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RESEARCH |
Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK
Correspondence should be addressed to J Carroll; Email: j.carroll{at}ucl.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Cyclin abundance is an important requirement for activation of cdk1 (Murray & Kirschner 1989). In mitotic cell cycles, new cyclin synthesis precedes each mitosis (Evans et al. 1983) and exit from mitosis is driven by a ubiquitin-dependent, proteasome-mediated destruction of cyclin B1 (Glotzer et al. 1991). In mouse oocytes arrested at the germinal vesicle (GV) stage, protein synthesis is not required for the activation of MPF or GVBD (Clarke & Masui 1983, Hashimoto & Kishimoto 1988, Downs 1990). In the absence of a requirement for new cyclin B synthesis it is thought that dephosphorylation of pre-MPF laid down during oogenesis is sufficient for MPF activation (Choi et al. 1991, Chesnel & Eppig 1995, Mitra & Schultz 1996). Protein synthesis is required to progress through meiosis I (MI), since protein synthesis inhibitors prevent normal MI spindle formation (Clarke & Masui 1983, Hashimoto & Kishimoto 1988, Hampl & Eppig 1995, Winston 1997). It is likely that cyclin B is one of the required proteins since the duration of MI and increase in MPF activity between GVBD and MI is correlated with the rate of cyclin synthesis (Hampl & Eppig 1995, Polanski et al. 1998). Furthermore, injection of cyclin B1 mRNA accelerates or delays MI depending on the length of the poly-A tail, a mechanism that regulates translational efficiency (Tay et al. 2000, Ledan et al. 2001). These studies suggest that the levels of cyclin B1 play an important role in determining the timing of events in MI, although the effects of cyclin B1 prior to GVBD are largely unexplored.
An additional aspect important for cdk1-cyclin B activity is its spatial distribution (Pines 1999, Takizawa & Morgan 2000). Cdk1-cyclin B is cytoplasmic during inter-phase and enters the nucleus late in prophase prior to nuclear envelope breakdown (NEBD) (Pines & Hunter 1991, Ookata et al. 1992, Hagting et al. 1998, Casas et al. 1999). In interphase, cdk1-cyclin B is retained in the cytoplasm in a chromosomal region maintenance protein 1 (CRM1)-dependent manner due to a CRM1-binding nuclear export sequence (Hagting et al. 1998, Toyoshima et al. 1998, Yang et al. 1998) in the cytoplasmic retention signal (Pines & Hunter 1994). In prophase, nuclear accumulation is driven by a phosphorylation-dependent decrease in CRM1-dependent export (Hagting et al. 1998, Yang et al. 1998) and increase in cyclin import (Hagting et al. 1999, Yang et al. 2001). After NEBD, cdk1-cyclin B associates with the mitotic apparatus, including the spindle poles, the microtubules and chromatin. These studies provide a concise account of the temporal and spatial aspects of cell cycle control during mitosis. To date, the spatial organisation of cdk1-cyclin B in meiosis in mammals has not been investigated.
In this study we have utilised a human cyclin B1-green fluorescent protein (GFP) fusion protein (Hagting et al. 1998, Clute & Pines 1999) to examine the effects of cyclin B1 abundance on GVBD, the dynamics of cyclin B1 distribution in relation to GVBD and the mechanisms underlying the localisation of cyclin B1 during MI in mouse oocytes.
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Materials and Methods |
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Microinjection
Oocytes were microinjected using a micropipette and Narishige manipulators mounted on a Leica or Nikon inverted microscope. Oocytes were placed in a drop of M2 + dbcAMP under paraffin oil in a lid of a 3 cm Falcon culture dish. Cyclin was diluted in injection buffer (120 mM KCl, 20 mM HEPES, pH 7.4). The oocyte was immobilised using a holding pipette and the injection pipette was pushed through the zona pellucida and against the plasma membrane. The pipette was inserted using a brief overcompensation of negative capacitance. Pressure was applied via a pico-pump attached to a nitrogen cylinder. The amount of solution injected was calculated using an eye-piece graticule to obtain an estimate of the diameter of the injection bolus. Oocyte volume was estimated to be 250 pl.
Confocal and fluorescence microscopy
Confocal microscopy was performed using a BioRad micro radiance confocal scan head mounted on a Zeiss axiovert 100TV. Oocytes were placed in a heated chamber (35–37 °C) and cyclin B1-GFP was excited using the 488 nm line of an argon laser. Fluorescence was collected through a 20 or 40 x 0.75 NA objective. Laser power was set to 1 or 3% of maximum and images were collected at intervals of 5 or 10 min. Each image was averaged two to three times. For conventional imaging, oocytes were placed in a heated chamber as described above on the stage of a Zeiss Axiovert 100TV. Excitation of cyclin B1-GFP (490 nm), GFP itself (490 nm) and rhodamine dextran (550 nm) was via a monochromater controlled by the Metafluor software (Universal Imaging). Fluorescence was collected through a 20 or 40 x 0.75 NA objective. Images were acquired using a Princeton Instruments MicroMax interline cooled CCD camera (Roper Scientific, Buckinghamshire, UK). Camera exposure times varied between 50 and 100 ms. Data were collected and analysed using Universal Imaging Metafluor and Metamorph software.
Kinase assays
MPF and MAP kinase activities were measured using histone H1 and myelin basic protein (MBP) kinase assays respectively. The MBP assay has been shown previously to correlate with MAP kinase activity in mouse oocytes as determined by more specific gel or immunoprecipitation-based assays (Verlhac et al. 1993). The protocol was similar to that described elsewhere (Kubiak et al. 1993, Moos et al. 1995). Five eggs (unless stated otherwise) in 2 µl HEPES-buffered KSOM were transferred in 3 µl storing solution (10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 mM p-nitrophenyl phosphate, 20 mM ß-glycerophosphate, 0.1 mM sodium orthovanadate, 5 mM EGTA) and immediately frozen on dry ice. The samples were subjected to three freeze–thaw cycles, diluted twice by the addition of two times concentrated kinase buffer containing 60 µg/ml leupeptin, 60 µg/ml aprotinin, 24 mM p-nitrophenyl phosphate, 90 mM ß-glycerophosphate, 4.6 mM sodium ortho-vanadate, 24 mM EGTA, 24 mM MgCl2, 0.2 mM EDTA, 4 mM NaF, 1.6 mM dithiothreitol, 2 mg/ml polyvinyl alcohol, 40 mM MOPS, 0.6 mM ATP, 2 mg/ml histone H1 (HIII-S from calf thymus; Sigma, St Louis, MO, USA), 0.5 mg/ml MBP (from guinea pig brain; Sigma) and 0.25 mCi/ml [32P]ATP. The samples were then incubated at 30 °C for 30 min. The reaction was stopped by the addition of two times SDS sample buffer (0.125M Tris–HCl, 4% SDS, 20% glycerol, 10% mercaptoethanol, 0.002% bromophenol blue) and boiled for 3–5 min. The samples were then analysed with SDS-PAGE followed by autoradiography. The autoradiographs were imaged using the Fuji Bas-1000 phosphorimager system and analysed with TINA 2.0 software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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). The acceleration was found to be dose-dependent: 50% GVBD taking 150, 60, 45 and 30 min for controls (n = 33) and cyclin injections of 10 pg (n = 44), 20 pg (n = 35) and 40 pg (n = 29) respectively (Fig. 1Ai). Similarly, the maximum level of GVBD for controls, 10 pg, 20 pg and 40 pg injections occurred after 180, 90, 60 and 45 min respectively (Fig. 1Ai).
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). MPF started to increase within 30 min of injection of 40 pg cyclin B1-GFP and had reached a maximum 25-fold increase over basal by 60 min after injection. In control oocytes, MPF activation was markedly slower and more modest with a twofold increase after 60 min and a fourfold increase after 120 min (Fig. 1Aiii). The induction of MPF activity and GVBD also caused an accelerated time of activation of MAP kinase (compare Fig. 1Aii
[PDB]
and iii). After cyclin injection, MAP kinase increased gradually from 30 min reaching a tenfold increase by 120 min after injection (Fig. 1Aii
[PDB]
). At this time (120 min), MAP kinase activity remained at basal levels in the controls. Starting around 120 min, the MAP kinase activity increased eightfold by 180 min (Fig. 1Aiii). Thus, although cyclin injection accelerated the induction of MAP kinase activity, it had little effect on the extent of the increase in activity.
Cyclin B1-GFP has a concentration-dependent effect on extrusion of the first polar body
The effects of cyclin B1 on first polar body formation were twofold. First, lower amounts (10 and 20 pg) initiated polar body formation earlier (8.5–9 h) than in control oocytes (10 h) (Fig. 1B). The second effect was that increasing concentrations of cyclin caused a dose-dependent decrease in the ability of oocytes to extrude a polar body (Fig. 1B). These data are consistent with those of Ledan et al.(2001), where over-expression of cyclin B1 caused a similar phenotype. The experiments in the present study extend these observations by determining the effect of a range of concentrations of cyclin B1, rather than relying on different lengths of poly-A tail to provide different amounts of cyclin B1. In oocytes injected with 10 pg, similar proportions of oocytes extruded the first polar body compared with controls (80%, 21/25 and 85%, 24/28 respectively). After injection of 20 pg, 50% (12/24) of oocytes extruded a polar body (P < 0.05 compared with controls) while after injection of 40 pg this was reduced to 5% (1/19; P < 0.01 compared with controls).
Cyclin B1 overrides cAMP-mediated arrest at the GV stage
Previously, Ledan et al.(2001) showed that over-expression of cyclin B1 was sufficient to override cAMP-mediated arrest. Our observation that exogenous cyclin B1 accelerated the rate of GVBD suggested that cyclin B1 abundance is a potent determinant of the timing of entry into MI. To determine if levels of cyclin that accelerate GVBD also override cAMP-mediated arrest we microinjected 40 pg cyclin B1-GFP protein into oocytes maintained in 250 µM dbcAMP (Fig. 2A). Cyclin B1-GFP stimulated GVBD at a time similar to the onset of maturation of injected controls cultured in the absence of dbcAMP (Fig. 2A). Fewer oocytes injected and maintained in the presence of dbcAMP underwent GVBD (see Fig. 2A). After 3 h, 60% (41/69) of the cyclin B1-injected oocytes had undergone GVBD in the presence of dbcAMP which was significantly less than the 90% that had undergone GVBD in its absence (P < 0.01). Removal of dbcAMP from the arrested oocytes resulted in GVBD within 60 min (87%, 21/24) (Fig. 2B).
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arrow). These accumulations of cyclin were present in all oocytes and are likely to be the oocyte equivalent of the centrosomes (microtubule organising centres) to which cyclin B1-GFP binds in prophase in somatic cells (Hagting et al. 1998). Cyclin B1-GFP can first be seen entering the nucleus after 20 min and reached a peak at about 40 min (Fig. 3A and B). GVBD followed peak nuclear accumulation within 10 min. At GVBD, cyclin B1-GFP redistributed in the cytoplasm and some remained associated with the condensed chromatin. Closer examination of the distribution of cyclin within the nucleus suggested that it does not bind to nucleoli and there is no evidence for preferential loading of cyclin-GFP onto the condensing chromatin (see 20 and 40 min time-points in Fig. 3A). At 40 min, when nuclear cyclin is at a peak, it appeared to be uniformly distributed throughout the nucleus. It is possible that the high level of cyclin-GFP in the nucleus at this stage effectively masked any localisation within the nucleus. The first indication that cyclin B1-GFP localises to the chromatin was after GVBD when the nuclear cyclin B1-GFP redistributes to the cytoplasm. To determine whether cyclin B1-GFP localises to the chromosomes at other stages, MII oocytes were injected with 20 pg cyclin B1-GFP and examined using confocal microscopy. The spindle poles and microtubules and chromatin were decorated with cyclin B1-GFP (Fig. 3C).
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). These data showed that the dextran was initially excluded from the nucleus and simply diffuses into the nuclear region after GVBD. There were no perinuclear accumulations of fluorescence or preferential accumulation of fluorescence in the GV prior to GVBD (Fig. 3D).
Cyclin B1 is actively exported from the GV
Cyclin B1 in somatic cells is retained in the cytoplasm by a nuclear export signal. We examined whether similar mechanisms were controlling cyclin B1 distribution in MI. To visualise a nuclear export mechanism, cyclin B1-GFP was microinjected directly into the GV of oocytes maintained in dbcAMP. Imaging experiments revealed that the nuclear cyclin was exported from the GV from the time of the first measurement, 5 min after the start of recording (Fig. 4A and D). The gradual loss of cyclin supports the idea that it is actively exported rather than being a result of GVBD, where loss from the nucleus would be expected to be more abrupt. In addition, bright field observation of the oocyte after the completion of the experiment revealed that the GV remained intact (not shown).
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). As a further test that the decrease in cyclin B1-GFP fluorescence from the nucleus was not due to simple diffusion, 70kDa fluorescein dextran was injected into the nucleus. The 70kDa dextran was retained in the nucleus for the duration of the 2-h observation (Fig. 4C). Thus the export from the nucleus is a feature specific to cyclin B1-GFP.
Inhibition of nuclear export accelerates accumulation of nuclear cyclin B1-GFP
To examine the kinetics and mechanism of cyclin B1-GFP nuclear import during MI, leptomycin B was used to inhibit CRM1-dependent nuclear export (Fornerod et al. 1997, Hagting et al. 1998, Kudo et al. 1998, Yang et al. 1998) while monitoring nuclear import. When cyclin B1-GFP-injected oocytes (2–3 pg) were incubated with 20nM leptomycin B, nuclear accumulation of the cyclin B1-GFP was apparent from the time of the first recording 5–10 min after injection (Fig. 5A and B). In contrast to this immediate increase in nuclear cyclin B1-GFP, oocytes undergoing GVBD in the absence of leptomycin B did not show nuclear accumulation in the first 20 min of the experiment (Fig. 5A and B). This observation was quantified by determining the change in fluorescence intensity in the GV that takes place over a 10 min interval at the start of the experiment in the presence and absence of leptomycin B using the formula (Fl10min –Fl0 min)/Fl0 min; Fl, fluorescence intensity unit. This analysis confirmed that in the first 10 min of the experiment there was significantly greater nuclear accumulation of cyclin B1-GFP in the presence of leptomycin compared with controls (Fig. 5C). In addition, we measured the change in fluorescence in the GV in control oocytes just prior to GVBD to determine whether inhibition of export is sufficient to explain the rapid accumulation just prior to GVBD. The data showed that the change in fluorescence was greater just prior to GVBD than that seen in the presence of leptomycin B soon after release from dbcAMP (Fig. 5C). This suggested that nuclear accumulation involves factors other than inhibition of export, most likely an increase in import.
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Discussion |
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Exogenous cyclin stimulates progression through meiosis
The ability of cyclin B1-GFP to accelerate the rate of GVBD has demonstrated that GVBD in mouse oocytes is sensitive to the abundance of cyclin. The accelerated rate of GVBD was accompanied by an increase in MPF activity, some fivefold greater than the level of MPF that accompanied GVBD in control oocytes. These high levels of MPF are likely to explain the ability of exogenous cyclin B1 to override cAMP-mediated arrest (Ledan et al. 2001, present study). The sensitivity of GVBD to cyclin B1 abundance has been reported in other species including Xenopus (Pines & Hunt 1987, Westendorf et al. 1989, Li et al. 1997) and bovine (Levesque & Sirard 1996, Robert et al. 2002) but, in these species, it was consistent with the need for new cyclin B synthesis to induce GVBD. In contrast, mouse oocytes do not require new protein synthesis to undergo GVBD (Clarke & Masui 1983) and have been reported to have a sevenfold abundance of cyclin B1 over cdk1 (Kanatsu-Shinohara et al. 2000). Nevertheless, despite the apparent abundance of cyclin B1 in fully grown GV stage oocytes, our data show that exogenous cyclin B1-GFP is a potent activator of MPF activity.
How does excess exogenous cyclin B1 lead to the activation of MPF? One possibility is that, despite the reported excess of cyclin B, much of it may be sequestered (Westendorf et al. 1989, de Vantéry et al. 1997, Beckhelling et al. 2003, Terasaki et al. 2003). A small increase in ‘free’ exogenous cyclin B1 may provide a sufficient increase in pre-MPF available for cdc25 to initiate the autocatalytic reaction leading to complete MPF activation (Hoffmann et al. 1993, Lincoln et al. 2002). This is consistent with the idea that activation of cdk1 behaves as a bi-stable switch, thereby ensuring oscillations between an interphase and M-phase state (Pomerening et al. 2003). This cyclin abundance-stimulated switch in activity provides a reasonable explanation for the early onset of MPF activity seen in our studies. The switch-like activation of proteins that regulate the cell cycle is also seen in the activation of MAP kinase (Ferrell & Machleder 1998). This is consistent with our findings showing that MAP kinase was activated to the same extent irrespective of the level of MPF activity.
The fivefold increase in MPF activity over that seen in uninjected oocytes suggests that injected cyclin B1-GFP is contributing to the high levels of cdk1 activity seen. The extent of cdk1 activity may be regulated in part by the lack of a negative feedback normally provided by cdk1-dependent activation of the anaphase promoting complex (APC) (Pomerening et al. 2003). In MI, APC activity is suppressed for 4–5 h after the initial activation of cdk1, probably via the activation of the mos/MAP kinase pathway and the spindle assembly checkpoint (Brunet et al. 2003, Tunquist & Maller 2003, Wassmann et al. 2003). As such, in the absence of the inactivation pathway, the positive feedback pathways that lead to MPF activation may progress to completion with the extent of activation being related to the amount of cyclin B1 available for driving cdk1 activity.
Cyclin B1 availability controls the duration of MI
Cyclin injection also had effects on the timing of the first meiotic division. The ability of low doses of cyclin (10 and 20 pg) to accelerate the timing of extrusion of the first polar body is similar to previous studies where cyclin B was increased using cyclin B1 mRNA (Polanski et al. 1998, Ledan et al. 2001). The present data are in agreement with the finding that progression through MI is dependent on translation of cyclin B (Hampl & Eppig 1995, Polanski et al. 1998). In addition to the acceleration of polar body extrusion at low doses, we found a dose-dependent inhibition of polar body extrusion. The saturation of mechanisms for cyclin destruction during MI (Kobayashi et al. 1991) is the most likely explanation of this inhibitory effect of excess cyclin B1. This is supported by recent observations in mouse oocytes over-expressing cyclin B1-GFP showing that where destruction is delayed or insufficient, polar body extrusion does not take place (Ledan et al. 2001). In Xenopus oocytes cyclin destruction is not necessary for the transition from MI to MII (Peter et al. 2001, Taieb et al. 2001). In the mouse, the effects of excess cyclin B1 and more recent studies using nondestructible cyclin B1-GFP (Herbert et al. 2003) suggest that cyclin destruction is an important requirement in the progression from MI to MII.
Cyclin B1 undergoes nuclear translocation prior to GVBD
In GV stage mouse oocytes, cyclin B1-GFP was localised to the cytoplasm and excluded from the GV. In most oocytes, there were two accumulations of cyclin B1-GFP in the close proximity of the GV. These are likely to represent the oocyte centrosomes, also referred to as the microtubule organising centres since mouse oocytes do not contain centrioles. In somatic cells, Xenopus eggs and starfish oocytes, the centrioles are considered to be the site at which cdk1-cyclin B1 is first activated (Beckhelling et al. 2003, Jackman et al. 2003, Terasaki et al. 2003). Additional aggregates of cyclin B have been reported throughout the cytoplasm in Xenopus, clam and starfish oocytes (Westendorf et al. 1989, Beckhelling et al. 2003, Terasaki et al. 2003). These aggregates are detected by immunofluorescence and in starfish oocytes have also been detected using starfish or human cyclin B1-GFP fusion proteins (Terasaki et al. 2003). Using the same human fusion protein we have not observed any such aggregates in GV stage mouse oocytes. It will be interesting to determine whether the lack of any aggregates in mouse oocytes has any functional effects on the kinetics of MPF activation, as would be suggested by recent modelling studies (Slepchenko & Terasaki 2003).
Cyclin B1-GFP was retained in the cytoplasm of GV stage oocytes through an active nuclear-export mechanism, consistent with findings in other systems (Hagting et al. 1989, 1999, Yang et al. 1998). Cyclin B1-GFP injected into the GV was exported while a similar size dextran was retained. In addition, the nuclear export inhibitor, leptomycin B, accelerated accumulation of cyclin B1-GFP in the GV. These experiments demonstrated that cyclin B1 cycles between the GV and the cytoplasm and that cyclin B1 is retained in the cytoplasm of GV stage oocytes by a rate of export that outpaces the rate of import.
In the 10–15 min preceding GVBD, the balance was shifted such that import became dominant with the result that cyclin B1-GFP accumulates in the GV. This accumulation is due to active transport rather than passive diffusion through leaky nuclear pores (Lenart et al. 2003) since it accumulates to levels that exceed those in the cytoplasm. This is consistent with recent elegant experiments in starfish oocytes showing that the accumulation of cyclin B1-GFP precedes an increase in nuclear pore permeability by imaging the distribution of a 70kDa dextran simultaneously with cyclin B1-GFP (Terasaki et al. 2003). The rapid accumulation of cyclin B1-GFP just prior to GVBD suggests that, in addition to inhibition of nuclear export, an increase in import is necessary for driving cyclin B1-GFP translocation to the GV.
The tight temporal relationship between cyclin B1-GFP accumulation in the GV and GVBD suggests that nuclear accumulation of cyclin B1 is required for the rapid onset of GVBD. This is further supported by the observation that, in oocytes that remained arrested in the presence of dbcAMP, the cyclin B1-GFP remained cytoplasmic. In Xenopus oocytes, there is direct evidence that nuclear accumulation of cyclin B is necessary for GVBD since exogenous cyclin B is sufficient to drive GVBD in the absence of progester-one but a phosphorylation mutant (cyclin B1ala) that does not undergo nuclear translocation (Li et al. 1997) is not. The addition of an nuclear localizing sequence to cyclin Bala restores nuclear accumulation and the capacity to stimulate GVBD (Li et al. 1997). The fact that nuclear accumulation of cyclin B1-GFP precedes GVBD or NEBD is consistent with the role of cdk1-cyclin B1 in phosphorylation of nuclear lamins, an important step in initiation of nuclear envelope disassembly (Peter et al. 1990).
These studies highlight the importance of dynamic changes in the localization of cyclin B1 in the control of GVBD and MI. Since requirements for cyclin B synthesis have been met in GV stage oocytes, localisation may be one of the major mechanisms governing the timing of GVBD. In future studies it will be important to understand how the hormonal changes that induce GVBD in vivo link up with the mechanisms that stimulate the activation and relocalisation of cyclin B1-cdk1.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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), 20 (
) or 40 pg (
) cyclin B1-GFP compared with controls (•) (Ai). H1 kinase and MBP assays for MPF and MAP kinase activities for oocytes injected with 40 pg cyclin B1-GFP (Aii) and control oocytes are presented as the fold increase over basal (Aiii). Note that the kinase activities increase sooner and to a higher level after injection of cyclin B1-GFP. (B) The effect of cyclin B1-GFP on polar body extrusion was shown (a.u., arbitrary units). Lower doses of 10 and 20 pg accelerated polar body extrusion while higher doses increased the proportion of oocytes arrested at MI. Data are pooled from two to three independent experiments with a total of 44, 35, 29 and 33 oocytes for the 10 pg, 20 pg, 40 pg and uninjected control groups respectively.
