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Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis

Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis JCB: ARTICLE Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis 1 2,3 1 2,3 1 Suzanne Madgwick, David V. Hansen, Mark Levasseur, Peter K. Jackson, and Keith T. Jones Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle, Newcastle NE2 4HH, England, UK 2 3 Department of Pathology and Program in Cancer Biology, Stanford University School of Medicine, Stanford, CA 94305 uring interkinesis, a metaphase II (MetII) spindle Emi2 antisense morpholino knockdown during oo- is built immediately after the completion of mei- cyte maturation did not affect polar body (PB) extrusion. D osis I. Oocytes then remain MetII arrested until However, in interkinesis the central spindle microtubules fertilization. In mouse, we fi nd that early mitotic inhibi- from meiosis I persisted for a short time, and a MetII spindle tor 2 (Emi2), which is an anaphase-promoting complex failed to assemble. The chromatin in the oocyte quickly de- inhibitor, is involved in both the establishment and the condensed and a nucleus formed. All of these effects were maintenance of MetII arrest. In MetII oocytes, Emi2 needs caused by the essential role of Emi2 in stabilizing cyclin to be degraded for oocytes to exit meiosis, and such deg- B1 after the fi rst PB extrusion because in Emi2 knockdown radation, as visualized by fl uorescent protein tagging, oocytes a MetII spindle was recovered by Emi2 rescue or occurred tens of minutes ahead of cyclin B1. by expression of nondegradable cyclin B1 after meiosis I. Introduction Oocytes arrest at metaphase of the second meiotic division both M-phase (maturation)–promoting factor (MPF) activity (MetII) before fertilization because of an activity termed cyto- (CDK1/cyclin B1) and cohesin, which holds sister chromatids static factor (CSF; Masui, 2000; Tunquist and Maller, 2003; together (Nixon et al., 2002; Madgwick et al., 2004). Resumption 2+ Jones, 2005). Sperm break this arrest via a Ca signal (Jones, of meiosis in mammalian oocytes is achieved by a sperm-borne 1998; Stricker, 1999; Runft et al., 2002), and in so doing, oo- phospholipase C activity (Saunders et al., 2002; Knott et al., 2+ cytes complete the second meiotic division before entering the 2005), which generates an oscillatory Ca signal, switching on cdc20 embryonic cell cycles. APC (Nixon et al., 2002; Madgwick et al., 2004) through CSF activity, a terminology that was fi rst defi ned several a signaling pathway involving calmodulin-dependent protein decades ago (Masui and Market, 1971), is now known to consti- kinase II (CamKII; Markoulaki et al., 2003, 2004; Madgwick tute an inhibitor of the anaphase-promoting complex/cyclosome et al., 2005). This signaling process is conserved and was fi rst (APC; Tunquist and Maller, 2003). The APC is an E3 ubiquitin demonstrated in frog eggs (Lorca et al., 1993, 1994). Activation ligase whose activity is required for the metaphase–anaphase of the APC in MetII oocytes induces the destruction of MPF transition to polyubiquitinate key cell cycle proteins, thereby and sister chromatid cohesion through the polyubiquitination of earmarking them for immediate proteolysis through associa- cyclin B1 and securin, respectively (Morgan, 1999; Zachariae tion with its key coactivator cdc20 (Fang et al., 1998; Kramer and Nasmyth, 1999; Peters, 2002). Loss of cyclin B1 causes et al., 1998; Harper et al., 2002; Eytan et al., 2006). The reduced a reduction in MPF, and the loss of securin frees separase cdc20 APC activity in MetII oocytes prevents the destruction of to act on the kleisin component of cohesin (Zachariae and Nasmyth, 1999; Peters, 2002; Blow and Tanaka, 2005; Nasmyth and Haering, 2005). Correspondence to Keith T. Jones: [email protected]; or Suzanne Madgwick: Many proteins have been associated with the establishment [email protected] D.V. Hansen and P.K. Jackson’s current address is the Department of Tumor and/or maintenance of CSF activity. Factors responsible for Biology and Angiogenesis, Genentech, Inc., South San Francisco, CA 94080. setting up a second meiotic spindle after completion of meiosis I Abbreviations used in this paper: APC, anaphase-promoting complex/ do not, a priori, have to be the same as those that are responsible cyclosome; Bub, budding uninhibited by benzimidazole; CSF, cytostatic factor; Emi, early mitotic inhibitor protein; GV, germinal vesicle; GVBD, GV break- for maintaining arrest. Indeed, proteins have been described down; Mad, mitotic arrest defi cient; MISS, MAPK-interacting and spindle- that are involved in establishing MetII, but not in maintaining stabilizing protein; MO, morpholino; MPF, M-phase (maturation)–promoting arrest once it has been achieved (Tunquist and Maller, 2003). factor; PB, polar body. © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 174, No. 6, September 11, 2006 791–801 http://www.jcb.org/cgi/doi/10.1083/jcb.200604140 JCB 791 THE JOURNAL OF CELL BIOLOGY The mechanism of CSF is most well characterized in the frog, As there are no fi rm candidates suggested to be involved where various groups have fi rmly defi ned the c-Mos–MAPK– in the establishment of CSF arrest in mouse, we have investi- 90-kD ribosomal protein S6 kinase (p90rsk)–budding uninhib- gated the potential of mouse Emi2 in this process. We fi nd that ited by benzimidazole 1 (Bub1) pathway in establishing CSF Emi2 has activity consistent with CSF, and its degradation in activity (Sagata et al., 1989; Abrieu et al., 1996; Bhatt and real time is ahead of any change in cyclin B1. We also demon- Ferrell, 1999; Gross et al., 1999, 2000; Tunquist et al., 2002). strate that a function of Emi2 is in restabilizing cyclin B1 upon Other activities that are fundamentally involved in the estab- exit from anaphase I and that, in this study, it contributes to the lishment of CSF in frog include cyclin E/Cdk2 (Tunquist et al., formation a bipolar spindle. Oocytes matured without Emi2 do 2002) and mitotic arrest defi cient 2 (Mad2; Tunquist et al., not assemble MetII spindles, and, in the absence of cyclin B1, 2003). However, once established, p90rsk, Mad2, Bub1, and eventually decondense their chromatin. cyclin E/Cdk2 are all dispensable for the maintenance of CSF activity (Bhatt and Ferrell, 1999; Tunquist et al., 2002, 2003). Results So, how is CSF activity maintained in the frog? Current 2+ evidence suggests it is through early mitotic inhibitor 2 (Emi2)/ Stable Emi2 is rapidly degraded by Ca Emi-related protein 1 (Liu and Maller, 2005; Rauh et al., 2005; To measure real-time changes in Emi2 levels in oocytes we gen- Schmidt et al., 2005; Tung et al., 2005; Hansen et al., 2006). Emi2, erated cRNA to mouse Emi2 coupled to Venus fl uorescent pro- which acts to inhibit the APC accumulated during oocyte matura- tein (Emi2-V), which is a yellow variant of GFP. MetII mouse tion, is present and stable in CSF frog egg extracts, but is rapidly oocytes were microinjected with this construct at a dose of ei- 2+ degraded on Ca addition (Schmidt et al., 2005). Degradation of ther 0.15 or 0.5 pg, and then cultured for a few hours to allow Emi2 is induced by phosphorylation through CamKII (Liu and for Emi2 expression. We Western blotted oocytes expressing Maller, 2005; Rauh et al., 2005; Hansen et al., 2006) and, thus, Emi2-V with a polyclonal antibody against Emi2 to determine would be predicted to occur ahead of APC activation and cyclin the amount of Emi2-V expression relative to endogenous protein. B1 degradation, although this has not been tested. More than one band was detected on oocyte blots using this In mouse oocytes, the mechanism of CSF arrest is less antibody; ho wever, one band at 85 kD migrated at the same well understood. As the mouse Emi2 homologue appears to molecular mass as in vitro–translated Emi2 (and this band was have a similar function in maintaining CSF activity (Shoji et al., later knocked down by Emi2 morpholino [MO]). At the 0.15-pg 2006), it would be logical to predict that the mechanism of dose, Emi2-V levels were less than endogenous protein after CSF establishment is also conserved between frog and mouse. 2 h, whereas the 0.5-pg dose was expressed to about the same Ho wever, this is not so. Oocytes from a triple Rsk (1,2,3) knockout level as endogenous protein (Fig. 1 A). mouse arrest normally at MetII (Dumont et al., 2005), demon- Emi2-V was very stable in MetII-arrested oocytes, but 2+ strating that p90Rsk is not involved in mouse CSF arrest. became rapidly unstable when cytosolic Ca increased. With Furthermore, using dominant-negative mutants Tsurumi et al., either 0.15 pg (Fig. 1 B) or 0.5 pg (not depicted) injections, we (2004) confi rmed that the spindle checkpoint proteins Bub1 and observed no degradation of Emi2-V in MetII oocytes after block- Mad2 are not required for either the establishment or the main- ing further synthesis by washing into cycloheximide- containing tenance of mouse CSF. Although c-Mos is known to be involved media. However, when this experiment was repeated, but by 2+ 2+ in CSF maintenance, it does not appear to be involved in the washing into Sr -containing media to induce spermlike Ca establishment of CSF arrest because c-Mos −/− oocytes remain spiking (Bos-Mikich et al., 1997), we observed dose- dependent at MetII for 2–4 h before extruding a second PB (PB2; Verlhac effects on Emi2-V. At the higher 0.5-pg dose, we observed no et al., 1996). loss in Emi2-V signal; instead, Emi2-V levels steadily increased Figure 1. Emi2-V is stable in MetII oocytes, 2+ but is rapidly degraded by Ca . (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. *, nonspecifi c band. (B) Emi2-V (0.15 pg cRNA) expression levels on addition of cycloheximide (CHX; n = 12). (C and D) Emi2-V (C, 0.5 pg cRNA; D, 0.15 pg cRNA) 2+ expression levels after washing into Sr media at the times indicated (C, n = 14; D, n = 15). (E) Cyclin B1-V (0.09 pg cRNA) 2+ expression levels after washing into Sr media at the times indicated (n = 15). T and T repre- E C sent the time from complete degradation of the Venus fl uorochrome (T , Emi2; T , cyclin B1) to E C PB2 extrusion in oocytes. PB2 was observed at the times indicated. A is representative of two independent Western blots. (B–E) are represen- tative traces of n oocytes that have been used from two to four independent experiments. 792 JCB • VOLUME 174 • NUMBER 6 • 2006 (Fig. 1 C). However, at the lower 0.15-pg dose Emi2-V was rap- confi rm the immediate loss of endogenous Emi2 signal in idly degraded (Fig. 1 D). oocytes, which is especially important given that Shoji et al. (2006) The high dose of Emi2 maintained oocytes in a MetII had reported very little loss in Emi2 signal at a 6-h time point 2+ arrest. Oocytes injected with 0.5 pg Emi2-V cRNA showed no after activation with Sr . Oocytes were activated by washing 2+ morphological signs of meiotic resumption, which is consistent into Sr media, and samples from a pool of oocytes were removed with the maintained Emi2-V levels in these oocytes (Fig. 1 C). at various time points and probed for either cyclin B1 or Emi2 In contrast, oocytes that had been injected with 0.15 pg Emi2-V by Western blotting (Fig. 2 A). In these experiments, it was cRNA extruded a PB2 (Fig. 1 D). This suggests that Emi2-V apparent that loss in Emi2 protein w as rapid and complete by has physiological CSF activity and that the oocyte has a fi nite 30 min (in agreement with the rapid loss of Emi-V shown in capacity to degrade Emi2. Fig. 1 D). Interestingly, Emi2 levels increased again in pronu- cleate embryos (6-h time point), which is in agreement with Emi2 is degraded ahead of cyclin B1 Shoji et al. (2006) and suggests that it may have a further mitotic At the lower dose Emi2 injection, where we observed Emi2-V function (see Discussion). Similar to Emi2, we observed the loss degradation, the minimum in its degradation profi le after activa- of cyclin B1 at 30 min, as well as increased levels in pronucleate 2+ tion with Sr media was reached tens of minutes before PB2 embryos (Fig. 2 A), which is consistent with our observation that extrusion (Fig. 1 D, T ). However, we have previously reported the APC is switched off at this time (Nixon et al., 2002). that cyclin B1 degradation, visualized by coupling to GFP, is Because of the numbers of oocytes needed for Western only completed at the time of PB2 formation (Hyslop et al., blots and the tens of minutes of asynchrony in timing at which 2+ 2+ 2004). This suggests that the Emi2 and cyclin B1 degradation Ca spiking starts with Sr media, (Madgwick et al., 2004), profi les may not fully overlap. When cyclin B1 was expressed we could not reproducibly resolve Emi2 degradation ahead of in mouse oocytes with the same fl uorescent protein tag as Emi2 cyclin B1 by Western blotting groups of oocytes. Therefore, to (cyclin B1-Venus; cyclin B1-V), we observed the same degra- examine with more accuracy the immediate degradation pro- dation profi le as that previously found for cyclin B1-GFP, such fi les of cyclin B1 and Emi2, we decided to measure their simul- that a minimum in the cyclin B1-V profi le was reached within taneous degradation in the same oocyte. Cyclin B1 was coupled minutes of PB2 extrusion (Fig. 1 E, T ). to Cerulean fl uorescent protein (cyclin B1-C), which is a cyan Comparing the degradation profi les of Emi2-V and cyclin variant of GFP. There was no overlap in Venus and Cerulean B1-V suggests that Emi2 degradation is initiated ahead of cyclin signals, showing that both Emi2-V and cyclin B1-C, with ap- 2+ B1. Sr -induced Emi2 degradation begins immediately (Fig. 1 D), propriate fi lters, could be imaged simultaneously in the same whereas that of cyclin B1 begins 20 min later (Fig. 1 E). This oocyte (Fig. 2 B). In these experiments, it was evident that the would have to occur if MetII arrest is being mediated by Emi2- introduction of Emi2-V delayed cyclin B1 degradation (Fig. 1 E cdc20 induced inhibition of APC activity. Therefore, we wanted to and Fig. 2 C), which is consistent with Emi2-inhibiting APC Figure 2. Emi2 is degraded ahead of cyclin B1. (A) West- ern 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 bril- liant blue (CB) to show equivalent loading. Oocytes are 2+ activated by washing into Sr media for the times indi- cated. (B) Epifl uorescence images of MetII oocytes micro- injected with Emi2-V cRNA or cyclin B1-C cRNA, at the excitation wavelengths of Cerulean (430 nm) and Venus (500 nm). There is negligible overlap in signal between the two fl uorochromes, demonstrating that Emi2-V and cy- clin B1-C may be imaged in the same oocyte. Bar, 20 μm. (C) Simultaneous Venus (yellow) and Cerulean (blue) fl uo- rescence in a MetII oocyte expressing Emi2-V and cyclin 2+ B1-C, and washed into Sr media at time t = 0 min. Emi2-V degradation begins tens of minutes before that of cyclin B1. (A) The Western blot was repeated once with similar timings. C is representative of seven oocytes from two independent experiments. EMI2 AND MEIOSIS II ENTRY • MADGWICK ET AL. 793 Table I. Emi2 MOs used in this study MO Sequence Mouse Emi2 A GCC AGC CAC AGA GCA GGA AGC AAT ATG GAC TCC TCT Emi2 MO ATT GCT TCC TGC TCT GTG GCT GGC T 5mp MO ATT CCT TGC TGC TGT GTG CCT GCC T Inv MO TCG GTC GGT GTC TCG TCC TTC GTT A Emi2 MO was designed to target the 5′UTR of mouse Emi2 (underlined). A 5mp MO and an Inv MO were used as control MOs. 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 matura- tion makes it highly likely that Emi2 expression can be knocked out by an antisense approach. Therefore, we designed an anti- sense 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 (Table I); a 5-base mispair MO (5mp-MO), in which fi ve 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) Figure 3. Emi2-V degradation is independent of a spindle checkpoint. oocytes, which were then matured in vitro. Oocytes were held at Venus fl uorescence levels in a MetII oocyte expressing cRNA to cyclin B1-V 2+ the GV stage in milrinone-containing media for 2 h after MO (A; n = 12) or Emi2-V (B; n = 15) after the addition of Sr media contain- ing 100 ng/ml nocodazole at time t = 0 h. Both A and B are represent- injection; they were then released from GV arrest and allowed ative of oocytes collected in two independent experiments. to mature for 16 h. Blotting of GV-stage oocytes, Emi2 MO– matured oocytes, and uninjected control matured oocytes dem- activity. However, these coexpression studies revealed that onstrated that Emi2 protein levels increase between GV and Emi2 degradation began tens of minutes before that of cyclin MetII stage, and confi rmed the Emi2 knockdown in Emi2 MO– B1 (Fig. 2 C). Emi2-V levels were degraded by at least 50% injected oocytes (Fig. 4 A). before the start of cyclin B1-C degradation (n = 7). During maturation, oocytes were scored for the morpho- logical events of oocyte maturation, which are GV breakdown Emi2 degradation is independent (GVBD) and PB1 extrusion (Fig. 4 B). Both GVBD (Fig. 4 C) of a spindle checkpoint and PB1 extrusion (Fig. 4 D) occurred with normal timings cdc20 Degradation of cyclin B1 is dependent on APC activity, and in Emi2 MO–injected oocytes. However, after maturation, we in mouse oocytes it can be blocked by the induction of a spindle observed marked differences in the morphology of control checkpoint (Nixon et al., 2002; Madgwick et al., 2005). In con- oocytes and those injected with Emi2 MO. When control trast, Emi2 degradation should be checkpoint-independent be- oocytes (uninjected, 5mp-MO, and Inv-MO–injected) were cdc20 cause its degradation is independent of APC involvement. stained for chromatin, oocytes were morphologically normal. Incubation of Emi2- and cyclin B1-expressing oocytes with the They had a PB1 containing chromatin, which was produced spindle poison nocodazole blocks mouse oocytes from exiting on completion of the fi rst meiotic division, and a fully formed 2+ MetII arrest when washed into Sr media. As expected, the ad- MetII spindle (100%; uninjected oocytes, n = 60; 5mp-MO, dition of nocodazole completely stabilized cyclin B1 levels n = 32; Inv-MO, n = 25; Fig. 5 A). However, although oocytes (n = 15/15; Fig. 3 A). However, nocodazole had no effect on the injected with Emi2 MO did have a PB1 containing chromatin, rate of Emi2-V degradation (n = 12/12; Fig. 3 B). Such an ob- the chromatin in the oocyte was decondensed inside a nucleus servation is consistent with cyclin B1, but not Emi2 degrada- (93%; n = 110; Fig. 5 A). tion, being dependent on the APC. Despite the lack of effect of a 5mp-MO, it remained Therefore, in summary, we have obtained data that are en- possible that we had been extremely unlucky in the MO design, tirely consistent with a model of MetII arrest achieved by Emi2- such that the observed effects of Emi2 MO were caused by its mediated inhibition of cyclin B1 degradation. Also, the Emi2-V ability to block the expression of an unrelated protein involved construct generated is a physiologically active, useful tool for in MetII arrest. We thought this unlikely, given that a similar both establishing CSF activity and measuring its loss in real morphology of Emi2 knockdown oocytes has been reported re- 2+ time after a Ca signal. cently using a double-stranded RNAi approach (Shoji et al., 2006). 794 JCB • VOLUME 174 • NUMBER 6 • 2006 Figure 4. GVBD and PB1 extrusion occur nor- mally in Emi2 MO–injected oocytes. (A) West- ern blot of GV-stage, Emi2 MO– matured, and control matured oocytes; 75 oocytes were loaded per lane. *, nonspecifi c band. (B) Brightfi eld time lapse of an oocyte microin- jected with Emi2 MO and allowed to mature. GVBD and PB1 are marked. Bar, 20 μm. The percentage rate of GVBD (C) and PB1 extru- sion (D) at the times indicated for control oo- cytes (n = 32) and oocytes microinjected with Emi2 MO (n = 33). The Western blot in A is representative of two independent experi- ments. The data in C and D was pooled from two independent experiments. However, we decided it was important to confi rm the specifi city We determined whether Emi2 MO knockdown oocytes by a rescue to the control phenotype in Emi2-MO–injected were able to build a MetII bipolar spindle by staining oocytes oocytes by expression of exogenous Emi2. To recover Emi2, for both chromatin and tubulin at various times after PB1 extrusion. Emi2-V cRNA was microinjected into oocytes 2 h after micro- Oocytes were fi xed at 0.5, 1, and 2 h after PB1 extrusion. In injection of Emi2 MO and immediately before release from GV control uninjected oocytes, central spindle microtubules were arrest. This rescue is made possible because Emi2-V lacks the observed at 0.5 h after PB extrusion (Fig. 6 A), and over the next 5′UTR recognized by the MO. 1.5 h a MetII spindle formed, such that by 2 h after PB1 a fully Injection of Emi2-V cRNA alone into GV oocytes that formed MetII spindle was found in all oocytes (Fig. 6 B). In were matured induced a MetI arrest (n = 40; Fig. 5 A), which is Emi2-MO–injected oocytes, at 0.5 h after PB1 there was no dif- consistent with Emi2-V having CSF activity and the need to ference from controls, with central spindle microtubules evident keep Emi2 levels low until completion of the fi rst meiotic between the chromatin in the ooplasm and the PB1 (Fig. 6 A). division. Importantly, Emi2-V cRNA expression could rescue However, in contrast to control oocytes, in Emi2-MO–injected the effects of MO knockdown. Rescue oocytes could progress oocytes at both the 1 and the 2 h time points we observed no through meiosis I and arrest as controls with a fully formed MetII spindle; instead, the chromatin had remained essentially MetII spindle. A high dose of Emi2-V (0.5 pg; n = 45) rescued unaltered from the time of PB1 extrusion. The residual central the Emi2 MO phenotype, with an equal mix of either MetI or spindle microtubules remained between the chromatin in the MetII arrest, whereas oocytes microinjected with the lower oocyte and the PB, and by 2 h the chromatin appeared to be Emi2 dose showed a rescue with a predominantly MetII arrest beginning to decondense (Fig. 6 B). As the chromatin started (n = 45; Fig. 5 A). to decondense, spindle microtubule structure was lost, which is consistent with entry into interphase. By 6 h, all Emi2-MO– Lack of Emi2 results in a failure injected oocytes had a single nucleus containing fully decon- to assemble a metaphase II spindle densed chromatin. We did not assess if these oocytes underwent By scoring oocytes for decondensed chromatin at only one S-phase; however, they showed no obvious signs of degenera- time point (16 h), we were unable to pinpoint at which stage of tion over the 6-h time period from PB1 extrusion. Thus, we meiosis oocytes undergo chromatin decondensation. However, failed to observe the formation of a bipolar spindle in any Emi2- we noted that the vast majority of oocytes with decondensed MO–treated oocytes; instead, oocytes underwent full chromatin chromatin extruded only a single PB and contained only one decondensation. nucleus (88%; n = 102). This observation suggests either de- condensation of chromatin occurred after anaphase I, such Emi2 stabilizes cyclin B1 after that oocytes did not form a MetII spindle (Fig. 5 B, i), or, PB1 extrusion alternatively, that after establishment of a MetII spindle there Because the formation of a MetII spindle requires an increase in was no sister chromatid disjunction before decondensation the levels of CDK1 activity after PB1 extrusion, we reasoned (Fig. 5 B, ii). that in the absence of Emi2, levels of CDK1’s regulatory partner EMI2 AND MEIOSIS II ENTRY • MADGWICK ET AL. 795 Figure 5. Decondensed chromatin in Emi2 MO–injected matured oocytes. (A) Percentage of maturation rates in in vitro–matured oocytes after micro- injection at the GV stage of MOs and/or Emi2-V cRNA, as indicated. Figure 6. A bipolar MetII spindle does not form in Emi2 MO–injected Oocyte maturation was assessed at 16 h after release from GV arrest. oocytes. Tubulin (red) and chromatin (blue) staining in either Emi2 MO– Oocytes were stained with Hoechst and scored as either arrested at MetI microinjected or control oocytes at 0.5 (A), 2 (B), or 6 h (C) after PB1 or MetII or as having decondensed chromatin. (B) Emi2 MO oocytes typi- extrusion. (A) In control and Emi2 MO–injected oocytes, a central spindle cally extruded a PB1 and contained only one nucleus (88%; n = 102). This forms between segregated homologues at 0.5 h. (B) By 2 h after PB1 extru- single nucleus must result through chromatin decondensation either imme- sion, all control oocytes had a bipolar MetII spindle; however, Emi2 MO diately after PB1 extrusion (i) or after the formation of a MetII spindle that oocytes showed only microtubules that remained from the persistence of fails to segregate its sister chromatids (ii). The data in A was pooled from the central spindle. (C) By 6 h after PB1, chromatin in Emi2 MO–injected 12 independent experiments. See Oocytes with Emi2 knockdown extrude oocytes had completely decondensed inside a nucleus (N). Bar, 20 μm. a PB, but do not MetII arrest. Each image is representative of at least 10 oocytes. A–C are representa- tive of two, three, and seven independent experiments, respectively. cyclin B1 may not be reestablished. To test this directly, Emi2 MO or control GV oocytes were microinjected with cyclin with Emi2 MO at the GV stage showed much lower cyclin B1-V cRNA and matured in vitro. Cyclin B1-V levels were B1 levels than control in vitro–matured oocytes (Fig. 7 C). monitored in real time during maturation, along with brightfi eld Further more, cyclin B1 levels in individual oocytes were mea- microscopy to assess progression through meiosis I. As previ- sured by immunofl uorescence at 16 h after release from GV ously reported (Hyslop et al., 2004), in control oocytes there arrest in control and Emi2-MO–injected oocytes (Fig. 7, D and E). was a period of cyclin B1 degradation that lasted a few hours We found that cyclin B1 immunofl uorescence levels in Emi2 and was terminated on PB1 extrusion (Fig. 7 A). Immediately MO–treated oocytes was about one third that of control oocytes after PB1 extrusion in these oocytes, cyclin B1 levels rose to (Fig. 7 E), suggesting a relatively uniform level of knockdown. 24.0 ± 1.1% (n = 14) of the peak level before PB1 extrusion (Fig. 7 B). In Emi2-MO–injected oocytes, there was no differ- Nondegradable cyclin B1 and Mad2 ence from controls in the timing of initiation or in the rate of rescue a bipolar spindle in Emi2 cyclin B1-V degradation during meiosis I, which is in agreement knockdown oocytes with the observed lack of effect of the MO on the timing of PB1 Because the formation of a MetII spindle requires an elevation (Fig. 4 D). However, in these Emi2 MO oocytes there was no in the levels of cyclin B1, we reasoned that the Emi2 MO spin- reelevation of cyclin B1 after PB1 extrusion; cyclin B1-V levels dle defect may be recovered by the addition of cRNA to non- increased to just 1.6 ± 0.3% (n = 15) of the peak level before degradable cyclin B1. Furthermore, it should also be rescued cdc20 PB1 extrusion (Fig. 7 B). by inhibiting the APC by addition of Mad2. Cyclin B1 with In keeping with the real-time effects on exogenous cyclin deletion of 90 N-terminal amino acids (∆90 cyclin B1) lacks a B1-V expression, we also observed a loss of endogenous cyclin D-box and is therefore not a substrate of the APC (Glotzer et al., B1 after Emi2 knockdown. In vitro–matured oocytes injected 1991; Madgwick et al., 2004). Emi2-MO–injected oocytes 796 JCB • VOLUME 174 • NUMBER 6 • 2006 Figure 7. Cyclin B1 levels remain low after PB extrusion in Emi2 MO–matured oocytes. (A) Venus fl uorescence 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 indicated. Note the similar profi le of cyclin B1-V degradation in both oocytes, but the fail- ure of cyclin B1-V levels to rise in the oocyte injected with Emi2 MO after PB1. (B) Quan- tifi cation of the elevation in cyclin B1-V lev- els after PB1 extrusion in the control oocytes (n = 14) and Emi2 MO oocytes (n = 15) used in A. Endogenous cyclin B1 levels in matured oocytes detected by Western blotting (C) or immunofl uorescence (D and E). (C) A West- ern blot (WB) for cyclin B1 in control matured oocytes and oocytes matured after microinjec- tion of Emi2 MO at the GV stage (n = 40), and the corresponding membrane stained with Coomassie brilliant blue (CB) to demonstrate equivalent loading. In D, representative im- ages of cyclin B1 levels in matured oocytes (control) and in matured oocytes microinjected at the GV stage with Emi2 MO detected using a TRITC-conjugated secondary antibody. Mean fl uorescence readings are marked for both oocytes on the 8-bit scale bar. (E) Quantifi ca- tion of cyclin B1 immunofl uorescence levels in control (n = 25) and Emi2 MO–microinjected (n = 32) oocytes from D, relative to uninjected controls. A is representative of four indepen- dent experiments whose data is pooled in B. (C) The Western blot was repeated once. D is representative of two independent experiments whose data is pooled in E. either received no further treatment or were microinjected with presence of a bipolar spindle (Fig. 8 A). As observed in Fig. 6 B, 1.5 pg ∆90 cyclin B1 or Mad2 cRNA within 15 min of PB1 oocytes microinjected with Emi2 MO gave decondensing chro- extrusion. This injection had to be done immediately after PB1 matin with a lack of any bipolar spindle structure. However, extrusion because nondegradable cyclin B1 addition to oocytes as with uninjected oocytes, we observed a fully formed bipolar before PB1, as predicted by the sustained MPF activity, blocks MetII spindle in oocytes microinjected with Emi2 MO and PB extrusion (Herbert et al., 2003). A few hours of ∆90 cyclin either ∆90 cyclin B1 cRNA or Mad2 (Fig. 8 B). B1 cRNA expression generates cyclin B1 at a similar level to endogenous cyclin B1 in a MetII oocyte (Madgwick et al., Discussion 2004). This dose of Mad2 causes a metaphase I arrest in matur- ing oocytes (Homer et al., 2005) and prevents completion of We have demonstrated in live oocytes and in real time that upon 2+ meiosis in MetII oocytes (Madgwick et al., 2005). At 2 h after a Ca signal, Emi2 degradation occurs ahead of cyclin B1, PB1 extrusion, all oocytes were fi xed, stained, and scored for the independent of APC inhibition. The current study confi rmed Figure 8. Emi2 MO phenotype recover y using 𝚫 90 cyclin B1 or Mad2. (A) Percentage maturation rates in in vitro– matured oocytes after no treatment (n = 12), or microin- jection of Emi2 MO at the GV stage with or without further microinjection of ∆90 cyclin B1 or Mad2 after PB1 extru- sion (Emi2 MO only, n = 20; with ∆90 cyclin B1, n = 25; or with Mad2, n = 35). Oocytes were fi xed and stained 2 h after PB1 extrusion and scored for a bipolar spindle. (B) Tubulin (red) and chromatin (blue) staining in an oocyte microinjected with Emi2 MO at the GV stage and then ∆90 cyclin B1 cRNA 15 min after PB1 extrusion. Image is representative of 20/25 oocytes. Bar, 20 μm. Data is pooled from two independent experiments. EMI2 AND MEIOSIS II ENTRY • MADGWICK ET AL. 797 β-TRCP β-TRCP that Emi2 constitutes CSF activity in mouse MetII oocytes and known SCF (Skp1-Cullin-F-box ) motif (DSGX S) is responsible for the maintenance of MetII arrest (Shoji et al., and not the APC (Rauh et al., 2005; Tung et al., 2005). 2006). More importantly, we fi nd that Emi2 is necessary and suffi cient in stabilizing cyclin B1 levels at the completion of Establishment of MetII arrest meiosis I, and such stabilization is essential in establishing a We used a MO antisense oligonucleotide to ablate the Emi2 rise bipolar spindle. during mouse oocyte maturation and confi rmed a knockdown By defi nition, CSF activity must accumulate during oo- by Western blotting. By blocking the accumulation of Emi2, cdc20 cyte maturation, inhibit APC activity at MetII, and be inac- we have demonstrated that it is essential for stabilizing cyclin 2+ tivated on a Ca signal at fertilization. Although only recently B1 after MetI during interkinesis, and therefore essential for the identifi ed, the F-box protein Emi2 is very likely to constitute formation of a MetII spindle. Because of the known inhibitory CSF in frog eggs (Liu and Maller, 2005; Rauh et al., 2005; action of Emi2 on APC activity, we interpret this observation to Schmidt et al., 2005; Tung et al., 2005; Hansen et al., 2006). mean that in the absence of Emi2, the APC remains active, and cdc20 Emi2 inhibits the ubiquitin ligase activity of the APC thus cyclin B1 is continuously degraded. In agreement with this in vitro, and in keeping with the nature of CSF, it accumulates is our observation that nondegradable cyclin B1, but not full- during oocyte maturation, is present in CSF extracts, and is length cyclin B1, rescues a MetII arrest after Emi2 knockdown 2+ cdc20 rapidly degraded by Ca (Schmidt et al., 2005). (Fig. 7 vs. Fig. 8), as does the APC inhibitor Mad2. The cur- rent data have not ruled out some effect of Emi2 on cyclin B1 Maintenance of MetII arrest synthesis. However, if so, Emi2 would also be able to negatively We tested the characteristics of the mouse Emi2 homologue in regulate expression of the microinjected cyclin B1 cRNA (Fig. 7). MetII mouse oocytes. This achieved two goals. First, it allowed This lacks the cyclin B1 gene 3′UTR, which is important for us to demonstrate that Venus-tagged Emi2 is physiologically regulating translation through polyadenylation (Tay et al., 2000). active, and second, it provided us with data relating to the tim- Therefore, we would argue that Emi2 is most likely affecting the ing of Emi2 degradation on activation. Excess X. laevis Emi2 rate of cyclin B1 degradation, rather than synthesis. 2+ protein prevents Ca -induced CSF release in egg extracts The observed Emi2 MO phenotype, PB1 extrusion and (Schmidt et al., 2005); similarly, our MetII-arrested mouse oo- decondensed chromatin within a single pronucleus, was not 2+ cytes were refractory to Ca through modest rises in Emi2-V. caused by a nonspecifi c effect of the MO for two reasons. First, A lower Emi2-V dose, less than endogenous protein, did not we used two control MOs that were without effect. Second, we block escape from MetII arrest and allowed us to demonstrate rescued the Emi2 knockdown by readdition of Emi2 cRNA, that Emi2 is degraded ahead of cyclin B1. If Emi2 inhibits the an approach that was made possible by the fact that the res- APC in MetII arrest, it follows that Emi2 destruction must oc- cue cRNA did not contain the 5′UTR recognized by the MO. cur before the APC’s accelerated degradation of cyclin B1. In some instances, especially where rescue was done with the I ndeed, when both were simultaneously imaged in an oocyte higher dose of Emi2, oocytes went on to arrest at MetI. This 2+ after a Ca rise, Emi2 was degraded by at least 50% before would be consistent with Emi2 inhibiting the APC at this cyclin B1. An anti-Emi2 antibody also revealed Emi2 protein time and the requirement for APC activity in order for mouse in pronucleate embryos, which is in agreement with the obser- oocytes to complete the fi rst meiotic division (Herbert et al., vations of Shoji et al. (2006), demonstrating that Emi2 reaccu- 2003; Terret et al., 2003; Hyslop et al., 2004; Homer et al., mulates in one-cell embryos and, thus, interestingly, may have 2005). Interestingly, the effect of Emi2 knockdown on oocyte 2+ a mitotic function. We have previously reported on Ca spiking maturation is the same as that obtained when expression of during the fi rst mitotic division of mouse embryos (Kono et al., cyclin B1 is prevented by antisense during oocyte maturation 1996), suggesting that in this study Emi2 may be degraded by (Ledan et al., 2001). In oocytes matured with cyclin B1 anti- 2+ a similar Ca -dependent process. sense, MPF activity is not reestablished after PB1 extrusion, In MetII-arrested mouse oocytes, the APC is already ac- and a single pronucleus forms. 2+ tive, but the addition of a Ca signal speeds up cyclin B1 deg- The Emi2 phenotype observed on in vitro–matured oocytes radation sixfold through an increase in APC activity (Nixon to some extent resembles the siRNA Emi2 knockdown pheno- et al., 2002). In mouse, the high levels of cyclin B1 required for type obtained previously (Shoji et al., 2006), with decondensed MetII arrest are therefore achieved through equilibrium between chromatin in the parthenote. In that study, however, there was synthesis and degradation (Kubiak et al., 1993). Induction of a no analysis made of the spindle during the normal time course spindle-assembly checkpoint by nocodazole prevents cyclin B1 of maturation; instead, observations were made some 30 h after 2+ degradation, with or without the presence of a Ca signal microinjection. Therefore, in such circumstances it was not pos- (Nixon et al., 2002), showing that in mouse oocytes APC activ- sible to judge how the oocyte was progressing through meiosis I, ity can be held in check by both CSF and a spindle-assembly for example if a MetII spindle formed and remained stable for checkpoint. In mouse, the spindle-assembly checkpoint is dis- some hours before extruding a PB and exiting meiosis II. Also, tinct from CSF (Tsurumi et al., 2004). In contrast to cyclin B1, the diffi culty in interpretation at this single time point was exac- we found little evidence of Emi2 protein turnover in MetII- erbated in this strain of mouse by the degeneration of the PB1 arrested ooc ytes, suggesting that Emi2 itself is not a substrate of during the 30-h period. 2+ the APC. Indeed, upon a Ca signal, destruction of X. laevis The present experiments were designed to examine in Emi2 has been shown to occur via the phosphorylation of a detail the role of Emi2 during the normal timings of meiosis I. 798 JCB • VOLUME 174 • NUMBER 6 • 2006 It was found that Emi2 was essential to restabilize cyclin B1 Previously, all CSF candidates demonstrated in frog ap- during interkinesis, and this rise in cyclin B1 was necessary to peared to play either no role in CSF activity in mouse or are build a bipolar spindle. This is a previously unreported role for involved in only the maintenance of arrest once a MetII spin- Emi2 in either frog or mouse and may add clarity to the function dle is established. In this study, we have demonstrated that in of CSF in mouse. Given that species-specifi c differences appear the absence of Emi2, mouse oocytes fail to form a bipolar to exist, it is unclear whether this role in establishing MetII MetII spindle and, thus, fail to establish MetII arrest because arrest would be conserved in frog. In frog, several studies have of an inability to elevate cyclin B1 levels in interkinesis. elegantly identifi ed the c-Mos–MAPK–p90rsk–Bub1 pathway Therefore, these data, together with rescue of the Emi2 MO (Sagata et al., 1989; Kosako et al., 1994; Bhatt and Ferrell, phenotype using exogenous ∆90 cyclin B1 added after PB1 1999; Gross et al., 1999, 2000; Tunquist and Maller, 2003) and extrusion, imply that the driving force in establishing a bipolar the spindle checkpoint proteins Mad1 and Mad2 as essential for spindle at the end of meiosis is a restabilization of cyclin B1 the establishment of MetII arrest (Tunquist et al., 2003). Once by Emi2. established, Emi2 contributes to the maintenance of MetII arrest (Liu and Maller, 2005; Rauh et al., 2005; Schmidt et al., Materials and methods 2005; Tung et al., 2005; Hansen et al., 2006), and is perhaps All chemicals were obtained from Sigma-Aldrich, unless otherwise stated, also required for the establishment of MetII arrest; however, and were of tissue culture or embryo-tested grade where appropriate. this requires further investigation. When compared with reports in frog, the mechanism of Gamete collection and culture 4–6-wk-old MF1 mice (Harlan) were used. GV oocytes and MetII oocytes the establishment of MetII arrest in mouse is poorly understood. were collected from hormonally primed animals, as previously described In contrast to frog, there have been no fi rm candidates suggested (Nixon et al., 2002; Hyslop et al., 2004). For bench handling, microinjec- to be involved in establishment. Oocytes from c-Mos −/− mice tions, and imaging experiments, GV oocytes and MetII oocytes were cul- tured in medium M2. When necessary, oocytes were arrested at the GV initially arrest at MetII for 2–4 h, but then fail to maintain stage in medium M2 containing 1 μM milrinone. For longer term incuba- this arrest and exhibit spontaneous parthenogenetic activation tion, GV oocytes and maturing oocytes were held in a 5% CO -humidifi ed (Colledge et al., 1994; Hashimoto et al., 1994; Verlhac et al., incubator at 37°C and cultured in MEM (Invitrogen) with 20% fetal calf serum. Parthenogenetic activation of MetII oocytes was achieved by washing 1996). Verlhac et al. (1996) demonstrated convincingly that 2+ oocytes into Ca -free M2 media containing 10 mM SrCl (Bos-Mikich although c-Mos is required for the stabilization of MPF once et al., 1997; Madgwick et al., 2004). Cycloheximide was added to media MetII is achieved, neither c-Mos nor MAPK are involved in at a concentration of 10 μg/ml, and nocodazole was added at a concen- tration of 100 nM. In the case of nocodazole, oocytes were incubated for MPF reactivation during the MetI–MetII transition. These data 15 mins before parthenogenetic activation. suggest that c-Mos is part of the maintenance of MetII arrest, but not its establishment. In agreement with this, the addition Microinjection and imaging Microinjection of MOs and cRNA constructs were made as described pre- of MAPK inhibitors to MetII oocytes induces parthenogenetic viously on the heated stage of an inverted microscope (TE300; Nikon) activation (Phillips et al., 2002). Therefore, it is clear that the fi tted for epifl uorescence (Nixon et al., 2002). In brief, fabricated micro- c-Mos–MAPK pathway contributes to MetII arrest once it is pipettes were inserted into cells using the negative capacitance overcom- pensation facility on an electrophysiological amplifi er (World Precision established. In any event, MAPK activity declines after cyclin Instruments); this procedure ensures a high rate of survival (>90%). Images B1 degradation, so MAPK is not targeted immediately by the were recorded using a 20× objective, NA 0.75, and a charge-coupled 2+ Ca signal at fertilization (Verlhac et al., 1994). Similarly, the device camera (Micromax 1300Y; Sony). Filter sets were 430 ±10 nm and 500 ±10 excitation fi lters for Cerulean and Venus, respectively main downstream target of MAPK activity in frog appears to (Coherent) with a CFP/YFP 51017 BS&M dichroic mirror and emission have little role in MetII arrest in mouse because oocytes from fi lter (Chroma Technology Corp.). Hoechst staining was examined using p90rsk knockout mice (Dumont et al., 2005) still arrest at MetII. a 360 ±10.9 nm excitation fi lter (Chroma Technology Corp.). All live cell imaging was performed in medium M2 at 37°C. Confocal sections of Furthermore, oocytes injected with dominant-negative forms Hoechst and Texas red staining were performed on a confocal microscope of Bub1 and Mad2 still arrest at MetII (Tsurumi et al., 2004), (SP2; Leica) to capture images of the spindle microtubules and chromatin. suggesting that spindle checkpoint proteins are not involved in MetaMorph and MetaFluor imaging software (Universal Imaging Corp.) were used for image capture and data analysis. MetII arrest in mouse. Although not involved in the establishment of MetII arrest, cRNA constructs and MOs MAPK still appears to have a role in mouse oocytes before the Using primers specifi cally for the mouse homologue of the Emi2 gene (AK030012; National Center for Biotechnology Information), we ampli- formation of a MetII spindle through a MAPK-interacting and fi ed full-length mouse Emi2 by PCR from a mouse MetII-arrested oocyte spindle-stabilizing protein (MISS; Lefebvre et al., 2002). MISS cDNA library. In brief, the cDNA library was prepared by collecting 750 is present at meiosis I, but only becomes stable in meiosis II, MetII oocytes in sterile PBS. Oocytes were lysed immediately, and mRNA was isolated directly using a commercial kit according to the manufactur- where it is essential for the maintenance of spindle integrity. ers instruction (Dynabeads mRNA Direct kit; Dynal). Double-stranded Oocytes maturing in the absence of MISS reach MetII arrest, but cDNA was synthesized from RNA and amplifi ed by PCR using a cDNA with severe spindle disorganization, including monopolar spin- synthesis kit (SMART PCR cDNA Synthesis kit; CLONTECH Laboratories, Inc.). Mouse Emi2 was then cloned from the cDNA library into a modifi ed dles, numerous asters in the cytoplasm, or no spindle (Lefebvre pRN3 vector, designed to produce mRNA transcript C-terminally coupled et al., 2002). However, because spindle defects in these oocytes to Venus. Full-length cyclin B1 and ∆90 cyclin B1 were amplifi ed by PCR are not associated with a drop in cyclin B1 levels or histone H1 as previously described (Madgwick et al., 2004). Full-length cyclin B1 was cloned into two modifi ed pRN3 vectors designed to produce mRNA tran- activity, it is suggested that MISS does not have a direct role in scripts C-terminally coupled to Venus or Cerulean; ∆90 cyclin B1 was CSF arrest. Instead, MISS plays a role in microtubule dynamics cloned into a nonfl uorescent pRN3 vector (Madgwick et al., 2004). Mad2 through an interaction with a microtubule-associated protein. was manufactured as described in Madgwick et al. (2005). cRNA maximal EMI2 AND MEIOSIS II ENTRY • MADGWICK ET AL. 799 Eytan, E., Y. Moshe, I. Braunstein, and A. Hershko. 2006. Roles of the anaphase- stability was conferred in all constructs by the presence of a 5′ globin UTR promoting complex/cyclosome and of its activator Cdc20 in functional upstream and both 3′UTR and poly (A)–encoding tracts downstream of the substrate binding. Proc. Natl. Acad. Sci. USA. 103:2081–2086. gene. cRNA was synthesized using T3 mMESSAGE mMACHINE (Ambion) Fang, G., H. Yu, and M.W. Kirschner. 1998. The checkpoint protein MAD2 and dissolved in nuclease-free water to the required micropipette concen- and the mitotic regulator CDC20 form a ternary complex with the tration. An antisense MO (Genetools) was designed to recognize the anaphase-promoting complex to control anaphase initiation. Genes Dev. 5′UTR of Emi2. Two further controls were also designed, the invert of this 12:1871–1883. region and a 5mp (for sequences see Table I). All MOs were used at a Glotzer, M., A.W. Murray, and M.W. Kirschner. 1991. Cyclin is degraded by the micropipette concentration of 2 mM. ubiquitin pathway. Nature. 349:132–138. Gross, S.D., M.S. Schwab, A.L. Lewellyn, and J.L. Maller. 1999. Induction of Anti-Emi2 antibody purifi cation metaphase arrest in cleaving Xenopus embryos by the protein kinase An MBP fusion of the human Emi2 N terminus (residues 1–400; hEmi2N) p90Rsk. Science. 286:1365–1367. and a GST fusion of the mouse Emi2 N terminus (residues 1–398; mEmi2N) Gross, S.D., M.S. Schwab, F.E. Taieb, A.L. Lewellyn, Y.-W. Qian, and J.L. were bacterially expressed and purifi ed using standard protocols, with the Maller. 2000. The critical role of the MAP kinase pathway in meiosis II exception that all procedures involving GST-mEmi2N were conducted in in Xenopus oocytes is mediated by p90Rsk. Curr. Biol. 10:430–438. the presence of 0.01% Triton X-100 to maintain protein solubility. Sera Hansen, D.V., J.J. Tung, and P.K. Jackson. 2006. CaMKII and Polo-like kinase 1 from rabbits immunized against MBP-hEmi2N were screened for immuno- sequentially phosphorylate the cytostatic factor Emi2/XErp1 to trigger its blot cross- reactivity toward in vitro-translated mouse Emi2. GST-mEmi2N, destruction and meiotic exit. Proc. Natl. Acad. Sci. USA. 103:608–613. immobilized on a column of CNBr-activated Sepharose 4B (17–0430-01; Harper, J.W., J.L. Burton, and M.J. Solomon. 2002. The anaphase-promoting GE Healthcare), was used to affi nity purify anti-mEmi2 antibodies from complex: it’s not just for mitosis any more. Genes Dev. 16:2179–2206. cross-reactive sera. Antibodies were eluted from the column using 100 mM Hashimoto, N., N. Watanabe, Y. Furuta, H. Tamemoto, N. Sagata, M. Yokoyama, glycine, pH 2.5. K. Okazaki, M. Nagayoshi, N. Takedat, Y. Ikawatll, and S. Aizawai. 1994. Parthenogenetic activation of oocytes in c-mos-defi cient mice. Nature. Immunoblotting 370:68–71. For Emi2, lysates from oocytes were incubated overnight with anti-mEmi2 Herbert, M., M. Levasseur, H. Homer, K. Yallop, A. Murdoch, and A. McDougall. in the range of 2.5 to 10 μg/ml. Nonfat milk (5%) was used as a blocking 2003. Homologue disjunction in mouse oocytes requires proteolysis of solution. Anti–rabbit IgG (P0448; DakoCytomation; Figs. 1 and 2; NA934; securin and cyclin B1. Nat. Cell Biol. 5:1023–1025. GE Healthcare; Fig. 4) and ECL Plus (RPN2132; GE Healthcare) were used Homer, H.A., A. McDougall, M. Levasseur, K. Yallop, A.P. Murdoch, and M. as secondary detection reagents. Herbert. 2005. Mad2 prevents aneuploidy and premature proteolysis For cyclin B1, lysates from oocytes were incubated overnight with of cyclin B and securin during meiosis I in mouse oocytes. Genes Dev. anti-cyclin B1(1:400; Abcam; ab72). Nonfat milk (5%) was used as a 19:202–207. blocking solution. Anti–mouse IgG (P0447; DakoCytomation) and ECL Plus Hyslop, L., V. Nixon, M. Levasseur, F. Chapman, K. Chiba, A. McDougall, J. 2+ were used as secondary detection reagents. Venables, D. Elliott, and K.T. Jones. 2004. Ca -promoted cyclin B1 degradation in mouse oocytes requires the establishment of a metaphase arrest. Dev. Biol. 269:206–219. Immunostaining of oocytes 2+ Immunostaining of cyclin B1 was performed as previously described (Reis Jones, K.T. 1998. Ca oscillations in the activation of the egg and development et al., 2006). In brief, oocytes were fi xed and permeabilized by an incuba- of the embryo in mammals. Int. J. Dev. Biol. 42:1–10. 2+ tion in 3.7% paraformaldehyde in PBS (30 min), followed by incubation in Jones, K.T. 2005. Mammalian egg activation: from Ca spiking to cell cycle 3.7% paraformaldehyde, 2% Triton X-100 in PBS (30 min). Fixed oocytes progression. Reproduction. 130:813–823. were then washed extensively in 1% PVP and 1% BSA in PBS. For spindle Knott, J.G., M. Kurokawa, R.A. Fissore, R.M. Schultz, and C.J. Williams. 2005. staining, oocytes were incubated with rat anti-tubulin antibody (YL1/2, Transgenic RNA interference reveals role for mouse sperm phospholi- 2+ 1:40, Abcam). 5 μg/ml Texas red conjugated anti–rat goat IgG (Abcam) pase Cζ in triggering Ca oscillations during fertilization. Biol. Reprod. was used as a secondary antibody. To stain chromatin, oocytes received 72:992–996. an additional incubation in 10 μg/ml Hoechst 33258. For cyclin B1 stain- Kono, T., K.T. Jones, A. Bos-Mikich, D.G. Whittingham, and J. Carroll. 1996. 2+ ing, oocytes were incubated with mouse anti-cyclin B1 antibody (1:400; A cell cycle-associated change in Ca releasing activity leads to the 2+ generation of Ca transients in mouse embryos during the fi rst mitotic Abcam). TRITC-conjugated anti–mouse rabbit IgG/IgM/IgA was used as division. J. Cell Biol. 132:915–923. a secondary antibody (1:40; DakoCytomation). Kosako, H., Y. Gotoh, and E. Nishida. 1994. Mitogen-activated protein kinase We thank Jonathan Pines for the gift of Cerulean fl uorescent protein. kinase is required for the mos-induced metaphase arrest. J. Biol. Chem. This work is supported by a project grant (069236) and equipment 269:28354–28358. grant (065354) from the Wellcome Trust to K.T. Jones. Kramer, E.R., C. Gieffers, G. Holzl, M. Hengstschlager, and J.-M. Peters. 1998. Activation of the human anaphase-promoting complex by proteins of the Submitted: 21 April 2006 CDC20/Fizzy family. Curr. Biol. 8:1207–1210. Accepted: 8 August 2006 Kubiak, J.Z., M. 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Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis

Madgwick, Suzanne; Hansen, David V.; Levasseur, Mark; Jackson, Peter K.; Jones, Keith T.
The Journal of Cell Biology , Volume 174 (6) – Sep 11, 2006
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JCB: ARTICLE Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis 1 2,3 1 2,3 1 Suzanne Madgwick, David V. Hansen, Mark Levasseur, Peter K. Jackson, and Keith T. Jones Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle, Newcastle NE2 4HH, England, UK 2 3 Department of Pathology and Program in Cancer Biology, Stanford University School of Medicine, Stanford, CA 94305 uring interkinesis, a metaphase II (MetII) spindle Emi2 antisense morpholino knockdown during oo- is built immediately after the completion of mei- cyte maturation did not affect polar body (PB) extrusion. D osis I. Oocytes then remain MetII arrested until However, in interkinesis the central spindle microtubules fertilization. In mouse, we fi nd that early mitotic inhibi- from meiosis I persisted for a short time, and a MetII spindle tor 2 (Emi2), which is an anaphase-promoting complex failed to assemble. The chromatin in the oocyte quickly de- inhibitor, is involved in both the establishment and the condensed and a nucleus formed. All of these effects were maintenance of MetII arrest. In MetII oocytes, Emi2 needs caused by the essential role of Emi2 in stabilizing cyclin to be degraded for oocytes to exit meiosis, and such deg- B1 after the fi rst PB extrusion because in Emi2 knockdown radation, as visualized by fl uorescent protein tagging, oocytes a MetII spindle was recovered by Emi2 rescue or occurred tens of minutes ahead of cyclin B1. by expression of nondegradable cyclin B1 after meiosis I. Introduction Oocytes arrest at metaphase of the second meiotic division both M-phase (maturation)–promoting factor (MPF) activity (MetII) before fertilization because of an activity termed cyto- (CDK1/cyclin B1) and cohesin, which holds sister chromatids static factor (CSF; Masui, 2000; Tunquist and Maller, 2003; together (Nixon et al., 2002; Madgwick et al., 2004). Resumption 2+ Jones, 2005). Sperm break this arrest via a Ca signal (Jones, of meiosis in mammalian oocytes is achieved by a sperm-borne 1998; Stricker, 1999; Runft et al., 2002), and in so doing, oo- phospholipase C activity (Saunders et al., 2002; Knott et al., 2+ cytes complete the second meiotic division before entering the 2005), which generates an oscillatory Ca signal, switching on cdc20 embryonic cell cycles. APC (Nixon et al., 2002; Madgwick et al., 2004) through CSF activity, a terminology that was fi rst defi ned several a signaling pathway involving calmodulin-dependent protein decades ago (Masui and Market, 1971), is now known to consti- kinase II (CamKII; Markoulaki et al., 2003, 2004; Madgwick tute an inhibitor of the anaphase-promoting complex/cyclosome et al., 2005). This signaling process is conserved and was fi rst (APC; Tunquist and Maller, 2003). The APC is an E3 ubiquitin demonstrated in frog eggs (Lorca et al., 1993, 1994). Activation ligase whose activity is required for the metaphase–anaphase of the APC in MetII oocytes induces the destruction of MPF transition to polyubiquitinate key cell cycle proteins, thereby and sister chromatid cohesion through the polyubiquitination of earmarking them for immediate proteolysis through associa- cyclin B1 and securin, respectively (Morgan, 1999; Zachariae tion with its key coactivator cdc20 (Fang et al., 1998; Kramer and Nasmyth, 1999; Peters, 2002). Loss of cyclin B1 causes et al., 1998; Harper et al., 2002; Eytan et al., 2006). The reduced a reduction in MPF, and the loss of securin frees separase cdc20 APC activity in MetII oocytes prevents the destruction of to act on the kleisin component of cohesin (Zachariae and Nasmyth, 1999; Peters, 2002; Blow and Tanaka, 2005; Nasmyth and Haering, 2005). Correspondence to Keith T. Jones: [email protected]; or Suzanne Madgwick: Many proteins have been associated with the establishment [email protected] D.V. Hansen and P.K. Jackson’s current address is the Department of Tumor and/or maintenance of CSF activity. Factors responsible for Biology and Angiogenesis, Genentech, Inc., South San Francisco, CA 94080. setting up a second meiotic spindle after completion of meiosis I Abbreviations used in this paper: APC, anaphase-promoting complex/ do not, a priori, have to be the same as those that are responsible cyclosome; Bub, budding uninhibited by benzimidazole; CSF, cytostatic factor; Emi, early mitotic inhibitor protein; GV, germinal vesicle; GVBD, GV break- for maintaining arrest. Indeed, proteins have been described down; Mad, mitotic arrest defi cient; MISS, MAPK-interacting and spindle- that are involved in establishing MetII, but not in maintaining stabilizing protein; MO, morpholino; MPF, M-phase (maturation)–promoting arrest once it has been achieved (Tunquist and Maller, 2003). factor; PB, polar body. © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 174, No. 6, September 11, 2006 791–801 http://www.jcb.org/cgi/doi/10.1083/jcb.200604140 JCB 791 THE JOURNAL OF CELL BIOLOGY The mechanism of CSF is most well characterized in the frog, As there are no fi rm candidates suggested to be involved where various groups have fi rmly defi ned the c-Mos–MAPK– in the establishment of CSF arrest in mouse, we have investi- 90-kD ribosomal protein S6 kinase (p90rsk)–budding uninhib- gated the potential of mouse Emi2 in this process. We fi nd that ited by benzimidazole 1 (Bub1) pathway in establishing CSF Emi2 has activity consistent with CSF, and its degradation in activity (Sagata et al., 1989; Abrieu et al., 1996; Bhatt and real time is ahead of any change in cyclin B1. We also demon- Ferrell, 1999; Gross et al., 1999, 2000; Tunquist et al., 2002). strate that a function of Emi2 is in restabilizing cyclin B1 upon Other activities that are fundamentally involved in the estab- exit from anaphase I and that, in this study, it contributes to the lishment of CSF in frog include cyclin E/Cdk2 (Tunquist et al., formation a bipolar spindle. Oocytes matured without Emi2 do 2002) and mitotic arrest defi cient 2 (Mad2; Tunquist et al., not assemble MetII spindles, and, in the absence of cyclin B1, 2003). However, once established, p90rsk, Mad2, Bub1, and eventually decondense their chromatin. cyclin E/Cdk2 are all dispensable for the maintenance of CSF activity (Bhatt and Ferrell, 1999; Tunquist et al., 2002, 2003). Results So, how is CSF activity maintained in the frog? Current 2+ evidence suggests it is through early mitotic inhibitor 2 (Emi2)/ Stable Emi2 is rapidly degraded by Ca Emi-related protein 1 (Liu and Maller, 2005; Rauh et al., 2005; To measure real-time changes in Emi2 levels in oocytes we gen- Schmidt et al., 2005; Tung et al., 2005; Hansen et al., 2006). Emi2, erated cRNA to mouse Emi2 coupled to Venus fl uorescent pro- which acts to inhibit the APC accumulated during oocyte matura- tein (Emi2-V), which is a yellow variant of GFP. MetII mouse tion, is present and stable in CSF frog egg extracts, but is rapidly oocytes were microinjected with this construct at a dose of ei- 2+ degraded on Ca addition (Schmidt et al., 2005). Degradation of ther 0.15 or 0.5 pg, and then cultured for a few hours to allow Emi2 is induced by phosphorylation through CamKII (Liu and for Emi2 expression. We Western blotted oocytes expressing Maller, 2005; Rauh et al., 2005; Hansen et al., 2006) and, thus, Emi2-V with a polyclonal antibody against Emi2 to determine would be predicted to occur ahead of APC activation and cyclin the amount of Emi2-V expression relative to endogenous protein. B1 degradation, although this has not been tested. More than one band was detected on oocyte blots using this In mouse oocytes, the mechanism of CSF arrest is less antibody; ho wever, one band at 85 kD migrated at the same well understood. As the mouse Emi2 homologue appears to molecular mass as in vitro–translated Emi2 (and this band was have a similar function in maintaining CSF activity (Shoji et al., later knocked down by Emi2 morpholino [MO]). At the 0.15-pg 2006), it would be logical to predict that the mechanism of dose, Emi2-V levels were less than endogenous protein after CSF establishment is also conserved between frog and mouse. 2 h, whereas the 0.5-pg dose was expressed to about the same Ho wever, this is not so. Oocytes from a triple Rsk (1,2,3) knockout level as endogenous protein (Fig. 1 A). mouse arrest normally at MetII (Dumont et al., 2005), demon- Emi2-V was very stable in MetII-arrested oocytes, but 2+ strating that p90Rsk is not involved in mouse CSF arrest. became rapidly unstable when cytosolic Ca increased. With Furthermore, using dominant-negative mutants Tsurumi et al., either 0.15 pg (Fig. 1 B) or 0.5 pg (not depicted) injections, we (2004) confi rmed that the spindle checkpoint proteins Bub1 and observed no degradation of Emi2-V in MetII oocytes after block- Mad2 are not required for either the establishment or the main- ing further synthesis by washing into cycloheximide- containing tenance of mouse CSF. Although c-Mos is known to be involved media. However, when this experiment was repeated, but by 2+ 2+ in CSF maintenance, it does not appear to be involved in the washing into Sr -containing media to induce spermlike Ca establishment of CSF arrest because c-Mos −/− oocytes remain spiking (Bos-Mikich et al., 1997), we observed dose- dependent at MetII for 2–4 h before extruding a second PB (PB2; Verlhac effects on Emi2-V. At the higher 0.5-pg dose, we observed no et al., 1996). loss in Emi2-V signal; instead, Emi2-V levels steadily increased Figure 1. Emi2-V is stable in MetII oocytes, 2+ but is rapidly degraded by Ca . (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. *, nonspecifi c band. (B) Emi2-V (0.15 pg cRNA) expression levels on addition of cycloheximide (CHX; n = 12). (C and D) Emi2-V (C, 0.5 pg cRNA; D, 0.15 pg cRNA) 2+ expression levels after washing into Sr media at the times indicated (C, n = 14; D, n = 15). (E) Cyclin B1-V (0.09 pg cRNA) 2+ expression levels after washing into Sr media at the times indicated (n = 15). T and T repre- E C sent the time from complete degradation of the Venus fl uorochrome (T , Emi2; T , cyclin B1) to E C PB2 extrusion in oocytes. PB2 was observed at the times indicated. A is representative of two independent Western blots. (B–E) are represen- tative traces of n oocytes that have been used from two to four independent experiments. 792 JCB • VOLUME 174 • NUMBER 6 • 2006 (Fig. 1 C). However, at the lower 0.15-pg dose Emi2-V was rap- confi rm the immediate loss of endogenous Emi2 signal in idly degraded (Fig. 1 D). oocytes, which is especially important given that Shoji et al. (2006) The high dose of Emi2 maintained oocytes in a MetII had reported very little loss in Emi2 signal at a 6-h time point 2+ arrest. Oocytes injected with 0.5 pg Emi2-V cRNA showed no after activation with Sr . Oocytes were activated by washing 2+ morphological signs of meiotic resumption, which is consistent into Sr media, and samples from a pool of oocytes were removed with the maintained Emi2-V levels in these oocytes (Fig. 1 C). at various time points and probed for either cyclin B1 or Emi2 In contrast, oocytes that had been injected with 0.15 pg Emi2-V by Western blotting (Fig. 2 A). In these experiments, it was cRNA extruded a PB2 (Fig. 1 D). This suggests that Emi2-V apparent that loss in Emi2 protein w as rapid and complete by has physiological CSF activity and that the oocyte has a fi nite 30 min (in agreement with the rapid loss of Emi-V shown in capacity to degrade Emi2. Fig. 1 D). Interestingly, Emi2 levels increased again in pronu- cleate embryos (6-h time point), which is in agreement with Emi2 is degraded ahead of cyclin B1 Shoji et al. (2006) and suggests that it may have a further mitotic At the lower dose Emi2 injection, where we observed Emi2-V function (see Discussion). Similar to Emi2, we observed the loss degradation, the minimum in its degradation profi le after activa- of cyclin B1 at 30 min, as well as increased levels in pronucleate 2+ tion with Sr media was reached tens of minutes before PB2 embryos (Fig. 2 A), which is consistent with our observation that extrusion (Fig. 1 D, T ). However, we have previously reported the APC is switched off at this time (Nixon et al., 2002). that cyclin B1 degradation, visualized by coupling to GFP, is Because of the numbers of oocytes needed for Western only completed at the time of PB2 formation (Hyslop et al., blots and the tens of minutes of asynchrony in timing at which 2+ 2+ 2004). This suggests that the Emi2 and cyclin B1 degradation Ca spiking starts with Sr media, (Madgwick et al., 2004), profi les may not fully overlap. When cyclin B1 was expressed we could not reproducibly resolve Emi2 degradation ahead of in mouse oocytes with the same fl uorescent protein tag as Emi2 cyclin B1 by Western blotting groups of oocytes. Therefore, to (cyclin B1-Venus; cyclin B1-V), we observed the same degra- examine with more accuracy the immediate degradation pro- dation profi le as that previously found for cyclin B1-GFP, such fi les of cyclin B1 and Emi2, we decided to measure their simul- that a minimum in the cyclin B1-V profi le was reached within taneous degradation in the same oocyte. Cyclin B1 was coupled minutes of PB2 extrusion (Fig. 1 E, T ). to Cerulean fl uorescent protein (cyclin B1-C), which is a cyan Comparing the degradation profi les of Emi2-V and cyclin variant of GFP. There was no overlap in Venus and Cerulean B1-V suggests that Emi2 degradation is initiated ahead of cyclin signals, showing that both Emi2-V and cyclin B1-C, with ap- 2+ B1. Sr -induced Emi2 degradation begins immediately (Fig. 1 D), propriate fi lters, could be imaged simultaneously in the same whereas that of cyclin B1 begins 20 min later (Fig. 1 E). This oocyte (Fig. 2 B). In these experiments, it was evident that the would have to occur if MetII arrest is being mediated by Emi2- introduction of Emi2-V delayed cyclin B1 degradation (Fig. 1 E cdc20 induced inhibition of APC activity. Therefore, we wanted to and Fig. 2 C), which is consistent with Emi2-inhibiting APC Figure 2. Emi2 is degraded ahead of cyclin B1. (A) West- ern 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 bril- liant blue (CB) to show equivalent loading. Oocytes are 2+ activated by washing into Sr media for the times indi- cated. (B) Epifl uorescence images of MetII oocytes micro- injected with Emi2-V cRNA or cyclin B1-C cRNA, at the excitation wavelengths of Cerulean (430 nm) and Venus (500 nm). There is negligible overlap in signal between the two fl uorochromes, demonstrating that Emi2-V and cy- clin B1-C may be imaged in the same oocyte. Bar, 20 μm. (C) Simultaneous Venus (yellow) and Cerulean (blue) fl uo- rescence in a MetII oocyte expressing Emi2-V and cyclin 2+ B1-C, and washed into Sr media at time t = 0 min. Emi2-V degradation begins tens of minutes before that of cyclin B1. (A) The Western blot was repeated once with similar timings. C is representative of seven oocytes from two independent experiments. EMI2 AND MEIOSIS II ENTRY • MADGWICK ET AL. 793 Table I. Emi2 MOs used in this study MO Sequence Mouse Emi2 A GCC AGC CAC AGA GCA GGA AGC AAT ATG GAC TCC TCT Emi2 MO ATT GCT TCC TGC TCT GTG GCT GGC T 5mp MO ATT CCT TGC TGC TGT GTG CCT GCC T Inv MO TCG GTC GGT GTC TCG TCC TTC GTT A Emi2 MO was designed to target the 5′UTR of mouse Emi2 (underlined). A 5mp MO and an Inv MO were used as control MOs. 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 matura- tion makes it highly likely that Emi2 expression can be knocked out by an antisense approach. Therefore, we designed an anti- sense 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 (Table I); a 5-base mispair MO (5mp-MO), in which fi ve 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) Figure 3. Emi2-V degradation is independent of a spindle checkpoint. oocytes, which were then matured in vitro. Oocytes were held at Venus fl uorescence levels in a MetII oocyte expressing cRNA to cyclin B1-V 2+ the GV stage in milrinone-containing media for 2 h after MO (A; n = 12) or Emi2-V (B; n = 15) after the addition of Sr media contain- ing 100 ng/ml nocodazole at time t = 0 h. Both A and B are represent- injection; they were then released from GV arrest and allowed ative of oocytes collected in two independent experiments. to mature for 16 h. Blotting of GV-stage oocytes, Emi2 MO– matured oocytes, and uninjected control matured oocytes dem- activity. However, these coexpression studies revealed that onstrated that Emi2 protein levels increase between GV and Emi2 degradation began tens of minutes before that of cyclin MetII stage, and confi rmed the Emi2 knockdown in Emi2 MO– B1 (Fig. 2 C). Emi2-V levels were degraded by at least 50% injected oocytes (Fig. 4 A). before the start of cyclin B1-C degradation (n = 7). During maturation, oocytes were scored for the morpho- logical events of oocyte maturation, which are GV breakdown Emi2 degradation is independent (GVBD) and PB1 extrusion (Fig. 4 B). Both GVBD (Fig. 4 C) of a spindle checkpoint and PB1 extrusion (Fig. 4 D) occurred with normal timings cdc20 Degradation of cyclin B1 is dependent on APC activity, and in Emi2 MO–injected oocytes. However, after maturation, we in mouse oocytes it can be blocked by the induction of a spindle observed marked differences in the morphology of control checkpoint (Nixon et al., 2002; Madgwick et al., 2005). In con- oocytes and those injected with Emi2 MO. When control trast, Emi2 degradation should be checkpoint-independent be- oocytes (uninjected, 5mp-MO, and Inv-MO–injected) were cdc20 cause its degradation is independent of APC involvement. stained for chromatin, oocytes were morphologically normal. Incubation of Emi2- and cyclin B1-expressing oocytes with the They had a PB1 containing chromatin, which was produced spindle poison nocodazole blocks mouse oocytes from exiting on completion of the fi rst meiotic division, and a fully formed 2+ MetII arrest when washed into Sr media. As expected, the ad- MetII spindle (100%; uninjected oocytes, n = 60; 5mp-MO, dition of nocodazole completely stabilized cyclin B1 levels n = 32; Inv-MO, n = 25; Fig. 5 A). However, although oocytes (n = 15/15; Fig. 3 A). However, nocodazole had no effect on the injected with Emi2 MO did have a PB1 containing chromatin, rate of Emi2-V degradation (n = 12/12; Fig. 3 B). Such an ob- the chromatin in the oocyte was decondensed inside a nucleus servation is consistent with cyclin B1, but not Emi2 degrada- (93%; n = 110; Fig. 5 A). tion, being dependent on the APC. Despite the lack of effect of a 5mp-MO, it remained Therefore, in summary, we have obtained data that are en- possible that we had been extremely unlucky in the MO design, tirely consistent with a model of MetII arrest achieved by Emi2- such that the observed effects of Emi2 MO were caused by its mediated inhibition of cyclin B1 degradation. Also, the Emi2-V ability to block the expression of an unrelated protein involved construct generated is a physiologically active, useful tool for in MetII arrest. We thought this unlikely, given that a similar both establishing CSF activity and measuring its loss in real morphology of Emi2 knockdown oocytes has been reported re- 2+ time after a Ca signal. cently using a double-stranded RNAi approach (Shoji et al., 2006). 794 JCB • VOLUME 174 • NUMBER 6 • 2006 Figure 4. GVBD and PB1 extrusion occur nor- mally in Emi2 MO–injected oocytes. (A) West- ern blot of GV-stage, Emi2 MO– matured, and control matured oocytes; 75 oocytes were loaded per lane. *, nonspecifi c band. (B) Brightfi eld time lapse of an oocyte microin- jected with Emi2 MO and allowed to mature. GVBD and PB1 are marked. Bar, 20 μm. The percentage rate of GVBD (C) and PB1 extru- sion (D) at the times indicated for control oo- cytes (n = 32) and oocytes microinjected with Emi2 MO (n = 33). The Western blot in A is representative of two independent experi- ments. The data in C and D was pooled from two independent experiments. However, we decided it was important to confi rm the specifi city We determined whether Emi2 MO knockdown oocytes by a rescue to the control phenotype in Emi2-MO–injected were able to build a MetII bipolar spindle by staining oocytes oocytes by expression of exogenous Emi2. To recover Emi2, for both chromatin and tubulin at various times after PB1 extrusion. Emi2-V cRNA was microinjected into oocytes 2 h after micro- Oocytes were fi xed at 0.5, 1, and 2 h after PB1 extrusion. In injection of Emi2 MO and immediately before release from GV control uninjected oocytes, central spindle microtubules were arrest. This rescue is made possible because Emi2-V lacks the observed at 0.5 h after PB extrusion (Fig. 6 A), and over the next 5′UTR recognized by the MO. 1.5 h a MetII spindle formed, such that by 2 h after PB1 a fully Injection of Emi2-V cRNA alone into GV oocytes that formed MetII spindle was found in all oocytes (Fig. 6 B). In were matured induced a MetI arrest (n = 40; Fig. 5 A), which is Emi2-MO–injected oocytes, at 0.5 h after PB1 there was no dif- consistent with Emi2-V having CSF activity and the need to ference from controls, with central spindle microtubules evident keep Emi2 levels low until completion of the fi rst meiotic between the chromatin in the ooplasm and the PB1 (Fig. 6 A). division. Importantly, Emi2-V cRNA expression could rescue However, in contrast to control oocytes, in Emi2-MO–injected the effects of MO knockdown. Rescue oocytes could progress oocytes at both the 1 and the 2 h time points we observed no through meiosis I and arrest as controls with a fully formed MetII spindle; instead, the chromatin had remained essentially MetII spindle. A high dose of Emi2-V (0.5 pg; n = 45) rescued unaltered from the time of PB1 extrusion. The residual central the Emi2 MO phenotype, with an equal mix of either MetI or spindle microtubules remained between the chromatin in the MetII arrest, whereas oocytes microinjected with the lower oocyte and the PB, and by 2 h the chromatin appeared to be Emi2 dose showed a rescue with a predominantly MetII arrest beginning to decondense (Fig. 6 B). As the chromatin started (n = 45; Fig. 5 A). to decondense, spindle microtubule structure was lost, which is consistent with entry into interphase. By 6 h, all Emi2-MO– Lack of Emi2 results in a failure injected oocytes had a single nucleus containing fully decon- to assemble a metaphase II spindle densed chromatin. We did not assess if these oocytes underwent By scoring oocytes for decondensed chromatin at only one S-phase; however, they showed no obvious signs of degenera- time point (16 h), we were unable to pinpoint at which stage of tion over the 6-h time period from PB1 extrusion. Thus, we meiosis oocytes undergo chromatin decondensation. However, failed to observe the formation of a bipolar spindle in any Emi2- we noted that the vast majority of oocytes with decondensed MO–treated oocytes; instead, oocytes underwent full chromatin chromatin extruded only a single PB and contained only one decondensation. nucleus (88%; n = 102). This observation suggests either de- condensation of chromatin occurred after anaphase I, such Emi2 stabilizes cyclin B1 after that oocytes did not form a MetII spindle (Fig. 5 B, i), or, PB1 extrusion alternatively, that after establishment of a MetII spindle there Because the formation of a MetII spindle requires an increase in was no sister chromatid disjunction before decondensation the levels of CDK1 activity after PB1 extrusion, we reasoned (Fig. 5 B, ii). that in the absence of Emi2, levels of CDK1’s regulatory partner EMI2 AND MEIOSIS II ENTRY • MADGWICK ET AL. 795 Figure 5. Decondensed chromatin in Emi2 MO–injected matured oocytes. (A) Percentage of maturation rates in in vitro–matured oocytes after micro- injection at the GV stage of MOs and/or Emi2-V cRNA, as indicated. Figure 6. A bipolar MetII spindle does not form in Emi2 MO–injected Oocyte maturation was assessed at 16 h after release from GV arrest. oocytes. Tubulin (red) and chromatin (blue) staining in either Emi2 MO– Oocytes were stained with Hoechst and scored as either arrested at MetI microinjected or control oocytes at 0.5 (A), 2 (B), or 6 h (C) after PB1 or MetII or as having decondensed chromatin. (B) Emi2 MO oocytes typi- extrusion. (A) In control and Emi2 MO–injected oocytes, a central spindle cally extruded a PB1 and contained only one nucleus (88%; n = 102). This forms between segregated homologues at 0.5 h. (B) By 2 h after PB1 extru- single nucleus must result through chromatin decondensation either imme- sion, all control oocytes had a bipolar MetII spindle; however, Emi2 MO diately after PB1 extrusion (i) or after the formation of a MetII spindle that oocytes showed only microtubules that remained from the persistence of fails to segregate its sister chromatids (ii). The data in A was pooled from the central spindle. (C) By 6 h after PB1, chromatin in Emi2 MO–injected 12 independent experiments. See Oocytes with Emi2 knockdown extrude oocytes had completely decondensed inside a nucleus (N). Bar, 20 μm. a PB, but do not MetII arrest. Each image is representative of at least 10 oocytes. A–C are representa- tive of two, three, and seven independent experiments, respectively. cyclin B1 may not be reestablished. To test this directly, Emi2 MO or control GV oocytes were microinjected with cyclin with Emi2 MO at the GV stage showed much lower cyclin B1-V cRNA and matured in vitro. Cyclin B1-V levels were B1 levels than control in vitro–matured oocytes (Fig. 7 C). monitored in real time during maturation, along with brightfi eld Further more, cyclin B1 levels in individual oocytes were mea- microscopy to assess progression through meiosis I. As previ- sured by immunofl uorescence at 16 h after release from GV ously reported (Hyslop et al., 2004), in control oocytes there arrest in control and Emi2-MO–injected oocytes (Fig. 7, D and E). was a period of cyclin B1 degradation that lasted a few hours We found that cyclin B1 immunofl uorescence levels in Emi2 and was terminated on PB1 extrusion (Fig. 7 A). Immediately MO–treated oocytes was about one third that of control oocytes after PB1 extrusion in these oocytes, cyclin B1 levels rose to (Fig. 7 E), suggesting a relatively uniform level of knockdown. 24.0 ± 1.1% (n = 14) of the peak level before PB1 extrusion (Fig. 7 B). In Emi2-MO–injected oocytes, there was no differ- Nondegradable cyclin B1 and Mad2 ence from controls in the timing of initiation or in the rate of rescue a bipolar spindle in Emi2 cyclin B1-V degradation during meiosis I, which is in agreement knockdown oocytes with the observed lack of effect of the MO on the timing of PB1 Because the formation of a MetII spindle requires an elevation (Fig. 4 D). However, in these Emi2 MO oocytes there was no in the levels of cyclin B1, we reasoned that the Emi2 MO spin- reelevation of cyclin B1 after PB1 extrusion; cyclin B1-V levels dle defect may be recovered by the addition of cRNA to non- increased to just 1.6 ± 0.3% (n = 15) of the peak level before degradable cyclin B1. Furthermore, it should also be rescued cdc20 PB1 extrusion (Fig. 7 B). by inhibiting the APC by addition of Mad2. Cyclin B1 with In keeping with the real-time effects on exogenous cyclin deletion of 90 N-terminal amino acids (∆90 cyclin B1) lacks a B1-V expression, we also observed a loss of endogenous cyclin D-box and is therefore not a substrate of the APC (Glotzer et al., B1 after Emi2 knockdown. In vitro–matured oocytes injected 1991; Madgwick et al., 2004). Emi2-MO–injected oocytes 796 JCB • VOLUME 174 • NUMBER 6 • 2006 Figure 7. Cyclin B1 levels remain low after PB extrusion in Emi2 MO–matured oocytes. (A) Venus fl uorescence 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 indicated. Note the similar profi le of cyclin B1-V degradation in both oocytes, but the fail- ure of cyclin B1-V levels to rise in the oocyte injected with Emi2 MO after PB1. (B) Quan- tifi cation of the elevation in cyclin B1-V lev- els after PB1 extrusion in the control oocytes (n = 14) and Emi2 MO oocytes (n = 15) used in A. Endogenous cyclin B1 levels in matured oocytes detected by Western blotting (C) or immunofl uorescence (D and E). (C) A West- ern blot (WB) for cyclin B1 in control matured oocytes and oocytes matured after microinjec- tion of Emi2 MO at the GV stage (n = 40), and the corresponding membrane stained with Coomassie brilliant blue (CB) to demonstrate equivalent loading. In D, representative im- ages of cyclin B1 levels in matured oocytes (control) and in matured oocytes microinjected at the GV stage with Emi2 MO detected using a TRITC-conjugated secondary antibody. Mean fl uorescence readings are marked for both oocytes on the 8-bit scale bar. (E) Quantifi ca- tion of cyclin B1 immunofl uorescence levels in control (n = 25) and Emi2 MO–microinjected (n = 32) oocytes from D, relative to uninjected controls. A is representative of four indepen- dent experiments whose data is pooled in B. (C) The Western blot was repeated once. D is representative of two independent experiments whose data is pooled in E. either received no further treatment or were microinjected with presence of a bipolar spindle (Fig. 8 A). As observed in Fig. 6 B, 1.5 pg ∆90 cyclin B1 or Mad2 cRNA within 15 min of PB1 oocytes microinjected with Emi2 MO gave decondensing chro- extrusion. This injection had to be done immediately after PB1 matin with a lack of any bipolar spindle structure. However, extrusion because nondegradable cyclin B1 addition to oocytes as with uninjected oocytes, we observed a fully formed bipolar before PB1, as predicted by the sustained MPF activity, blocks MetII spindle in oocytes microinjected with Emi2 MO and PB extrusion (Herbert et al., 2003). A few hours of ∆90 cyclin either ∆90 cyclin B1 cRNA or Mad2 (Fig. 8 B). B1 cRNA expression generates cyclin B1 at a similar level to endogenous cyclin B1 in a MetII oocyte (Madgwick et al., Discussion 2004). This dose of Mad2 causes a metaphase I arrest in matur- ing oocytes (Homer et al., 2005) and prevents completion of We have demonstrated in live oocytes and in real time that upon 2+ meiosis in MetII oocytes (Madgwick et al., 2005). At 2 h after a Ca signal, Emi2 degradation occurs ahead of cyclin B1, PB1 extrusion, all oocytes were fi xed, stained, and scored for the independent of APC inhibition. The current study confi rmed Figure 8. Emi2 MO phenotype recover y using 𝚫 90 cyclin B1 or Mad2. (A) Percentage maturation rates in in vitro– matured oocytes after no treatment (n = 12), or microin- jection of Emi2 MO at the GV stage with or without further microinjection of ∆90 cyclin B1 or Mad2 after PB1 extru- sion (Emi2 MO only, n = 20; with ∆90 cyclin B1, n = 25; or with Mad2, n = 35). Oocytes were fi xed and stained 2 h after PB1 extrusion and scored for a bipolar spindle. (B) Tubulin (red) and chromatin (blue) staining in an oocyte microinjected with Emi2 MO at the GV stage and then ∆90 cyclin B1 cRNA 15 min after PB1 extrusion. Image is representative of 20/25 oocytes. Bar, 20 μm. Data is pooled from two independent experiments. EMI2 AND MEIOSIS II ENTRY • MADGWICK ET AL. 797 β-TRCP β-TRCP that Emi2 constitutes CSF activity in mouse MetII oocytes and known SCF (Skp1-Cullin-F-box ) motif (DSGX S) is responsible for the maintenance of MetII arrest (Shoji et al., and not the APC (Rauh et al., 2005; Tung et al., 2005). 2006). More importantly, we fi nd that Emi2 is necessary and suffi cient in stabilizing cyclin B1 levels at the completion of Establishment of MetII arrest meiosis I, and such stabilization is essential in establishing a We used a MO antisense oligonucleotide to ablate the Emi2 rise bipolar spindle. during mouse oocyte maturation and confi rmed a knockdown By defi nition, CSF activity must accumulate during oo- by Western blotting. By blocking the accumulation of Emi2, cdc20 cyte maturation, inhibit APC activity at MetII, and be inac- we have demonstrated that it is essential for stabilizing cyclin 2+ tivated on a Ca signal at fertilization. Although only recently B1 after MetI during interkinesis, and therefore essential for the identifi ed, the F-box protein Emi2 is very likely to constitute formation of a MetII spindle. Because of the known inhibitory CSF in frog eggs (Liu and Maller, 2005; Rauh et al., 2005; action of Emi2 on APC activity, we interpret this observation to Schmidt et al., 2005; Tung et al., 2005; Hansen et al., 2006). mean that in the absence of Emi2, the APC remains active, and cdc20 Emi2 inhibits the ubiquitin ligase activity of the APC thus cyclin B1 is continuously degraded. In agreement with this in vitro, and in keeping with the nature of CSF, it accumulates is our observation that nondegradable cyclin B1, but not full- during oocyte maturation, is present in CSF extracts, and is length cyclin B1, rescues a MetII arrest after Emi2 knockdown 2+ cdc20 rapidly degraded by Ca (Schmidt et al., 2005). (Fig. 7 vs. Fig. 8), as does the APC inhibitor Mad2. The cur- rent data have not ruled out some effect of Emi2 on cyclin B1 Maintenance of MetII arrest synthesis. However, if so, Emi2 would also be able to negatively We tested the characteristics of the mouse Emi2 homologue in regulate expression of the microinjected cyclin B1 cRNA (Fig. 7). MetII mouse oocytes. This achieved two goals. First, it allowed This lacks the cyclin B1 gene 3′UTR, which is important for us to demonstrate that Venus-tagged Emi2 is physiologically regulating translation through polyadenylation (Tay et al., 2000). active, and second, it provided us with data relating to the tim- Therefore, we would argue that Emi2 is most likely affecting the ing of Emi2 degradation on activation. Excess X. laevis Emi2 rate of cyclin B1 degradation, rather than synthesis. 2+ protein prevents Ca -induced CSF release in egg extracts The observed Emi2 MO phenotype, PB1 extrusion and (Schmidt et al., 2005); similarly, our MetII-arrested mouse oo- decondensed chromatin within a single pronucleus, was not 2+ cytes were refractory to Ca through modest rises in Emi2-V. caused by a nonspecifi c effect of the MO for two reasons. First, A lower Emi2-V dose, less than endogenous protein, did not we used two control MOs that were without effect. Second, we block escape from MetII arrest and allowed us to demonstrate rescued the Emi2 knockdown by readdition of Emi2 cRNA, that Emi2 is degraded ahead of cyclin B1. If Emi2 inhibits the an approach that was made possible by the fact that the res- APC in MetII arrest, it follows that Emi2 destruction must oc- cue cRNA did not contain the 5′UTR recognized by the MO. cur before the APC’s accelerated degradation of cyclin B1. In some instances, especially where rescue was done with the I ndeed, when both were simultaneously imaged in an oocyte higher dose of Emi2, oocytes went on to arrest at MetI. This 2+ after a Ca rise, Emi2 was degraded by at least 50% before would be consistent with Emi2 inhibiting the APC at this cyclin B1. An anti-Emi2 antibody also revealed Emi2 protein time and the requirement for APC activity in order for mouse in pronucleate embryos, which is in agreement with the obser- oocytes to complete the fi rst meiotic division (Herbert et al., vations of Shoji et al. (2006), demonstrating that Emi2 reaccu- 2003; Terret et al., 2003; Hyslop et al., 2004; Homer et al., mulates in one-cell embryos and, thus, interestingly, may have 2005). Interestingly, the effect of Emi2 knockdown on oocyte 2+ a mitotic function. We have previously reported on Ca spiking maturation is the same as that obtained when expression of during the fi rst mitotic division of mouse embryos (Kono et al., cyclin B1 is prevented by antisense during oocyte maturation 1996), suggesting that in this study Emi2 may be degraded by (Ledan et al., 2001). In oocytes matured with cyclin B1 anti- 2+ a similar Ca -dependent process. sense, MPF activity is not reestablished after PB1 extrusion, In MetII-arrested mouse oocytes, the APC is already ac- and a single pronucleus forms. 2+ tive, but the addition of a Ca signal speeds up cyclin B1 deg- The Emi2 phenotype observed on in vitro–matured oocytes radation sixfold through an increase in APC activity (Nixon to some extent resembles the siRNA Emi2 knockdown pheno- et al., 2002). In mouse, the high levels of cyclin B1 required for type obtained previously (Shoji et al., 2006), with decondensed MetII arrest are therefore achieved through equilibrium between chromatin in the parthenote. In that study, however, there was synthesis and degradation (Kubiak et al., 1993). Induction of a no analysis made of the spindle during the normal time course spindle-assembly checkpoint by nocodazole prevents cyclin B1 of maturation; instead, observations were made some 30 h after 2+ degradation, with or without the presence of a Ca signal microinjection. Therefore, in such circumstances it was not pos- (Nixon et al., 2002), showing that in mouse oocytes APC activ- sible to judge how the oocyte was progressing through meiosis I, ity can be held in check by both CSF and a spindle-assembly for example if a MetII spindle formed and remained stable for checkpoint. In mouse, the spindle-assembly checkpoint is dis- some hours before extruding a PB and exiting meiosis II. Also, tinct from CSF (Tsurumi et al., 2004). In contrast to cyclin B1, the diffi culty in interpretation at this single time point was exac- we found little evidence of Emi2 protein turnover in MetII- erbated in this strain of mouse by the degeneration of the PB1 arrested ooc ytes, suggesting that Emi2 itself is not a substrate of during the 30-h period. 2+ the APC. Indeed, upon a Ca signal, destruction of X. laevis The present experiments were designed to examine in Emi2 has been shown to occur via the phosphorylation of a detail the role of Emi2 during the normal timings of meiosis I. 798 JCB • VOLUME 174 • NUMBER 6 • 2006 It was found that Emi2 was essential to restabilize cyclin B1 Previously, all CSF candidates demonstrated in frog ap- during interkinesis, and this rise in cyclin B1 was necessary to peared to play either no role in CSF activity in mouse or are build a bipolar spindle. This is a previously unreported role for involved in only the maintenance of arrest once a MetII spin- Emi2 in either frog or mouse and may add clarity to the function dle is established. In this study, we have demonstrated that in of CSF in mouse. Given that species-specifi c differences appear the absence of Emi2, mouse oocytes fail to form a bipolar to exist, it is unclear whether this role in establishing MetII MetII spindle and, thus, fail to establish MetII arrest because arrest would be conserved in frog. In frog, several studies have of an inability to elevate cyclin B1 levels in interkinesis. elegantly identifi ed the c-Mos–MAPK–p90rsk–Bub1 pathway Therefore, these data, together with rescue of the Emi2 MO (Sagata et al., 1989; Kosako et al., 1994; Bhatt and Ferrell, phenotype using exogenous ∆90 cyclin B1 added after PB1 1999; Gross et al., 1999, 2000; Tunquist and Maller, 2003) and extrusion, imply that the driving force in establishing a bipolar the spindle checkpoint proteins Mad1 and Mad2 as essential for spindle at the end of meiosis is a restabilization of cyclin B1 the establishment of MetII arrest (Tunquist et al., 2003). Once by Emi2. established, Emi2 contributes to the maintenance of MetII arrest (Liu and Maller, 2005; Rauh et al., 2005; Schmidt et al., Materials and methods 2005; Tung et al., 2005; Hansen et al., 2006), and is perhaps All chemicals were obtained from Sigma-Aldrich, unless otherwise stated, also required for the establishment of MetII arrest; however, and were of tissue culture or embryo-tested grade where appropriate. this requires further investigation. When compared with reports in frog, the mechanism of Gamete collection and culture 4–6-wk-old MF1 mice (Harlan) were used. GV oocytes and MetII oocytes the establishment of MetII arrest in mouse is poorly understood. were collected from hormonally primed animals, as previously described In contrast to frog, there have been no fi rm candidates suggested (Nixon et al., 2002; Hyslop et al., 2004). For bench handling, microinjec- to be involved in establishment. Oocytes from c-Mos −/− mice tions, and imaging experiments, GV oocytes and MetII oocytes were cul- tured in medium M2. When necessary, oocytes were arrested at the GV initially arrest at MetII for 2–4 h, but then fail to maintain stage in medium M2 containing 1 μM milrinone. For longer term incuba- this arrest and exhibit spontaneous parthenogenetic activation tion, GV oocytes and maturing oocytes were held in a 5% CO -humidifi ed (Colledge et al., 1994; Hashimoto et al., 1994; Verlhac et al., incubator at 37°C and cultured in MEM (Invitrogen) with 20% fetal calf serum. Parthenogenetic activation of MetII oocytes was achieved by washing 1996). Verlhac et al. (1996) demonstrated convincingly that 2+ oocytes into Ca -free M2 media containing 10 mM SrCl (Bos-Mikich although c-Mos is required for the stabilization of MPF once et al., 1997; Madgwick et al., 2004). Cycloheximide was added to media MetII is achieved, neither c-Mos nor MAPK are involved in at a concentration of 10 μg/ml, and nocodazole was added at a concen- tration of 100 nM. In the case of nocodazole, oocytes were incubated for MPF reactivation during the MetI–MetII transition. These data 15 mins before parthenogenetic activation. suggest that c-Mos is part of the maintenance of MetII arrest, but not its establishment. In agreement with this, the addition Microinjection and imaging Microinjection of MOs and cRNA constructs were made as described pre- of MAPK inhibitors to MetII oocytes induces parthenogenetic viously on the heated stage of an inverted microscope (TE300; Nikon) activation (Phillips et al., 2002). Therefore, it is clear that the fi tted for epifl uorescence (Nixon et al., 2002). In brief, fabricated micro- c-Mos–MAPK pathway contributes to MetII arrest once it is pipettes were inserted into cells using the negative capacitance overcom- pensation facility on an electrophysiological amplifi er (World Precision established. In any event, MAPK activity declines after cyclin Instruments); this procedure ensures a high rate of survival (>90%). Images B1 degradation, so MAPK is not targeted immediately by the were recorded using a 20× objective, NA 0.75, and a charge-coupled 2+ Ca signal at fertilization (Verlhac et al., 1994). Similarly, the device camera (Micromax 1300Y; Sony). Filter sets were 430 ±10 nm and 500 ±10 excitation fi lters for Cerulean and Venus, respectively main downstream target of MAPK activity in frog appears to (Coherent) with a CFP/YFP 51017 BS&M dichroic mirror and emission have little role in MetII arrest in mouse because oocytes from fi lter (Chroma Technology Corp.). Hoechst staining was examined using p90rsk knockout mice (Dumont et al., 2005) still arrest at MetII. a 360 ±10.9 nm excitation fi lter (Chroma Technology Corp.). All live cell imaging was performed in medium M2 at 37°C. Confocal sections of Furthermore, oocytes injected with dominant-negative forms Hoechst and Texas red staining were performed on a confocal microscope of Bub1 and Mad2 still arrest at MetII (Tsurumi et al., 2004), (SP2; Leica) to capture images of the spindle microtubules and chromatin. suggesting that spindle checkpoint proteins are not involved in MetaMorph and MetaFluor imaging software (Universal Imaging Corp.) were used for image capture and data analysis. MetII arrest in mouse. Although not involved in the establishment of MetII arrest, cRNA constructs and MOs MAPK still appears to have a role in mouse oocytes before the Using primers specifi cally for the mouse homologue of the Emi2 gene (AK030012; National Center for Biotechnology Information), we ampli- formation of a MetII spindle through a MAPK-interacting and fi ed full-length mouse Emi2 by PCR from a mouse MetII-arrested oocyte spindle-stabilizing protein (MISS; Lefebvre et al., 2002). MISS cDNA library. In brief, the cDNA library was prepared by collecting 750 is present at meiosis I, but only becomes stable in meiosis II, MetII oocytes in sterile PBS. Oocytes were lysed immediately, and mRNA was isolated directly using a commercial kit according to the manufactur- where it is essential for the maintenance of spindle integrity. ers instruction (Dynabeads mRNA Direct kit; Dynal). Double-stranded Oocytes maturing in the absence of MISS reach MetII arrest, but cDNA was synthesized from RNA and amplifi ed by PCR using a cDNA with severe spindle disorganization, including monopolar spin- synthesis kit (SMART PCR cDNA Synthesis kit; CLONTECH Laboratories, Inc.). Mouse Emi2 was then cloned from the cDNA library into a modifi ed dles, numerous asters in the cytoplasm, or no spindle (Lefebvre pRN3 vector, designed to produce mRNA transcript C-terminally coupled et al., 2002). However, because spindle defects in these oocytes to Venus. Full-length cyclin B1 and ∆90 cyclin B1 were amplifi ed by PCR are not associated with a drop in cyclin B1 levels or histone H1 as previously described (Madgwick et al., 2004). Full-length cyclin B1 was cloned into two modifi ed pRN3 vectors designed to produce mRNA tran- activity, it is suggested that MISS does not have a direct role in scripts C-terminally coupled to Venus or Cerulean; ∆90 cyclin B1 was CSF arrest. Instead, MISS plays a role in microtubule dynamics cloned into a nonfl uorescent pRN3 vector (Madgwick et al., 2004). Mad2 through an interaction with a microtubule-associated protein. was manufactured as described in Madgwick et al. (2005). cRNA maximal EMI2 AND MEIOSIS II ENTRY • MADGWICK ET AL. 799 Eytan, E., Y. Moshe, I. Braunstein, and A. Hershko. 2006. Roles of the anaphase- stability was conferred in all constructs by the presence of a 5′ globin UTR promoting complex/cyclosome and of its activator Cdc20 in functional upstream and both 3′UTR and poly (A)–encoding tracts downstream of the substrate binding. Proc. Natl. Acad. Sci. USA. 103:2081–2086. gene. cRNA was synthesized using T3 mMESSAGE mMACHINE (Ambion) Fang, G., H. Yu, and M.W. Kirschner. 1998. The checkpoint protein MAD2 and dissolved in nuclease-free water to the required micropipette concen- and the mitotic regulator CDC20 form a ternary complex with the tration. An antisense MO (Genetools) was designed to recognize the anaphase-promoting complex to control anaphase initiation. Genes Dev. 5′UTR of Emi2. Two further controls were also designed, the invert of this 12:1871–1883. region and a 5mp (for sequences see Table I). All MOs were used at a Glotzer, M., A.W. Murray, and M.W. Kirschner. 1991. Cyclin is degraded by the micropipette concentration of 2 mM. ubiquitin pathway. Nature. 349:132–138. Gross, S.D., M.S. Schwab, A.L. Lewellyn, and J.L. Maller. 1999. Induction of Anti-Emi2 antibody purifi cation metaphase arrest in cleaving Xenopus embryos by the protein kinase An MBP fusion of the human Emi2 N terminus (residues 1–400; hEmi2N) p90Rsk. Science. 286:1365–1367. and a GST fusion of the mouse Emi2 N terminus (residues 1–398; mEmi2N) Gross, S.D., M.S. Schwab, F.E. Taieb, A.L. Lewellyn, Y.-W. Qian, and J.L. were bacterially expressed and purifi ed using standard protocols, with the Maller. 2000. The critical role of the MAP kinase pathway in meiosis II exception that all procedures involving GST-mEmi2N were conducted in in Xenopus oocytes is mediated by p90Rsk. Curr. Biol. 10:430–438. the presence of 0.01% Triton X-100 to maintain protein solubility. Sera Hansen, D.V., J.J. Tung, and P.K. Jackson. 2006. CaMKII and Polo-like kinase 1 from rabbits immunized against MBP-hEmi2N were screened for immuno- sequentially phosphorylate the cytostatic factor Emi2/XErp1 to trigger its blot cross- reactivity toward in vitro-translated mouse Emi2. GST-mEmi2N, destruction and meiotic exit. Proc. Natl. Acad. Sci. USA. 103:608–613. immobilized on a column of CNBr-activated Sepharose 4B (17–0430-01; Harper, J.W., J.L. Burton, and M.J. Solomon. 2002. The anaphase-promoting GE Healthcare), was used to affi nity purify anti-mEmi2 antibodies from complex: it’s not just for mitosis any more. Genes Dev. 16:2179–2206. cross-reactive sera. Antibodies were eluted from the column using 100 mM Hashimoto, N., N. Watanabe, Y. Furuta, H. Tamemoto, N. Sagata, M. Yokoyama, glycine, pH 2.5. K. Okazaki, M. Nagayoshi, N. Takedat, Y. Ikawatll, and S. Aizawai. 1994. Parthenogenetic activation of oocytes in c-mos-defi cient mice. Nature. Immunoblotting 370:68–71. For Emi2, lysates from oocytes were incubated overnight with anti-mEmi2 Herbert, M., M. Levasseur, H. Homer, K. Yallop, A. Murdoch, and A. McDougall. in the range of 2.5 to 10 μg/ml. Nonfat milk (5%) was used as a blocking 2003. Homologue disjunction in mouse oocytes requires proteolysis of solution. Anti–rabbit IgG (P0448; DakoCytomation; Figs. 1 and 2; NA934; securin and cyclin B1. Nat. Cell Biol. 5:1023–1025. GE Healthcare; Fig. 4) and ECL Plus (RPN2132; GE Healthcare) were used Homer, H.A., A. McDougall, M. Levasseur, K. Yallop, A.P. Murdoch, and M. as secondary detection reagents. Herbert. 2005. Mad2 prevents aneuploidy and premature proteolysis For cyclin B1, lysates from oocytes were incubated overnight with of cyclin B and securin during meiosis I in mouse oocytes. Genes Dev. anti-cyclin B1(1:400; Abcam; ab72). Nonfat milk (5%) was used as a 19:202–207. blocking solution. Anti–mouse IgG (P0447; DakoCytomation) and ECL Plus Hyslop, L., V. Nixon, M. Levasseur, F. Chapman, K. Chiba, A. McDougall, J. 2+ were used as secondary detection reagents. Venables, D. Elliott, and K.T. Jones. 2004. Ca -promoted cyclin B1 degradation in mouse oocytes requires the establishment of a metaphase arrest. Dev. Biol. 269:206–219. Immunostaining of oocytes 2+ Immunostaining of cyclin B1 was performed as previously described (Reis Jones, K.T. 1998. Ca oscillations in the activation of the egg and development et al., 2006). In brief, oocytes were fi xed and permeabilized by an incuba- of the embryo in mammals. Int. J. Dev. Biol. 42:1–10. 2+ tion in 3.7% paraformaldehyde in PBS (30 min), followed by incubation in Jones, K.T. 2005. Mammalian egg activation: from Ca spiking to cell cycle 3.7% paraformaldehyde, 2% Triton X-100 in PBS (30 min). Fixed oocytes progression. Reproduction. 130:813–823. were then washed extensively in 1% PVP and 1% BSA in PBS. For spindle Knott, J.G., M. Kurokawa, R.A. Fissore, R.M. Schultz, and C.J. Williams. 2005. staining, oocytes were incubated with rat anti-tubulin antibody (YL1/2, Transgenic RNA interference reveals role for mouse sperm phospholi- 2+ 1:40, Abcam). 5 μg/ml Texas red conjugated anti–rat goat IgG (Abcam) pase Cζ in triggering Ca oscillations during fertilization. Biol. Reprod. was used as a secondary antibody. To stain chromatin, oocytes received 72:992–996. an additional incubation in 10 μg/ml Hoechst 33258. For cyclin B1 stain- Kono, T., K.T. Jones, A. Bos-Mikich, D.G. Whittingham, and J. Carroll. 1996. 2+ ing, oocytes were incubated with mouse anti-cyclin B1 antibody (1:400; A cell cycle-associated change in Ca releasing activity leads to the 2+ generation of Ca transients in mouse embryos during the fi rst mitotic Abcam). TRITC-conjugated anti–mouse rabbit IgG/IgM/IgA was used as division. J. Cell Biol. 132:915–923. a secondary antibody (1:40; DakoCytomation). Kosako, H., Y. Gotoh, and E. Nishida. 1994. Mitogen-activated protein kinase We thank Jonathan Pines for the gift of Cerulean fl uorescent protein. kinase is required for the mos-induced metaphase arrest. J. Biol. Chem. This work is supported by a project grant (069236) and equipment 269:28354–28358. grant (065354) from the Wellcome Trust to K.T. Jones. Kramer, E.R., C. Gieffers, G. Holzl, M. Hengstschlager, and J.-M. Peters. 1998. Activation of the human anaphase-promoting complex by proteins of the Submitted: 21 April 2006 CDC20/Fizzy family. Curr. Biol. 8:1207–1210. Accepted: 8 August 2006 Kubiak, J.Z., M. 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Published: Sep 11, 2006

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