First meiotic anaphase requires Cep55-dependent inhibitory cyclin-dependent kinase 1 phosphorylation

To prevent aneuploidy, the timing of anaphase must be finely tuned; such fine-tuning is the responsibility of the spindle assembly checkpoint (SAC) (Foley and Kapoor, 2013). In both mitosis and meiosis, anaphase occurs when the chromosomal glue, cohesin, is cleaved by the cysteine protease, separase (Hauf et al., 2001; Kudo et al., 2006; Terret et al., 2003). Activation of separase occurs following release from its inhibitory chaperone, securin (Waizenegger et al., 2002), brought about through securin proteolysis orchestrated by the anaphase-promoting complex (APC) acting in concert with its cell-division cycle protein 20 (Cdc20) coactivator (Homer, 2013; Pesin and Orr-Weaver, 2008; Herbert et al., 2003). As separase is also inhibited by cyclin-dependent kinase 1 (Cdk1) (Stemmann et al., 2001), anaphase also requires APC-Cdc20-mediated destruction of the Cdk1 activator, cyclin B1 (Pesin and Orr-Weaver, 2008; Wolf et al., 2006). In addition to cyclin B1 availability, Cdk1 activity is controlled by inhibitory Wee1/Myt1 kinase (Wee1B in oocytes)-mediated phosphorylation of Cdk1 residues Thr14 and Tyr15, which are removed by Cdc25 phosphatases (Cdc25B in oocytes) (Han et al., 2005; Malumbres, 2014; Adhikari and Liu, 2014; Lincoln et al., 2002). Inhibitory Cdk1 phosphorylation reinforces mitotic exit in cellular extracts, but this involves altered proteolysis (D'Angiolella et al., 2007; Forester et al., 2007). APC-Cdc20-mediated proteolysis is therefore the principal anaphase driver during mitosis (Meadows and Millar, 2015; Sullivan and Morgan, 2007). Thus, by controlling the timing of APC-Cdc20 activation, the mitotic SAC has stringent control over proteolysis and, by extension, anaphase timing, to prevent aneuploidy.

Meiotic maturation in mouse oocytes involves two consecutive M phases, meiosis I (MI) and meiosis II (MII), without intervening nuclear reformation, culminating in MII arrest (Greaney et al., 2018). As in mitosis, anaphase I and exit from MI in mouse oocytes require APC-Cdc20-mediated proteolysis of securin and cyclin B1 (Greaney et al., 2018; Herbert et al., 2003; Jones and Lane, 2013). Although an SAC exists in oocytes (Hached et al., 2011; Homer et al., 2005a,b; Wassmann et al., 2003), MI in oocytes is nevertheless prone to error, especially with ageing (Greaney et al., 2018; Jones and Lane, 2013). One model for explaining this paradox is that the oocyte SAC lacks stringency (Greaney et al., 2018; Gui and Homer, 2012; Kolano et al., 2012; Lane et al., 2012; Sebestova et al., 2012), but why this should be is not clear.

During exit from mitosis, the final steps of cytokinesis (abscission) occur after nuclear envelope reformation and require Cep55 (Fabbro et al., 2005; Gershony et al., 2014; Zhao et al., 2006). Cep55 is a centrosome- and midbody-associated coiled-coil protein (Fabbro et al., 2005) that orchestrates the final stages of membrane abscission by directing the midbody recruitment of key proteins required for cytokinesis completion, such as tumour-susceptibility gene 101 (TSG101) and ALG2-interacting protein X (ALIX) (Martinez-Garay et al., 2006; Morita et al., 2007; van der Horst et al., 2009; Zhao et al., 2006). Consequently, when Cep55 is depleted in somatic cells, mitosis arrests at a late post-anaphase stage (Fabbro et al., 2005; van der Horst et al., 2009; Zhao et al., 2006).

Here, we set out to investigate Cep55 function during MI in mouse oocytes, given that nuclear envelopes do not reform during exit from MI. We found that anaphase I and subsequent MI exit events failed following Cep55 depletion, despite normal SAC inactivation and intact APC-Cdc20-mediated proteolysis. This was the result of sustained Cdk1 activity secondary to reduced inhibitory Cdk1 phosphorylation, identifying a crucial proteolysis-independent requirement for anaphase I. Thus, anaphase I requires separate proteolytic and non-proteolytic inputs, contrasting with the exclusively proteolysis-driven mitotic anaphase pathway. Because the SAC can robustly regulate the timing of anaphase onset only if anaphase is completely controlled by APC-Cdc20-mediated proteolysis, this puts chromosome segregation at risk in oocytes.

MI begins with breakdown of the oocyte nucleus (termed germinal vesicle breakdown, GVBD) and concludes with the first polar body extrusion (PBE). Western blotting showed that Cep55 levels were very low in early MI and became prominent by late MI (Fig. 1A). To investigate Cep55 function, we depleted Cep55 by microinjecting siRNAs previously shown to target murine Cep55 effectively in somatic cells (Kalimutho et al., 2018). Cep55-targeting siRNAs depleted ∼80% of endogenous protein (Fig. 1B,C) and resulted in complete loss of Cep55 signals in immunostained oocytes (Fig. S1).

To investigate the impact of Cep55 knockdown on MI progression, we undertook timelapse imaging. Similarly high rates (∼80%) of GVBD were observed in Cep55- and mock-depleted oocytes (Fig. 1D) 2 h after washing from the GVBD inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Notably, however, GVBD was markedly accelerated within the first 30 min post-release from IBMX (Fig. 1D). Importantly, therefore, we performed all subsequent comparisons of MI progression with strict reference to the time of GVBD. Exit from MI (PBE) occurred by 9-10 h post-GVBD in ∼80% of controls (Fig. 1E). Strikingly, however, only ∼20% of Cep55-depleted oocytes underwent PBE by the same time post-GVBD (Fig. 1E). This was specific to Cep55 depletion because co-expression of exogenous Cep55 from microinjected siRNA-resistant Cep55 cRNA completely restored PBE rates to ∼80% (Fig. 1F). Thus, Cep55 knockdown accelerated entry into MI but markedly inhibited MI exit.

Given the requirement of Cep55 for mitotic abscission (Fabbro et al., 2005; Zhao et al., 2006), we presumed that PBE inhibition following Cep55 knockdown was the result of a late post-anaphase I defect. To investigate this, we undertook timelapse imaging of fluorescently labelled spindles and chromosomes in live oocytes (Wei et al., 2018). Bipolar spindles assembled normally by 5-6 h post-GVBD and chromosome alignment at the spindle equator in Cep55-depleted oocytes was similar to that in mock-depleted controls (Fig. 1G; Fig. S1, Movies 1 and 2). Mock-depleted oocytes then progressed to anaphase I and PBE at around 7-9 h post-GVBD (Fig. 1E,G). Surprisingly, however, in Cep55-depleted oocytes, chromosome separation did not occur in ∼80% of oocytes, even after 19 h post-GVBD, well beyond the time that controls underwent anaphase I (Fig. 1E,G; Movies 1 and 2). Using chromosome spreads, we confirmed that homologous chromosome pairs had indeed remained intact in MI-arrested Cep55-depleted oocytes (Fig. 1H). Thus, Cep55 knockdown induced metaphase I arrest in oocytes, contrasting with the post-anaphase abscission defect in mitosis (Zhao et al., 2006).

We reasoned that anaphase I failure could be caused by inhibition of APC-Cdc20, as proteolysis of securin and cyclin B1 are understood to mediate chromosome segregation (Herbert et al., 2003; Homer, 2013; Meadows and Millar, 2015; Pesin and Orr-Weaver, 2008). Because APC-Cdc20 activity is inhibited by the SAC until chromosomes attach to spindle microtubules via kinetochores (Foley and Kapoor, 2013), we first asked whether the SAC remained chronically active following Cep55 depletion. An active SAC is signified by enrichment of the SAC protein, Mad2, at kinetochores (Gui and Homer, 2012; Waters et al., 1998; Lane et al., 2012). In Cep55-depleted oocytes, as in controls, unattached kinetochores during early MI (1-2 h post-GVBD when kinetochores are mostly unattached) recruited high levels of Mad2 (Fig. 2A). By 6-7 h post-GVBD in controls, after the spindle had bipolarised and chromosomes were placed under tension at the spindle equator (Fig. 1G; Fig. S1), Mad2 levels were largely undetectable, which is indicative of high kinetochore-microtubule occupancy and SAC silencing (Fig. 2A). Significantly, in Cep55-depleted oocytes, kinetochore Mad2 levels also dissipated over the same period, indicating that the SAC was being silenced normally (Fig. 2A). This was consistent with our earlier timelapse data which clearly showed that normal bipolar spindles assembled after Cep55 depletion and that chromosomes aligned at the spindle equator (Fig. 1G; Fig. S1), thereby satisfying conditions for silencing the SAC.

To investigate whether residual SAC activity might still be present despite normal Mad2 loss from kinetochores and chromosome alignment, we treated metaphase I-arrested Cep55 knockdown oocytes using a specific inhibitor (reversine) of Mps1, which is required for the SAC in oocytes (Hached et al., 2011). We found that reversine could not induce anaphase I or MI exit (Fig. 2B,C). Thus, anaphase I failure following Cep55 knockdown was not the result of persistent SAC activation.

Because SAC silencing should allow APC-Cdc20 to become active (Pesin and Orr-Weaver, 2008; Foley and Kapoor, 2013), the failure to induce anaphase I despite forced SAC inactivation (Fig. 2B,C) was surprising, leading us to ask whether APC-Cdc20 activity might be impacted in an SAC-independent manner by Cep55 depletion. To investigate this, we used timelapse fluorescence imaging of securin-GFP, whose destruction provides a readout of APC-Cdc20 activity (Homer et al., 2005b; Lane and Jones, 2014), to determine whether APC-Cdc20 was becoming active. Notably, however, a decline in securin-GFP fluorescence, reflecting onset of APC-Cdc20-mediated proteolysis, clearly occurred in Cep55-depleted oocytes and began at the same time (about 5-6 h post-GVBD) as in controls (Fig. 3A,B), consistent with above data showing on-time inactivation of the SAC. Furthermore, there was no difference in either extent or rate of proteolysis between the two groups (Fig. 3C,D). In keeping with our findings using exogenous securin-GFP, we also found that the majority of endogenous securin underwent destruction by 8 h post-GVBD in both Cep55-depleted and control oocytes (Fig. 3E). We confirmed that proteolysis was APC-mediated by using an APC-specific inhibitor, APCIN (Wei et al., 2018), which induced protein stabilisation in both mock- and Cep55-depleted oocytes (Fig. 3F).

It was unexpected that SAC inactivation and proteolysis remained completely intact despite metaphase I arrest. We therefore sought to confirm that anaphase I failure and ongoing proteolysis were co-existing within the same oocytes. To do this, we monitored securin-GFP while simultaneously imaging spindles and chromosomes in the same oocytes. We found that securin-GFP levels increased during the first 5-6 h post-GVBD, coincident with bipolar spindle assembly and chromosome alignment in both Cep55- and mock-depleted oocytes (Fig. 3G). Although proteolysis then occurred in both controls and Cep55-depleted oocytes, chromosome separation occurred in the former but not the latter (Fig. 3G; Movies 3 and 4). We repeated these experiments using cyclin B1-GFP instead of securin-GFP and obtained identical results (Fig. S2; Movies 5 and 6). Thus, following Cep55 depletion, anaphase I does not occur despite normal SAC inactivation and intact proteolysis, showing that anaphase I is not exclusively driven by proteolysis in oocytes.

Separase activity is modulated by securin as well as Cdk1 activity levels (Stemmann et al., 2001). Because securin destruction occurred normally after Cep55 depletion (Fig. 3), we reasoned that Cdk1 inactivation might be perturbed. Strikingly, we found that Cdk1 activity remained markedly higher in late MI following Cep55 knockdown compared with controls (Fig. 4A,B). Because sustained Cdk1 activity was not a result of failed cyclin B1 destruction (Fig. S2), we next investigated whether the other mechanism for inactivating Cdk1, inhibitory Tyr15 phosphorylation (pY15), was affected. Significantly, we found that levels of pY15-phosphorylated Cdk1 (p-Cdk1) in late MI were dramatically reduced after Cep55 knockdown compared with controls (Fig. 4C,D).

These data suggested that anaphase I was being prevented by persistent Cdk1 activity brought about by suboptimal inhibitory phosphorylation. If this were the case, then forcing Cdk1 inactivation should induce anaphase I. We therefore treated MI-arrested Cep55 knockdown oocytes with the small molecule Cdk1 inhibitor, flavopiridol (Wei et al., 2018). Significantly, flavopiridol induced anaphase I and PBE in >70% of Cep55-depleted oocytes compared with only 20% after DMSO treatment (Fig. 4E,F; Movies 7 and 8).

Thus, persistent Cdk1 activity and reduced Cdk1 phosphoinhibition are associated with metaphase I arrest after Cep55 knockdown, and forced Cdk1 inactivation over-rides this arrest to induce anaphase I. Significantly, this pathway is not under SAC control, as reversine could not overcome metaphase I arrest (Fig. 2B,C).

The need for Cdk1 phosphoinhibition to inactivate Cdk1 sufficiently for anaphase I initiation was unexpected, as it is widely believed that cyclin B1 destruction is sufficient (Herbert et al., 2003). Further supporting a role for phosphoinhibition, p-Cdk1 levels exhibited a prominent rise specifically in late MI in untreated oocytes (Fig. 5A). Somewhat surprisingly, levels of Wee1B kinase, which mediates inhibitory Cdk1 phosphorylation (Oh et al., 2011), did not increase by late MI; however, levels of Cdc25B phosphatase, which removes Wee1B-induced inhibitory phosphorylation (Adhikari and Liu, 2014), decreased concurrently with increased p-Cdk1 (Fig. 5A). Taken together, these data suggest that anaphase I requires Wee1B-dependent Cdk1 phosphoinhibition secondary to reduced phosphatase activity, rather than to increased inhibitory kinase activity.

If inhibitory phosphorylation is required for Cdk1 inactivation during late MI, then disrupting Wee1B in late MI should compromise MI exit. Because Wee1B regulates Cdk1 during G2 arrest (Han et al., 2005), we avoided approaches (e.g. knockdown) that would disrupt Wee1B from the GV stage to avoid incurring early-stage defects that could impact late MI events. Instead, we used small molecule inhibitors that could be introduced specifically in late MI. We treated oocytes at 5 h post-GVBD with either the Wee1-specific inhibitor MK1775 (Strauss et al., 2018) or DMSO solvent. Timelapse imaging showed that 70-80% of DMSO-treated oocytes underwent anaphase I followed by PBE by 9-10 h post-GVBD, as before (Fig. 5B,C; Movie 9). Strikingly, >90% of MK1775-treated oocytes failed to undergo either anaphase I or PBE, even by 15 h post-GVBD (Fig. 5B,C and Movie 10). Thus, inhibition of Wee1B specifically in late MI prevented anaphase I.

We reasoned that Cep55 depletion could lead to reduced p-Cdk1 levels in late MI by causing a shift from a normal Cdc25B-suppressed state to relative Cdc25B overactivity. If so, then reducing Cdc25B activity in Cep55-depleted oocytes during late MI should restore the balance and promote anaphase I. To test this, we treated Cep55-depleted oocytes with a specific Cdc25 inhibitor, NSC95397, late in MI. As validation of NSC95397, we found that (at the dose used) GVBD was significantly reduced and p-Cdk1 levels increased, proving efficient phosphatase inhibition (Fig. S3). Strikingly, inhibiting Cdc25 using NSC95397 induced anaphase I in ∼70% of Cep55-depleted oocytes compared with only ∼20% following DMSO treatment (Fig. 5D,E; Movies 11 and 12).

Next, we asked whether changes in Cdc25B protein abundance might account for altered activity following Cep55 depletion. Notably, accompanying the reduction in p-Cdk1 following Cep55 depletion, we found that Cdc25B underwent a marked increase whereas Wee1B was unaffected (Fig. 5F,G). Thus, loss of Cep55 promoted Cdc25B stability in late MI that resulted in sustained Cdk1 activity and anaphase I failure.

We then asked whether overexpression of Cep55 would have the opposite effect to Cep55 depletion and lead to accelerated exit from MI. This was not the case, as the timings of anaphase I and PBE were completely unaffected when Cep55 was overexpressed from microinjected cRNA (Fig. S4), suggesting that phosphorylation-dependent pathways are not sufficient for driving exit events independently of proteolysis. We wondered whether Cep55 might influence Cdc25B stability through direct binding. This did not appear to be the case because Cdc25B exhibited diffuse cytoplasmic localisation (Fig. S5A), similar to previous findings (Oh et al., 2010), that was distinct from the Cep55 enrichment at spindle poles seen during both MI and MII (Fig. S1). Furthermore, Cep55 levels increased even further by MII without an accompanying decrease in Cdc25B (Fig. S5B). Together, these data suggest that the effect of Cep55 on Cdc25B is complex and might be restricted to a narrow window in late MI.

MI in oocytes is known to be prone to chromosome mis-segregation. Here, we show that Cdk1 phosphoinhibition secondary to Cdc25 suppression is necessary for initiating anaphase I in oocytes, independently of proteolysis. Because phosphoinhibition was not under control of the SAC in oocytes (Fig. 2B,C), these data show that anaphase I in oocytes is not exclusively regulated by proteolysis and, hence, by the SAC. Moreover, because the status of kinetochore-microtubule attachment/tension is relayed by the SAC, the timing of phosphoinhibition is not coordinated with chromosome alignment, rendering chromosome segregation at increased risk of going awry in oocytes. Our findings differ from previous reports linking inhibitory Cdk1 phosphorylation with exit from M phase because, in these cases, proteolysis was also impacted (D'Angiolella et al., 2007; Forester et al., 2007; Oh et al., 2011).

In contrast to the role of Cep55 in abscission, long after anaphase in mitosis (Fabbro et al., 2005; Zhao et al., 2006), in female MI, Cep55 is required to initiate anaphase I by destabilising Cdc25B to promote Cdk1 inactivation. Cep55 appears to subdue Cdk1 in other situations, as Cdk1 is prematurely activated in breast cancer cells lacking Cep55 that are treated with anti-mitotic agents (Kalimutho et al., 2018). Furthermore, we observed that, although the overall capacity to enter M phase (marked by GVBD) was unchanged by Cep55 depletion, there was a doubling of GVBD rates within the first 30 min after release from IBMX (Fig. 1D), consistent with accelerated Cdk1 activation. We do not currently know the mechanism by which Cep55 induces Cdc25B instability in late MI. It may not be by direct binding, based on the lack of colocalisation in oocytes and our unpublished immunoprecipitation work involving somatic cells. Interestingly, destruction of Cdc25B mediated by the β-TrCP-Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase occurs at the metaphase-to-anaphase transition during mitosis (Thomas et al., 2010). One possibility is that Cep55 might promote Cdc25B-SCF interaction either directly or indirectly in late MI when Cep55 levels are maximal. Phosphorylation pathways do not appear to be sufficient to induce MI exit without additional input from proteolysis because Cep55 overexpression did not accelerate MI exit. Additionally, because Cep55 levels continued to increase into MII without a concomitant decrease in Cdc25B, there is probably an additional regulatory layer that limits the effects of Cep55 to late MI; perhaps the unique MII environment involving cytostatic factors has some role to play, but this remains to be investigated.

Our findings are consistent with a previous report showing that MI is disrupted following Cep55 depletion in oocytes (Xu et al., 2015). However, using oocytes that were fixed and immunostained at a single timepoint, that study suggested that Cep55 depletion induced spindle and chromosome alignment defects (Xu et al., 2015). Significantly, the impact of Cep55 depletion on GVBD (Fig. 1D, first 30 min) was not previously identified, and single-timepoint analyses were performed relative to time of transfer to IBMX-free medium rather than relative to GVBD (Xu et al., 2015), risking comparing oocytes at very different stages of MI. Here, using Cep55-specific siRNA sequences previously characterised in murine cells (Kalimutho et al., 2018) and extensive analyses of live oocytes (Fig. 1G, Fig. 3G, Fig. 4F, Fig. 5D) in addition to fixed oocytes (Fig. S1) in strict relationship to GVBD, we showed that spindle assembly and chromosome alignment remain intact following Cep55 depletion. Moreover, entirely in keeping with unperturbed attainment of metaphase I, we showed that SAC silencing occurs normally, as does APC-Cdc20-mediated proteolysis.

Surprisingly, the full extent of proteolysis that typically occurs during late MI was not sufficient to induce the degree of Cdk1 inactivation necessary for anaphase I, contrasting sharply with mitosis, in which anaphase requires only a fraction of cyclin B1 to be destroyed (Wolf et al., 2007, 2006). This could be related to cyclin B1 being present in large excess (∼sixfold) over Cdk1 in oocytes (Levasseur et al., 2019) versus the reverse in somatic cells (30-fold excess Cdk1 over cyclin B1) (Arooz et al., 2000). Under these conditions in oocytes, sufficient cyclin B1 might remain after proteolysis is complete to maintain Cdk1 above the threshold required for separase activation, necessitating Cep55-dependent phosphoinhibition.

All animals were housed in a specific pathogen-free environment in filter-top cages and fed a standard diet. All work involving animals complied with all relevant ethical regulations and was approved by the Animal Ethics Committee at the University of Queensland.

Oocytes were isolated from the ovaries of 3- to 4-week-old B6CBAF1 female mice 44–46 h after intraperitoneal injection of 7.5 international units (IU) of pregnant mare serum gonadotrophin (Pacific vet). Dissected ovaries were immediately transferred to the laboratory in prewarmed αMEM HEPES-buffered medium (Sigma-Aldrich) containing 50 μM 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich), which prevents oocytes from undergoing GVBD (Gui and Homer, 2012, 2013; Homer et al., 2009; Wei et al., 2018). Ovarian antral follicles were punctured in IBMX-treated medium in 35×10 mm² dishes using a 27G needle under direct vision on the stage of a stereomicroscope (M165C, Leica Microsystems). Only fully-grown cumulus-covered oocytes were isolated and used for further experiments. For longer-term culture and for all confocal imaging, oocytes were cultured in microdrops of M16 media (Sigma-Aldrich) under embryo-tested light mineral oil (Sigma-Aldrich) at 37°C in an atmosphere of 5% CO₂ in air (Gui and Homer, 2012, 2013; Homer et al., 2009, 2005b; Wei et al., 2018).

For microinjection (Gui and Homer, 2012, 2013; Homer et al., 2009; Wei et al., 2018), GV-stage oocytes in IBMX-treated medium were stabilised using suction applied through a hydraulic syringe to a prefabricated holding pipette (inner diameter 15 μm, outer diameter 75 μm, 35° bend; The Pipette Company). Microinjection needles were pulled from capillary tubes (0.86 mm inner diameter, 1.5 mm outer diameter; Harvard Apparatus) to a predetermined calibre using a vertical pipette puller (P30 vertical micropipette puller, Sutter Instruments). The tip of the microinjection pipette was advanced across the zona pellucida and oolemma into the cytoplasm of the oocyte, aided by a brief electrical pulse delivered by an intracellular electrometer (IE-25IA, Warner Instruments). A precise volume of test solution roughly equal to 5% of the oocyte volume was then delivered to the oocyte using a Pneumatic PicoPump (PV-820, World Precision Instruments). The rate of oocyte death following microinjection was consistently <10%.

The mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit (Ambion) was used to produce cRNA constructs by T3 promoter-driven in vitro transcription from linearised DNA templates (Gui and Homer, 2012, 2013; Homer et al., 2009, 2005b; Wei et al., 2018). Constructs used in this paper were histone 2B (H2B)-RFP, securin-GFP, cyclin B1-GFP and full-length murine Cep55 cRNAs. The latter was siRNA-resistant by virtue of lacking the 5′UTR targeted by the siRNA (see below). All plasmids were fully sequenced prior to transcription. Following in vitro transcription, cRNA size was verified on agarose gels and concentrations were determined using a spectrophotometer. Constructs were microinjected at the following concentrations: H2B-RFP at 250 ng μl⁻¹, securin-GFP at 200 ng μl⁻¹, cyclin B1-GFP at 200 ng μl⁻¹ and Cep55 at 1.5 µg μl⁻¹. Following microinjection at the GV stage, oocytes were held in 50 μM IBMX for at least 2 h to allow time for protein translation while maintaining GV arrest. Oocytes were then washed through 5-6 drops of IBMX-free media to allow resumption of maturation.

For depleting Cep55, GV-stage oocytes were microinjected with a previously published siRNA sequence that was designed to target the 5′UTR of murine Cep55: 5′-AGCUACUGAGCAGUAAGCAAACAT T-3′; 5′-AAUGUUUGCUUACUGCUCAGUAGCUUU-3′; control siRNA sequences were 5′-UUCUUCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′ (Gene Pharma, Shanghai, China) (Kalimutho et al., 2018). All siRNAs were injected at a final needle concentration of 100 µM. Following microinjection, oocytes were maintained in IBMX-treated medium for 24 h to allow time for protein knockdown.

Stock solutions of all small molecule inhibitors were made in DMSO at the highest possible concentration that enabled complete solubilisation, thereby minimising the volume of DMSO added to media. For inhibiting Cdk1 activity, flavopiridol (SelleckChem; 10 mM stock solution) was dissolved in medium for a final concentration of 5 μM, as used previously in mouse oocytes (Wei et al., 2018). The APC-Cdc20-specific inhibitor APCIN (Sigma-Aldrich; 50 mM stock solution) (Sackton et al., 2014; Wei et al., 2018) was used at 150 μM to inhibit proteolysis at 4 h post-GVBD before proteolysis had begun. To inhibit Wee1B activity late in MI (5-6 h post-GVBD), MK1775 (Strauss et al., 2018) (Celleckchem; 10 mM stock solution) was used at a concentration of 5 μM. To inhibit Cdc25B activity late in MI (5-6 h post-GVBD), NSC95397 (Cayman Chemical, USA; 10 mM stock solution) was used at a concentration of 400 nM after validation (see Fig. S3). As a control, DMSO, which was used to dissolve all inhibitors, was added to media at a final concentration that was the same concentration attained when inhibitors were added.

Individual oocytes were collected into tubes and immediately frozen on dry ice and stored at −80°C until lysis. Oocytes were lysed by four freeze-thaw cycles in 2 µl of 20 ng μl⁻¹ BSA in water. Assay buffer (8 µl; 50 mM β-glycerophosphate pH 7.35, 1.5 mM EGTA, 1 mM DTT and 10 mM MgCl2) was added and samples were prewarmed to 37°C. Unlabelled ATP, histone H1 (Sigma-Aldrich) and ɣP32 ATP (3000 µCi mmol⁻¹; Perkin Elmer) were mastermixed and added such that each reaction contained 1 µg of H1, 10 µCi of ɣP32 ATP and 5 µM unlabelled ATP in a final volume of 12 µl. Reactions were incubated for 70 min at 37°C and terminated by denaturation in SDS-PAGE loading buffer. Samples were resolved by SDS-PAGE and gels were stained with Coomassie Blue and vacuum dried. Exposures were collected using a phosphor storage screen. Kinase activity was quantified by scintillation counting excised gel pieces in Ultima Gold scintillant on a Packard Tri-Carb beta counter.

Western blotting was performed as described previously (Gui and Homer, 2012, 2013; Homer et al., 2009; Wei et al., 2018). Protein samples were prepared by washing oocytes with ultrapure water and lysing in LDS sample buffer (NuPAGE, Invitrogen). Samples were vigorously agitated and immediately snap-frozen at −80°C for storage until required. Samples were boiled at 95°C for 5 min following addition of reducing agent (NuPAGE, Invitrogen). Proteins were separated on 4-12% Bis-Tris gels (NuPAGE; Invitrogen Australia) and transferred to PVDF membranes (Immobilon-P, Millipore). Following transfer, membranes were blocked for 1 h at room temperature in 3% BSA in TBS (25 mM Trizma base, 150 mM sodium chloride) containing 0.05% Tween-20 (TBST). Primary antibody incubation was carried out overnight at 4°C in blocking buffer. The primary antibodies used were Cep55 [1:2000, made by the Khanna laboratory and described previously (Kalimutho et al., 2018)], Wee1B [1:800; a very kind gift from Marco Conti (Oh et al., 2011)], Cdc25B (New England Biolabs 9525#, 1:1000), securin (Abcam-ab3305; 1:1000), p-Cdk1 (Cell Signalling-9111S; 1:1000) and vinculin (Sigma Aldrich-V9131; 1:2000). Following three 5 min washes in TBST, membranes were incubated in the appropriate HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (1:1000; Bio-Rad) for 1 h at room temperature. Chemiluminescence detection was performed using Western Lightning ECL-Pro (Perkin Elmer) and imaged using Image Quant LAS500 (GE Healthcare).

Oocytes were fixed and stained as described previously (Gui and Homer, 2012, 2013; Homer et al., 2009). Briefly, oocytes were washed in PHEM buffer (pH 7.0) and prepermeabilised in 0.25% Triton-X in PHEM. Oocytes were then fixed in 3.7% paraformaldehyde solution in PHEM for 20 min. Oocytes were blocked overnight in 3% BSA in PBS containing 0.05% Tween-20 at 4°C. Primary antibody incubation with anti-Cep55 antibody (1:200) was carried out for 1 h at 37°C. Primary antibody incubation with anti-MAD2 antibody (1:200) was carried out for 4 h at 37°C. Following three 5 min washes in PBS containing 0.5% BSA and 0.05% Tween-20, oocytes were incubated with the appropriate Alexa Fluor 488-conjugated secondary antibodies (1:200; ThermoFisher) for 1 h at 37°C. Oocytes were then washed three times before staining for DNA by incubating in Hoechst 33342 (10 µg ml⁻¹; Sigma) for 5 min.

Imaging was performed using a Leica TCS SP8 confocal microscope equipped with a 20×0.75 NA Apochromat water-immersion objective fitted with an automated pump cap (water immersion microdispenser, Leica; automated pump mp-x controller, Bartels Mikrotechnik) as described previously (Wei et al., 2018).

Timelapse microscopy was used to image spindles and chromosomes simultaneously (Wei et al., 2018). To visualise microtubules, silicon rhodamine (SiR)-Tubulin dye (Cytoskeleton, Denver, CO) (Lukinavičius et al., 2014) was added to media at a final concentration of 100 nM, as validated previously in mouse oocytes (Wei et al., 2018). Oocytes were imaged in 1-2 μl microdrops of M16 medium in glass-bottom dishes (35×10 mm dish; MatTek) under mineral oil enclosed within a purpose-built stage-mounted incubation chamber designed to maintain conditions of 37°C and 5% CO₂ in air. Temperature fluctuation was further buffered by enclosing the entire microscope, including the stage-mounted chamber, in a custom-designed polycarbonate incubator (Life Imaging Services) that maintained a stable internal temperature of 37°C. Automated image capture was driven by the Leica LAS X software. At the commencement of imaging, the positions of the oocytes in the z-axis were located. The complete stack was then derived by setting a stack thickness of 50 μm. Z-stacks were acquired with step intervals of 4 μm at 5 min (Flavopiridol treatment), 10 min (for securin-GFP and cyclin B1-GFP destruction measurement) or 30 min (all other experiments) intervals and a speed of 600 Hz. Using the Leica mark-and-find tool, we typically imaged multiple groups of oocytes in separate droplets in each experiment. The 561 nm (for H2B-RFP) and 633 nm (for SiR-Tubulin) laser lines were used at 0.5% and 3% intensity, respectively.

Post-acquisition image processing was performed using Leica LAS X software and images were assembled into panels using Adobe Illustrator (Adobe Systems). Fluorescence images were produced by Z-projecting the entire stack of fluorescence images from both channels and merging them with images in the bright-field channel.

For quantifying proteolysis, GV-stage oocytes were microinjected with securin-GFP or cyclin B1-GFP cRNA and allowed to express protein for at least 2 h while being maintained GV-arrested using IBMX. GFP fluorescence was detected using the 488 nm laser line at 1% intensity. Z-stacks of 50 μm thickness were acquired with step intervals of 5 μm at 30 min intervals at a speed of 600 Hz. After importing LAS X images into Image J software (NIH, Bethesda, MD), a maximum intensity projection of the entire Z-stack was performed, a region of interest was drawn around the entire oocyte and the fluorescence intensity within this area was measured over time. Graphs of fluorescence intensity against time were then normalised and plotted.

GraphPad Prism (GraphPad, USA) was used to calculate the mean and standard error of the mean (s.e.m.). Statistical comparisons were made using a two-tailed Student's t-test with Welch's correction. Graphs were prepared in GraphPad Prism. P values were represented as *P<0.05, **P<0.01, ***P<0.001; ns denotes P>0.05 (not significant). All experiments were repeated a minimum of three times.

We are very grateful to Marco Conti (University of California, San Francisco, CA, USA) for the very kind gift of anti-Wee1B antibody.