Abstract
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.
Highlights
In both mitosis and meiosis, a transition from metaphase to anaphase requires the activation of a large multisubunit E3 ubiquitin ligase called anaphase promoting complex/cyclosome (APC/C) [1]
With increased concentration of prodrug tosyl-l-arginine methyl ester (proTAME) to 50 μM, 14% of cells remained arrested in metaphase I, and in 100 μM proTAME, 100% of oocytes were synchronized (Figure 1B), which means that bovine oocytes require a 20× higher concentration of proTAME for efficient synchronization in meiosis I in comparison to the mouse oocytes
It was shown that proTAME is capable of inhibiting both APC/C activators, cell division cycle 20 (CDC20) and cadherin 1 (CDH1) [6]
Summary
In both mitosis and meiosis, a transition from metaphase to anaphase requires the activation of a large multisubunit E3 ubiquitin ligase called anaphase promoting complex/cyclosome (APC/C) [1]. The activation of APC/C in somatic cells, as well as in oocytes, is controlled by the activity of the spindle assembly checkpoint (SAC), which facilitates the formation of the mitotic checkpoint complex (MCC) composed of BUB1 related kinase 1 (BUBR1), mitotic arrest deficient 2 (MAD2), budding uninhibited by benzimidazole 3 (BUB3) and cell division cycle 20 (CDC20) proteins on unattached kinetochores [2,3,4]. Results from the same laboratory later showed that the tosyl-l-arginine methyl ester (TAME, or in a cell-permeable prodrug form called proTAME) blocks APC/C activation by its CDC20 and cadherin 1 (CDH1) activators [6]. The mechanism of TAME inhibition of APC/C is not completely understood, but it seems that TAME in part blocks the ability of free CDC20 to bind to the APC/C and promotes the autoubiquitination of already prebound CDC20 [7]
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