy. Our results are therefore important for understanding chromosome segregation control mechanisms, which might contribute to the premature termination of development or severe developmental and mental disorders of newborns.

Cellular Imaging Core Facility Central European Institute CEITEC Masaryk University 624 00 Brno Czech Republic

Central European Institute of Technology Department of Genetics and Reproduction Veterinary Research Institute 621 00 Brno Czech Republic

National Institute of Environmental Health Sciences NIH Durham NC 27709 USA

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Peters J.M. The anaphase promoting complex/cyclosome: A machine designed to destroy. Nat. Rev. Mol. Cell Biol. 2006;7:644–656. doi: 10.1038/nrm1988. PubMed DOI

Musacchio A. The Molecular Biology of Spindle Assembly Checkpoint Signaling Dynamics. Curr. Biol. 2015;25:R1002–R1018. doi: 10.1016/j.cub.2015.08.051. PubMed DOI

Marston A.L., Wassmann K. Multiple Duties for Spindle Assembly Checkpoint Kinases in Meiosis. Front. Cell Dev. Biol. 2017;5:109. doi: 10.3389/fcell.2017.00109. PubMed DOI PMC

McGuinness B.E., Anger M., Kouznetsova A., Gil-Bernabé A.M., Helmhart W., Kudo N.R., Wuensche A., Taylor S., Hoog C., Novak B., et al. Regulation of APC/C activity in oocytes by a BUB1-dependent spindle assembly checkpoint. Curr. Biol. 2009;19:369–380. doi: 10.1016/j.cub.2009.01.064. PubMed DOI

Verma R., Peters N.R., D’Onofrio M., Tochtrop G.P., Sakamoto K.M., Varadan R., Zhang M., Coffino P., Fushman D., Deshaies R.J., et al. Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain. Science. 2004;306:117–120. doi: 10.1126/science.1100946. PubMed DOI

Zeng X., Sigoillot F., Gaur S., Choi S., Pfaff K.L., Oh D.C., Hathaway N., Dimova N., Cuny G.D., King R.W. Pharmacologic inhibition of the anaphase-promoting complex induces a spindle checkpoint-dependent mitotic arrest in the absence of spindle damage. Cancer Cell. 2010;18:382–395. doi: 10.1016/j.ccr.2010.08.010. PubMed DOI PMC

Zeng X., King R.W. An APC/C inhibitor stabilizes CYCLIN B1 by prematurely terminating ubiquitination. Nat. Chem. Biol. 2012;8:383–392. doi: 10.1038/nchembio.801. PubMed DOI PMC

Lara-Gonzalez P., Taylor S.S. Cohesion fatigue explains why pharmacological inhibition of the APC/C induces a spindle checkpoint-dependent mitotic arrest. PLoS ONE. 2012;7:e49041. doi: 10.1371/journal.pone.0049041. PubMed DOI PMC

De K., Grubb T.M., Zalenski A.A., Pfaff K.E., Pal D., Majumder S., Summers M.K., Venere M. Hyperphosphorylation of CDH1 in glioblastoma cancer stem cells attenuates APC/CCDH1 activity and pharmacological inhibition of APC/CCDH1/CDC20 compromises viability. Mol. Cancer Res. 2019 doi: 10.1158/1541-7786.MCR-18-1361. PubMed DOI PMC

Raab M., Sanhaji M., Zhou S., Rödel F., El-Balat A., Becker S., Strebhardt K. Blocking Mitotic Exit of Ovarian Cancer Cells by Pharmaceutical Inhibition of the Anaphase-Promoting Complex Reduces Chromosomal Instability. Neoplasia. 2019;21:363–375. doi: 10.1016/j.neo.2019.01.007. PubMed DOI PMC

Maes A., Maes K., De Raeve H., De Smedt E., Vlummens P., Szablewski V., Devin J., Faict S., De Veirman K., Menu E., et al. The anaphase-promoting complex/cyclosome: A new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma. Br. J. Cancer. 2019 doi: 10.1038/s41416-019-0471-0. PubMed DOI PMC

Akera T., Trimm E., Lampson M.A. Molecular and Evolutionary Strategies of Meiotic Cheating by Selfish Centromeres. [(accessed on 31 August 2018)]; Available online: https://www.biorxiv.org/content/biorxiv/early/2018/08/30/405068.full.pdf. PubMed

Vázquez-Diez C., Paim L.M.G., FitzHarris G. Cell-Size-Independent Spindle Checkpoint Failure Underlies Chromosome Segregation Error in Mouse Embryos. Curr. Biol. 2019;29:865–873. doi: 10.1016/j.cub.2018.12.042. PubMed DOI

Reis A., Chang H.Y., Levasseur M., Jones K.T. APC CDH1 activity in mouse oocytes prevents entry into the first meiotic division. Nat. Cell Biol. 2006;8:539–540. doi: 10.1038/ncb1406. PubMed DOI PMC

Rattani A., Ballesteros Mejia R., Roberts K., Roig M.B., Godwin J., Hopkins M., Eguren M., Sanchez-Pulido L., Okaz E., Ogushi S., et al. APC/C (CDH1) Enables Removal of SHUGOSHIN-2 from the Arms of Bivalent Chromosomes by Moderating Cyclin-Dependent Kinase Activity. Curr. Biol. 2017;27:1462–1476. doi: 10.1016/j.cub.2017.04.023. PubMed DOI PMC

Bennabi I., Terret M.E., Verlhac M.H. Meiotic spindle assembly and chromosome segregation in oocytes. J. Cell Biol. 2016;215:611–619. doi: 10.1083/jcb.201607062. PubMed DOI PMC

Mogessie B., Scheffler K., Schuh M. Assembly and Positioning of the Oocyte Meiotic Spindle. Ann. Rev. Cell Dev. Biol. 2018;34:381–403. doi: 10.1146/annurev-cellbio-100616-060553. PubMed DOI

Holubcová Z., Blayney M., Elder K., Schuh M. Human oocytes. Error-prone chromosome-mediated spindle assembly favors chromosome segregation defects in human oocytes. Science. 2015;348:1143–1147. doi: 10.1126/science.aaa9529. PubMed DOI PMC

Haverfield J., Dean N.L., Nöel D., Rémillard-Labrosse G., Paradis V., Kadoch I.J., FitzHarris G. Tri-directional anaphases as a novel chromosome segregation defect in human oocytes. Hum. Reprod. 2017;32:1293–1303. doi: 10.1093/humrep/dex083. PubMed DOI PMC

Lane S.I.R., Morgan S.L., Wu T., Collins J.K., Merriman J.A., ElInati E., Turner J.M., Jones K.T. DNA damage induces a kinetochore-based ATM/ATR-independent SAC arrest unique to the first meiotic division in mouse oocytes. Development. 2017;144:3475–3486. doi: 10.1242/dev.153965. PubMed DOI PMC

Hached K., Xie S.Z., Buffin E., Cladière D., Rachez C., Sacras M., Sorger P.K., Wassmann K. MPS1 at kinetochores is essential for female mouse meiosis I. Development. 2011;138:2261–2271. doi: 10.1242/dev.061317. PubMed DOI

Kolano A., Brunet S., Silk A.D., Cleveland D.W., Verlhac M.H. Error-prone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension. Proc. Natl. Acad. Sci. USA. 2012;109:E1858–E1867. doi: 10.1073/pnas.1204686109. PubMed DOI PMC

Kudo N.R., Wassmann K., Anger M., Schuh M., Wirth K.G., Xu H., Helmhart W., Kudo H., McKay M., Maro B., et al. Resolution of chiasmata in oocytes requires separase-mediated proteolysis. Cell. 2006;126:135–146. doi: 10.1016/j.cell.2006.05.033. PubMed DOI

Kudo N.R., Anger M., Peters A.H., Stemmann O., Theussl H.C., Helmhart W., Kudo H., Heyting C., Nasmyth K. Role of cleavage by separase of the REC8 kleisin subunit of cohesin during mammalian meiosis I. J. Cell Sci. 2009;122:2686–2698. doi: 10.1242/jcs.035287. PubMed DOI PMC

Danylevska A., Kovacovicova K., Awadova T., Anger M. The frequency of precocious segregation of sister chromatids in mouse female meiosis I is affected by genetic background. Chromosome Res. 2014;22:365–373. doi: 10.1007/s10577-014-9428-6. PubMed DOI

Kovacovicova K., Awadova T., Mikel P., Anger M. In Vitro Maturation of Mouse Oocytes Increases the Level of KIF11/EG5 on Meiosis II Spindles. Biol. Reprod. 2016;95:18. doi: 10.1095/biolreprod.115.133900. PubMed DOI

Chromosome Division in Early Embryos-Is Everything under Control? And Is the Cell Size Important?