Prometaphase APCcdh1 activity prevents non-disjunction in mammalian oocytes

Abstract

SummaryThe first female meiotic division (MI) is uniquely prone to chromosome segregation errors through non-disjunction, resulting in trisomies and early pregnancy loss1. Here, we show a fundamental difference in the control of mammalian meiosis which may underlie such susceptibility. It involved a reversal in the well-established timing of activation of the AnaphasePromoting Complex (APC)2, 3 by its co-activators cdc20 and cdh1. APC cdh1 was active first, during prometaphase I, and was needed in order to allow homologue congression, since loss of cdh1 speeded up MI, leading to premature chromosome segregation and a non-disjunction phenotype. APC cdh1 targeted cdc20 for degradation but not securin and cyclin B1. These were degraded later in MI through APC cdc20 , making cdc20 re-synthesis essential for successful meiotic progression. The switch from APC cdh1 to APC cdc20 activity was controlled by increasing CDK1 and cdh1 loss. These findings demonstrate a fundamentally different mechanism of control for the first meiotic division in mammalian oocytes not observed in meioses of other species.The E3 ligase activity of the Anaphase-Promoting Factor (APC) bound to its co-activator cdh1 (APC cdh1 ) is commonly associated with late M-and early G1-phases of the cell cycle, where it contributes to M-phase exit by degradation of mitotic proteins, while simultaneously preventing precocious DNA replication3-6. APC cdh1 activity is also observed in germinal vesicle stage (GV) mouse oocytes, equivalent to late G2, where it contributes to cyclin B1 degradation and as such is required for maintenance of GV arrest7, 8.We wanted to establish if cdh1 had any role in meiosis I (MI) after GV breakdown (GVB), independent of its role in maintaining GV arrest. Therefore, we examined the ability of oocytes to progress through MI following microinjection with a cdh1 antisense morpholino (cdh1 MO ). Culture in milrinone-containing medium for 24 h following cdh1 MO microinjection is sufficient to reduce cdh1 levels by >90% (hereafter 'cdh1 knockdown oocytes') but maintain GV arrest in the majority of oocytes, with longer term culture (48h) needed to promote GVB7. In cdh1 knockdown oocytes, which maintained arrest over 24 h, we found that progression through MI was accelerated following milrinone wash-out. Oocytes extruded a polar body (PB), which forms on completion of MI, 1.5 h earlier than non-injected oocytes (Fig 1a). This effect was attributed specifically to loss of cdh1, since it Correspondence should be addressed to KTJ. (email: k.t.jones@ncl.ac.uk). AUTHOR CONTRIBUTIONS K.T.J. directed the work. A.R. and S.M. performed most the experiments; with HY.C making the initial observations on the effects of the cdh1 MO , I.N. performing some of the Westerns, and M.L. making some of the constructs. KTJ wrote the paper in consultation with A.R. and S.M. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. was not observed in mock cdh1 depleted oocytes through addition of a 5-base-mismatch cdh1 morpholino (5...

The APC/C in female mammalian meiosis I

“…In contrast, analysis of fluorescently labelled constructs in mouse oocytes showed that degradation of cyclin A2 did not occur any earlier than that of securin in late MI (McGuinness et al 2009, Jin et al 2010. This would be consistent with APC/C Cdh1 -mediated proteolysis restricting Cdc20 accumulation to late MI (Reis et al 2007), thereby preventing earlier prometaphase cyclin A2 degradation. It is noteworthy, however, that levels of endogenous cyclin A2 detected by immunoblotting in a recent paper appear to decline ahead of securin (see Fig.…”

“…Following the establishment of kMt attachments, SAC-mediated inhibition dissipates, thereby allowing activation of APC/C Cdc20 , which could then be tasked with degrading not only cyclin B1 and securin but also cyclin A2 and cyclin B2 (Touati et al 2012, Gui & Homer 2013. It should be noted that APC/C Cdh1 -mediated regulation of APC/C Cdc20 through Cdc20 proteolysis (Reis et al 2007) is not depicted here. Following exit from MI, Emi2 limits APC/C Cdc20 activity allowing for cyclin B1 accumulation and spindle stabilisation in MII (Madgwick et al 2006).…”

“…With increasing Cdk1 activation, APC/C Cdh1 activity is extinguished, allowing Cdc20 to accumulate and APC/C Cdc20 activity to rise -Cdc20 being subject to degradation by APC/C Cdh1 before this (Reis et al 2007, Homer 2011). In addition to delaying Cdc20 accumulation, recent data also highlight an important role for APC/C Cdh1 during prometaphase I spindle assembly in mouse oocytes.…”

“…Prometaphase APC/C Cdh1 activity and acentrosomal spindle assembly Following GVBD, APC/C Cdh1 continues to be active during early prometaphase I when slow-rising Cdk1 activity is thought to provide a permissive environment that is not inhibitory to APC/C Cdh1 (Reis et al 2007). With increasing Cdk1 activation, APC/C Cdh1 activity is extinguished, allowing Cdc20 to accumulate and APC/C Cdc20 activity to rise -Cdc20 being subject to degradation by APC/C Cdh1 before this (Reis et al 2007, Homer 2011).…”

Dynamics of protein phosphorylation during meiotic maturation

“…MPM2 antibody was deployed here to track serine and threonine phosphorylated protein epitopes known to be phosphorylated by M-phase kinases during mitosis [25,26]. This antibody has been used in our laboratory for the identification of M-phase protein phosphorylation in oocytes as they acquire meiotic competence and undergo meiotic progression [21,27], transition states in the cell cycle that are known to be accompanied by gradual activation of H1 kinase that occurs during GVBD [28]. These studies extend that work identifying unreported dynamics of the MPM2 labeled phosphoproteins.…”