Stable Emi2 is rapidly degraded by Ca2+
To measure real-time changes in Emi2 levels in oocytes we generated cRNA to mouse Emi2 coupled to Venus fluorescent protein (Emi2-V), which is a yellow variant of GFP. MetII mouse oocytes were microinjected with this construct at a dose of either 0.15 or 0.5 pg, and then cultured for a few hours to allow for Emi2 expression. We Western blotted oocytes expressing Emi2-V with a polyclonal antibody against Emi2 to determine the amount of Emi2-V expression relative to endogenous protein. More than one band was detected on oocyte blots using this antibody; however, one band at ~85 kD migrated at the same molecular mass as in vitro–translated Emi2 (and this band was later knocked down by Emi2 morpholino [MO]). At the 0.15-pg dose, Emi2-V levels were less than endogenous protein after 2 h, whereas the 0.5-pg dose was expressed to about the same level as endogenous protein ().
Figure 1. Emi2-V is stable in MetII oocytes, but is rapidly degraded by Ca2+. (A) Western blot of oocytes microinjected 2 h before with 0.15 or 0.5 pg of Emi2-V. 35 oocytes were loaded per lane. Endogenous Emi2 and Emi2-V are marked. *, nonspecific (more ...)
Emi2-V was very stable in MetII-arrested oocytes, but became rapidly unstable when cytosolic Ca2+
increased. With either 0.15 pg () or 0.5 pg (not depicted) injections, we observed no degradation of Emi2-V in MetII oocytes after blocking further synthesis by washing into cycloheximide-containing media. However, when this experiment was repeated, but by washing into Sr2+
-containing media to induce spermlike Ca2+
spiking (Bos-Mikich et al., 1997
), we observed dose-dependent effects on Emi2-V. At the higher 0.5-pg dose, we observed no loss in Emi2-V signal; instead, Emi2-V levels steadily increased (). However, at the lower 0.15-pg dose Emi2-V was rapidly degraded ().
The high dose of Emi2 maintained oocytes in a MetII arrest. Oocytes injected with 0.5 pg Emi2-V cRNA showed no morphological signs of meiotic resumption, which is consistent with the maintained Emi2-V levels in these oocytes (). In contrast, oocytes that had been injected with 0.15 pg Emi2-V cRNA extruded a PB2 (). This suggests that Emi2-V has physiological CSF activity and that the oocyte has a finite capacity to degrade Emi2.
Emi2 is degraded ahead of cyclin B1
At the lower dose Emi2 injection, where we observed Emi2-V degradation, the minimum in its degradation profile after activation with Sr2+
media was reached tens of minutes before PB2 extrusion (, TE
). However, we have previously reported that cyclin B1 degradation, visualized by coupling to GFP, is only completed at the time of PB2 formation (Hyslop et al., 2004
). This suggests that the Emi2 and cyclin B1 degradation profiles may not fully overlap. When cyclin B1 was expressed in mouse oocytes with the same fluorescent protein tag as Emi2 (cyclin B1-Venus; cyclin B1-V), we observed the same degradation profile as that previously found for cyclin B1-GFP, such that a minimum in the cyclin B1-V profile was reached within minutes of PB2 extrusion (, Tc
Comparing the degradation profiles of Emi2-V and cyclin B1-V suggests that Emi2 degradation is initiated ahead of cyclin B1. Sr2+
-induced Emi2 degradation begins immediately (), whereas that of cyclin B1 begins 20 min later (). This would have to occur if MetII arrest is being mediated by Emi2-induced inhibition of APCcdc20
activity. Therefore, we wanted to confirm the immediate loss of endogenous Emi2 signal in oocytes, which is especially important given that Shoji et al. (2006)
had reported very little loss in Emi2 signal at a 6-h time point after activation with Sr2+
. Oocytes were activated by washing into Sr2+
media, and samples from a pool of oocytes were removed at various time points and probed for either cyclin B1 or Emi2 by Western blotting (). In these experiments, it was apparent that loss in Emi2 protein was rapid and complete by 30 min (in agreement with the rapid loss of Emi-V shown in ). Interestingly, Emi2 levels increased again in pronucleate embryos (6-h time point), which is in agreement with Shoji et al. (2006)
and suggests that it may have a further mitotic function (see Discussion). Similar to Emi2, we observed the loss of cyclin B1 at 30 min, as well as increased levels in pronucleate embryos (), which is consistent with our observation that the APC is switched off at this time (Nixon et al., 2002
Figure 2. Emi2 is degraded ahead of cyclin B1. (A) Western blot (WB) of oocytes for Emi2 (top; n = 100 oocytes per lane) and cyclin B1 (n = 30 oocytes per lane), and corresponding membranes stained with Coomassie brilliant blue (CB) to show equivalent (more ...)
Because of the numbers of oocytes needed for Western blots and the tens of minutes of asynchrony in timing at which Ca2+
spiking starts with Sr2+
media, (Madgwick et al., 2004
), we could not reproducibly resolve Emi2 degradation ahead of cyclin B1 by Western blotting groups of oocytes. Therefore, to examine with more accuracy the immediate degradation profiles of cyclin B1 and Emi2, we decided to measure their simultaneous degradation in the same oocyte. Cyclin B1 was coupled to Cerulean fluorescent protein (cyclin B1-C), which is a cyan variant of GFP. There was no overlap in Venus and Cerulean signals, showing that both Emi2-V and cyclin B1-C, with appropriate filters, could be imaged simultaneously in the same oocyte (). In these experiments, it was evident that the introduction of Emi2-V delayed cyclin B1 degradation ( and ), which is consistent with Emi2-inhibiting APC activity. However, these coexpression studies revealed that Emi2 degradation began tens of minutes before that of cyclin B1 (). Emi2-V levels were degraded by at least 50% before the start of cyclin B1-C degradation (n
Emi2 degradation is independent of a spindle checkpoint
Degradation of cyclin B1 is dependent on APCcdc20
activity, and in mouse oocytes it can be blocked by the induction of a spindle checkpoint (Nixon et al., 2002
; Madgwick et al., 2005
). In contrast, Emi2 degradation should be checkpoint-independent because its degradation is independent of APCcdc20
involvement. Incubation of Emi2- and cyclin B1-expressing oocytes with the spindle poison nocodazole blocks mouse oocytes from exiting MetII arrest when washed into Sr2+
media. As expected, the addition of nocodazole completely stabilized cyclin B1 levels (n
= 15/15; ). However, nocodazole had no effect on the rate of Emi2-V degradation (n
= 12/12; ). Such an observation is consistent with cyclin B1, but not Emi2 degradation, being dependent on the APC.
Figure 3. Emi2-V degradation is independent of a spindle checkpoint. Venus fluorescence levels in a MetII oocyte expressing cRNA to cyclin B1-V (A; n = 12) or Emi2-V (B; n = 15) after the addition of Sr2+ media containing 100 ng/ml nocodazole (more ...)
Therefore, in summary, we have obtained data that are entirely consistent with a model of MetII arrest achieved by Emi2-mediated inhibition of cyclin B1 degradation. Also, the Emi2-V construct generated is a physiologically active, useful tool for both establishing CSF activity and measuring its loss in real time after a Ca2+ signal.
Oocytes with Emi2 knockdown extrude a PB, but do not MetII arrest
Emi2 levels are low in both Xenopus laevis and mouse oocytes before they are matured. This would be predicted, as high Emi2 levels during maturation may be deleterious and arrest oocytes at MetI. The increased Emi2 expression during oocyte maturation makes it highly likely that Emi2 expression can be knocked out by an antisense approach. Therefore, we designed an antisense MO to the 5′UTR immediately adjacent to the start codon of mouse Emi2 (Emi2 MO) and used an additional two MO's as controls (); a 5-base mispair MO (5mp-MO), in which five bases have been altered from the complementary sequence, and an inverted MO (Inv-MO).
To explore the role of Emi2 in the establishment of MetII arrest, we injected Emi2 MO into germinal vesicle (GV) oocytes, which were then matured in vitro. Oocytes were held at the GV stage in milrinone-containing media for 2 h after MO injection; they were then released from GV arrest and allowed to mature for 16 h. Blotting of GV-stage oocytes, Emi2 MO–matured oocytes, and uninjected control matured oocytes demonstrated that Emi2 protein levels increase between GV and MetII stage, and confirmed the Emi2 knockdown in Emi2 MO–injected oocytes ().
Figure 4. GVBD and PB1 extrusion occur normally in Emi2 MO–injected oocytes. (A) Western blot of GV-stage, Emi2 MO–matured, and control matured oocytes; 75 oocytes were loaded per lane. *, nonspecific band. (B) Brightfield time lapse of (more ...)
During maturation, oocytes were scored for the morphological events of oocyte maturation, which are GV breakdown (GVBD) and PB1 extrusion (). Both GVBD () and PB1 extrusion () occurred with normal timings in Emi2 MO–injected oocytes. However, after maturation, we observed marked differences in the morphology of control oocytes and those injected with Emi2 MO. When control oocytes (uninjected, 5mp-MO, and Inv-MO–injected) were stained for chromatin, oocytes were morphologically normal. They had a PB1 containing chromatin, which was produced on completion of the first meiotic division, and a fully formed MetII spindle (100%; uninjected oocytes, n = 60; 5mp-MO, n = 32; Inv-MO, n = 25; ). However, although oocytes injected with Emi2 MO did have a PB1 containing chromatin, the chromatin in the oocyte was decondensed inside a nucleus (93%; n = 110; ).
Figure 5. Decondensed chromatin in Emi2 MO–injected matured oocytes. (A) Percentage of maturation rates in in vitro–matured oocytes after microinjection at the GV stage of MOs and/or Emi2-V cRNA, as indicated. Oocyte maturation was assessed at 16 (more ...)
Despite the lack of effect of a 5mp-MO, it remained possible that we had been extremely unlucky in the MO design, such that the observed effects of Emi2 MO were caused by its ability to block the expression of an unrelated protein involved in MetII arrest. We thought this unlikely, given that a similar morphology of Emi2 knockdown oocytes has been reported recently using a double-stranded RNAi approach (Shoji et al., 2006
). However, we decided it was important to confirm the specificity by a rescue to the control phenotype in Emi2-MO–injected oocytes by expression of exogenous Emi2. To recover Emi2, Emi2-V cRNA was microinjected into oocytes 2 h after microinjection of Emi2 MO and immediately before release from GV arrest. This rescue is made possible because Emi2-V lacks the 5′UTR recognized by the MO.
Injection of Emi2-V cRNA alone into GV oocytes that were matured induced a MetI arrest (n = 40; ), which is consistent with Emi2-V having CSF activity and the need to keep Emi2 levels low until completion of the first meiotic division. Importantly, Emi2-V cRNA expression could rescue the effects of MO knockdown. Rescue oocytes could progress through meiosis I and arrest as controls with a fully formed MetII spindle. A high dose of Emi2-V (0.5 pg; n = 45) rescued the Emi2 MO phenotype, with an equal mix of either MetI or MetII arrest, whereas oocytes microinjected with the lower Emi2 dose showed a rescue with a predominantly MetII arrest (n = 45; ).
Lack of Emi2 results in a failure to assemble a metaphase II spindle
By scoring oocytes for decondensed chromatin at only one time point (16 h), we were unable to pinpoint at which stage of meiosis oocytes undergo chromatin decondensation. However, we noted that the vast majority of oocytes with decondensed chromatin extruded only a single PB and contained only one nucleus (88%; n = 102). This observation suggests either decondensation of chromatin occurred after anaphase I, such that oocytes did not form a MetII spindle (, i), or, alternatively, that after establishment of a MetII spindle there was no sister chromatid disjunction before decondensation (, ii).
We determined whether Emi2 MO knockdown oocytes were able to build a MetII bipolar spindle by staining oocytes for both chromatin and tubulin at various times after PB1 extrusion. Oocytes were fixed at 0.5, 1, and 2 h after PB1 extrusion. In control uninjected oocytes, central spindle microtubules were observed at 0.5 h after PB extrusion (), and over the next 1.5 h a MetII spindle formed, such that by 2 h after PB1 a fully formed MetII spindle was found in all oocytes (). In Emi2-MO–injected oocytes, at 0.5 h after PB1 there was no difference from controls, with central spindle microtubules evident between the chromatin in the ooplasm and the PB1 (). However, in contrast to control oocytes, in Emi2-MO–injected oocytes at both the 1 and the 2 h time points we observed no MetII spindle; instead, the chromatin had remained essentially unaltered from the time of PB1 extrusion. The residual central spindle microtubules remained between the chromatin in the oocyte and the PB, and by 2 h the chromatin appeared to be beginning to decondense (). As the chromatin started to decondense, spindle microtubule structure was lost, which is consistent with entry into interphase. By 6 h, all Emi2-MO–injected oocytes had a single nucleus containing fully decondensed chromatin. We did not assess if these oocytes underwent S-phase; however, they showed no obvious signs of degeneration over the 6-h time period from PB1 extrusion. Thus, we failed to observe the formation of a bipolar spindle in any Emi2-MO–treated oocytes; instead, oocytes underwent full chromatin decondensation.
Figure 6. A bipolar MetII spindle does not form in Emi2 MO–injected oocytes. Tubulin (red) and chromatin (blue) staining in either Emi2 MO–microinjected or control oocytes at 0.5 (A), 2 (B), or 6 h (C) after PB1 extrusion. (A) In control and Emi2 (more ...)
Emi2 stabilizes cyclin B1 after PB1 extrusion
Because the formation of a MetII spindle requires an increase in the levels of CDK1 activity after PB1 extrusion, we reasoned that in the absence of Emi2, levels of CDK1's regulatory partner cyclin B1 may not be reestablished. To test this directly, Emi2 MO or control GV oocytes were microinjected with cyclin B1-V cRNA and matured in vitro. Cyclin B1-V levels were monitored in real time during maturation, along with brightfield microscopy to assess progression through meiosis I. As previously reported (Hyslop et al., 2004
), in control oocytes there was a period of cyclin B1 degradation that lasted a few hours and was terminated on PB1 extrusion (). Immediately after PB1 extrusion in these oocytes, cyclin B1 levels rose to 24.0 ± 1.1% (n
= 14) of the peak level before PB1 extrusion (). In Emi2-MO–injected oocytes, there was no difference from controls in the timing of initiation or in the rate of cyclin B1-V degradation during meiosis I, which is in agreement with the observed lack of effect of the MO on the timing of PB1 (). However, in these Emi2 MO oocytes there was no reelevation of cyclin B1 after PB1 extrusion; cyclin B1-V levels increased to just 1.6 ± 0.3% (n
= 15) of the peak level before PB1 extrusion ().
Figure 7. Cyclin B1 levels remain low after PB extrusion in Emi2 MO–matured oocytes. (A) Venus fluorescence levels in maturing oocytes microinjected with cyclin B1-V cRNA (control) or cyclin B1-V cRNA and Emi2 MO. PB1 extrusion was observed at the times (more ...)
In keeping with the real-time effects on exogenous cyclin B1-V expression, we also observed a loss of endogenous cyclin B1 after Emi2 knockdown. In vitro–matured oocytes injected with Emi2 MO at the GV stage showed much lower cyclin B1 levels than control in vitro–matured oocytes (). Furthermore, cyclin B1 levels in individual oocytes were measured by immunofluorescence at 16 h after release from GV arrest in control and Emi2-MO–injected oocytes (). We found that cyclin B1 immunofluorescence levels in Emi2 MO–treated oocytes was about one third that of control oocytes (), suggesting a relatively uniform level of knockdown.
Nondegradable cyclin B1 and Mad2 rescue a bipolar spindle in Emi2 knockdown oocytes
Because the formation of a MetII spindle requires an elevation in the levels of cyclin B1, we reasoned that the Emi2 MO spindle defect may be recovered by the addition of cRNA to nondegradable cyclin B1. Furthermore, it should also be rescued by inhibiting the APCcdc20
by addition of Mad2. Cyclin B1 with deletion of 90 N-terminal amino acids (Δ90 cyclin B1) lacks a D-box and is therefore not a substrate of the APC (Glotzer et al., 1991
; Madgwick et al., 2004
). Emi2-MO–injected oocytes either received no further treatment or were microinjected with 1.5 pg Δ90 cyclin B1 or Mad2 cRNA within 15 min of PB1 extrusion. This injection had to be done immediately after PB1 extrusion because nondegradable cyclin B1 addition to oocytes before PB1, as predicted by the sustained MPF activity, blocks PB extrusion (Herbert et al., 2003
). A few hours of Δ90 cyclin B1 cRNA expression generates cyclin B1 at a similar level to endogenous cyclin B1 in a MetII oocyte (Madgwick et al., 2004
). This dose of Mad2 causes a metaphase I arrest in maturing oocytes (Homer et al., 2005
) and prevents completion of meiosis in MetII oocytes (Madgwick et al., 2005
). At 2 h after PB1 extrusion, all oocytes were fixed, stained, and scored for the presence of a bipolar spindle (). As observed in , oocytes microinjected with Emi2 MO gave decondensing chromatin with a lack of any bipolar spindle structure. However, as with uninjected oocytes, we observed a fully formed bipolar MetII spindle in oocytes microinjected with Emi2 MO and either Δ90 cyclin B1 cRNA or Mad2 ().
Figure 8. Emi2 MO phenotype recovery using Δ90 cyclin B1 or Mad2. (A) Percentage maturation rates in in vitro–matured oocytes after no treatment (n = 12), or microinjection of Emi2 MO at the GV stage with or without further microinjection (more ...)