237Development 125, 237-248 (1998)Printed in Great Britain © The Company of Biologists Limited 1998DEV3743

Analysis of the early embryonic cell cycles of Xenopus ; regulation of cell

cycle length by Xe-wee1 and Mos

Monica S. Murakami and George F. Vande Woude*

ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA*Author for correspondence (e-mail: woude@ncifcrf.gov)

Accepted 17 October 1997; published on WWW 17 December 1997

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In Xenopus, cdc2 tyrosine phosphorylation is detected inthe first 60-75 minute cell cycle but not in the next elevencell cycles (cycles 2-12) which are only 30 minutes long.Here we report that the wee1/cdc25 ratio increases beforethe first mitotic interphase. We show that the Xe-wee1protein is absent in stage VI oocytes and is expressed frommeiosis II until gastrulation. A dominant negative form ofXe-wee1 (KM wee1) reduced the level cdc2 tyrosinephosphorylation and length of the first cycle. However, theratio of wee1/cdc25 did not decrease after the first cycle andtherefore did not explain the lack of cdc2 tyrosinephosphorylation in, nor the rapidity of, cycles 2-12.Furthermore, there was no evidence for a wee1/myt1inhibitor in cycles 2-12. We examined the role of Mos in thefirst cycle because it is present during the first 20 minutesof this cycle. We arrested the rapid embryonic cell cycle

(cycle 2 or 3) with Mos and restarted the cell cycle withcalcium ionophore; the 30 minute cycle was converted intoa 60 minute cycle, with cdc2 tyrosine phosphorylation. Inaddition, the injection of a non-degradable Mos (MBP-Mos) into the first cycle resulted in a dramatic elongationof this cycle (to 140 minutes). MBP-Mos did not delay DNAreplication or the translation of cyclins A or B; it did,however, result in the marked accumulation of tyrosinephosphorylated cdc2. Thus, while the wee1/cdc25 ratiochanges during development, these changes may not bresponsible for the variety of cell cycles observed duringearly Xenopusembryogenesis. Our experiments indicatethat Mos/MAPK can also contribute to cell cycle length.

Keywords: Xenopus, Wee1, Mos, cdc2 tyrosine phosphorylation, cecycle

SUMMARY

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INTRODUCTION

In Xenopus, the standard somatic cell cycle is modified duroocyte maturation and early embryogenesis (Kirschner Gerhart, 1981; Newport and Kirschner, 1982, 1984). Oocmaturation consists of two consecutive M-phases withoutintervening S-phase and results in an egg arrested at metapof meiosis II. Fertilization releases the arrest and initiates first mitotic cycle, which has two atypical ‘gap’ phases and60-75 minutes long. The next eleven cycles are composealternating S- and M-phases and are only 30 minutes long. length of the first embryonic cell cycle is curious since the vstores of nutrients and the lack of transcriptional requiremeeliminate the need for growth or gap phases during eaembryogenesis (Gerhart, 1980). Here we begin to examthese early embryonic cell cycles in vivo.

The meiotic and mitotic cycles of Xenopusare similar in thatentry into these cycles is regulated by Maturation PromotFactor (MPF), a complex of cdc2 and cyclin B. The catalyactivity of cdc2 is regulated by oscillating levels of cyclin and phosphorylation on three critical residues (for review Murray and Kirschner, 1989; Norbury and Nurse, 199Phosphorylation on Thr 161 is required for full activation

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the kinase, however, this phosphorylation does not appeabe regulated during the cell cycle (for review see Morga1995). In contrast, the phosphorylation of cdc2 at Tyr 15 aThr 14 is inhibitory and tightly regulated during the cell cycl(for review see Dunphy, 1994). Immature Xenopusoocytescontain inactive stores of cyclin B complexed to cdc2 (prMPF), the absence of biological or enzymatic activity maintained by these inhibitory phosphorylations (Dunphy aNewport, 1989; Gautier and Maller, 1991). Entry into M-phasis regulated, in part, by the dephosphorylation of Tyr 15 aThr 14 executed by the cdc25 phosphatase (for review Dunphy, 1994). In Drosophila,these phosphatases (string andtwine) are developmentally regulated. The appropriaexpression of each isoform is critical for meiosis and thtransition from maternal to zygotic control (for review seEdgar, 1995; Orr-Weaver, 1994).

The inhibitory phosphorylations on Tyr 15 and Thr 14 arendered by at least two kinases; wee1 and myt1. All wehomologues isolated to date phosphorylate cdc2 exclusivelyTyr 15 (Booher et al., 1993; Campbell et al., 1995Featherstone and Russell, 1991; Igarashi et al., 1991; Leal., 1994a; McGowan and Russell, 1993; Mueller et al., 1995Parker et al., 1991, 1992; Parker and Piwnica-Worms, 19

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Russell and Nurse, 1987). The phosphorylation of Thr 14 well as Tyr 15) is mediated by myt1, a membrane associakinase that is a distinct member of the wee1 family (AthertoFessler et al., 1994; Kornbluth et al., 1994; Liu et al., 19Mueller et al., 1995b). The regulation of these inhibitokinases occurs primarily at the level of phosphorylation andtightly controlled during the cell cycle. The activity is high iinterphase and low in mitosis (McGowan and Russell, 19Parker et al., 1995; Watanabe et al., 1995). In Xenopus, thenegative regulation of these kinases is mediated, in partcyclin B/cdc2 (Mueller et al., 1995a,b; Tang et al., 1993).

In addition to MPF, oocyte maturation in Xenopusalsorequires the Mos protooncogene. In Xenopus, Mos is necessarfor entry into meiosis I (Sagata et al., 1988) and meiosis(Daar et al., 1991; Kanki and Donoghue, 1991). Mos is ainvolved in the suppression of DNA synthesis during meio(Furuno et al., 1994) and it is a key component of cytostafactor (CSF), which maintains the metaphase arrest at meII (Sagata et al., 1989). Fertilization of the egg causes destruction of Mos which follows the destruction of MPF bapprox. 20 minutes (Watanabe et al., 1991).

In this report we examine the regulation of the embryocell cycles in vivo. We show that the level of Xe-wee1 proteincreases during oocyte maturation, increasing the wee1/cdratio before the first mitotic interphase. A dominant negatwee1 (KM wee1) reduced the level of cdc2 tyrosiphosphorylation and shortened the length of the cell cyindicating that Xe-wee1 does, in part, contribute to the lenof this cycle. However, the changes in the wee1/cdc25 rcannot mediate all of the cell cycle modifications which occduring early Xenopusembryogenesis. We show that thactivation of the Mos/MAPK pathway in the early embryoncell cycle can result in a longer cell cycle.

MATERIALS AND METHODS

Cloning and mutagenesisA Xenopuswee1 homologue was isolated by screening an ovarcDNA library (Stratagene, La Jolla) at low stringency (30formamide) with the partial human wee1 cDNA (generously providby H. Okayama). At the amino acid level, Xe-wee1 shares 2identity with both S. pombeand S. cerevisiaewee1, 57% identity withthe human wee1 and 92% identity with the Xenopuswee1 isolated byMueller (1995a). The GenBank ascension number is AF035443.

The lysine at position 242, in the ATP binding motif, was mutatedan isoleucine by PCR-based site directed mutagenesis. mutagenized fragments were generated using the external veprimers (T3 and T7 from Promega) and the following mutageoligonucleotide (sense and antisense) – the mutated nucleotide bold and underlined; 5′GGTTGTTTCTATGTCATCATACGCTC-CAAGAAGCCATTGGC 3′. 200 pmol of each oligonucleotide waused with 1 µg target DNA, with 25 cycles of 45 seconds at 94°C, seconds at 50°C and 2 minutes at 72°C. The mutagenized fragmwere assembled in a second PCR reaction using only the T3 anvector primers with the two mutagenized fragments as template. resultant DNA was digested with BstEII and BamHI, this fragment wasused to replace the corresponding wild-type fragment. The enregion derived from the PCR mutagenesis procedure was sequencverify the point mutation.

Kinase assaysXe-wee1 kinase assaysIn vitro translated (TNT, Promega) Xe-wee1 was incubated with p

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immune or immune sera (Ab 725 or 1532 – 50 µl TNT reaction: 2µlantibody: 4 µg peptide where indicated). The Xe-wee1 immuncomplexes were collected on Protein-A Sepharose (Pharmacia) washed 3 times with Wash buffer (50 mM Hepes pH 7.5, 150 mNaCl, 5 mM EDTA, 5 mM EGTA) and once with Kinase buffer (20mM Hepes pH 7.5, 150 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 10µM ATP, 1 mM DTT, 10% sucrose). The Xe-wee1 immune complexewere incubated in Kinase buffer supplemented with 10 µCi [γ-32P]ATP (3000 Ci/mmol, Amersham) and kinase-inactivecyclinB/cdc2 complex (2 µl; generously provided by Margaret S. Leeand H. Piwnica-Worms) in a 25 µl reaction for 20 minutes at roomtemperature (RT). For the phosphoamino acid analysis, the kinaassays were resolved by SDS-PAGE and transferred to immobimembranes (Millipore). The band corresponding to cdc2 wasubjected to acid hydrolysis and 2D electrophoresis as describedBoyle et al (1991). The effect of Xe-wee1 on cyclin B/cdc2 histonH1 kinase activity was analyzed as described above except that Xe-wee1 immune complexes were incubated in Kinase buffer wi‘kinase active’ MPF (5 ng, UBI) for 15 minutes at RT, after whichtime 2 µg histone H1, 10 µCi [γ-32P]ATP and PKA inhibitor peptide(5 µM final) was added and the reaction was continued for anotherminutes at RT.

Histone H1 kinase assaysOocyte/embryo lysates were prepared in modified EB (80 mM β-glycerophosphate, 20 mM Hepes pH 7.5, 20 mM EGTA, 15 mMMgCl2, 1 mM sodium vanadate, 50 mM NaF, 20 mM sodiumpyrophosphate, 2 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, 50µg/ml leupeptin, 2 µg/ml pepstatin, 1 µM microcystin, 2.5 µMokadaic acid) at 10 µl/oocyte or embryo. The lysates were clarifiedby centrifugation at 15,000 r.p.m. for 15 minutes at 4ºC. 5 µl of theresulting supernatant was added to 20 µl of H1 kinase reaction mix(final concentrations, 20 mM Hepes pH 7.5, 5 mM EGTA, 10 mMMgCl2, 0.1 mM ATP, 5 µM PKA inhibitor peptide, 2 µg histone H1and 10 µCi [γ-32P]ATP). The reactions were incubated at roomtemperature for 15 minutes then resolved by SDS-PAGE.

DNA replication assaysIn vitro matured oocytes were injected with 1 µCi [α-32P]dCTP attime 0 minutes. At the appropriate interval, oocytes/eggs wecollected, solubilized in 1% SDS, 10 mM Tris, pH 7.0 and 10 mMEDTA. Lysates were incubated for 1 hour with 10 µg/ml proteinaseK, then the nucleic acids were extracted with phenol and precipitawith ethanol. Samples were resolved on a 0.8% TAE agarose which was subsequently dried and exposed for autoradiography.

Oocyte and embryo manipulationStage VI oocytes were isolated by manual dissection in 1× ModifiedBarth’s Saline (Specialty Media, Lavallette, NJ) from unprimefemales (XenopusI, Ann Arbor MI). Oocytes were cultured at 16°Cin Ooycte Culture Medium (60% Leibovitz-15 medium, 0.4 mM L-glutamine, 1% penicillin-streptomycin (Gibco) and 0.04% BSA)Maturation was induced with 10 µg/ml progesterone. GVBD wasscored by the appearance of a white spot in the animal hemisphand confirmed after fixation in 10% TCA. Nuclear and cytoplasmfractions were prepared following manual dissection of the germinvesicle in isolation medium (83 mM KCl, 17 mM NaCl, 6.5 mMNa2HPO4, 3.5 mM KH2PO4). All fractions were resuspended in EB(10 µl/oocyte, nuclei or cytoplasm) and clarified by centrifugationInjections into stage VI oocytes were done in OCM. MBP-Mos fusioprotein was prepared as described by Yew (1992) and injected tfinal concentration of approximately 9 ng/oocyte. RNAs wertranscribed and capped in vitro using the mMessage machine (Ambion), resuspended in DEPC treated H2O and injected at approx.40 ng/oocyte except as indicated.

Unfertilized eggs were isolated either by in vitro maturation ostage VI oocytes, or by injecting female frogs with 800 U Huma

239Early embryonic cell cycles in Xenopus

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Chorionic Gonadotropin 12-16 hours prior to isolation. For tgeneration of embryos, unfertilized eggs were washed twice in×MMR and fertilized with a macerated testis. 15-30 minutes affertilization the embryos were dejellied (in 2% cysteine/0.3× MMR)and injected with RNA at the one or two cell stage (in 1× MMR/5%Ficoll). In some experiments, the embryos were allowed to devenormally and the stages were determined according to NieuwkoopFaber (1967).

Eggs were dejellied prior to activation with ionophore. In vitrmatured oocytes, eggs and embryos were washed twice in 1× MMR,then incubated in A23187 calcium ionophore for 5-10 minutes at (Boehringer Mannheim; 0.5 µg/ml in 1× MMR for eggs and in vitromatured oocytes; 0.3 µg/ml A23187 in 0.3×MMR for embryos), thenwashed 3×in 0.1× MMR. Embryos were monitored carefully andtransferred to 0.1× MMR as soon as a change in cell shape wevident.

Western blotting and immunoprecipitationLysates were prepared in EB and 1-1.5 oocytes or embryo equivawere loaded per lane. For the analysis of Xe-wee1, two antibowere generated by immunizing rabbits with the following peptideKAAELQKQLNVEKFKTAMLERELQAAK (Ab 725) andCRGRKRLVGAKNARSLSFT (Ab 1532). Antibodies 1532 and 72were used at a dilution of 1:25 for immunoprecipitation and antibo725 was used at a dilution of 1:1000 for immunoblotting. Mos (S86 @ 1:200), MAPK (SC-154 @1:200) and cdc2 (SC-54@1:20antibodies were purchased from Santa Cruz Biotechnology. CyA1, B1 and B2 antisera (1:750) were a generous gift from J. Ma(University of Colorado). Immunoblotting was completed with aHRP-conjugated goat anti-rabbit or sheep secondary antib(Boehringer Mannheim) and ECL (Amersham) detection.

For the analysis of cdc2 tyrosine phosphorylation, lysates from 15 oocytes or embryos were incubated with 30-50µl of p13/suc1beads (Oncogene Science) overnight at 4ºC. Although the presenendogenous Xenopus suc1 might interfere with the completecollection of cdc2, the use of p13/suc1 beads has been reporteefficiently deplete extracts of cdc2 (Dunphy and Newport, 198Izumi and Maller, 1995). The beads were washed 3× in Wash buffer,subjected to SDS-PAGE and transferred to Immobilon membrawhich were incubated with the anti-phosphotyrosine antibody 4G(1:1000, generously provided by D. Morrison). The analysis wcompleted with an HRP-conjugated goat anti-mouse secondantibody and ECL detection.

RESULTS

A Xenopushomologue of wee1 was isolated from an ovaricDNA library (GenBank accession AF035443). As it shar92% identity, at the amino acid level, with the Xenopuswee1homologue isolated by Mueller et al. (1995a), we consideto be another isoform rather than a distinct family member. confirmed that this cDNA encoded a bona fide wee1 by sevcriteria. The in vitro translated Xe-wee1 cDNA was used in immune complex kinase assay with kinase-inactive cycB/cdc2 as a substrate. Xe-wee1 efficiently phosphorylacdc2 (Fig. 1A, lane 2) and phosphoamino acid analydemonstrated that cdc2 was phosphorylated exclusivelytyrosine (Fig. 1B). This phosphorylation was functional asinhibited the histone H1 kinase activity of active cyclinB/cdccomplexes (Fig. 1C, lanes 3 and 6). We analyzed the biologactivity of Xe-wee1 by injecting in vitro transcribed RNA intstage VI oocytes. Following incubation for 16 hours at 18°progesterone was added and oocyte maturation was monitoInjection of 12.5 ng of RNA, transcribed in the sense directio

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efficiently inhibited oocyte maturation, while injection of 50ng RNA, transcribed in the antisense direction, had virtuano effect (Fig. 1D). Thus, Xe-wee1 inhibits entry into meiosas well as mitosis (Mueller et al., 1995a).

Northern analysis of endogenous Xe-wee1 RNA in adutissues revealed a single transcript exclusive to the ovary (shown). The endogenous Xe-wee1 protein was characteriby western analysis using antibody 725, which detected tproteins in egg lysates of approximately 75and 85×103 Mr (Fig.1E, lane 3). Only the lower band represents Xe-wee1: the lowband was not present in uninjected stage VI oocytes, appeared upon injection of Xe-wee1 RNA (Fig. 1E, lanes 1 a2; Fig. 1F); the lower band was immunodepleted animmunoprecipitated with two different Xe-wee1 specifiantisera (Fig. 1E, lanes 5-10); and the lower band wrecognized by a third antibody generated against the enkinase domain (data not shown). Interestingly, western analyindicated that Xe-wee1 was not present in stage VI oocytbut it was readily detected after injection of full length Xewee1 RNA (Fig. 1F). Following injection of Xe-wee1 RNA,the protein product was found to be equally distributebetween the nucleus and cytoplasm (Fig. 1F, lanes 4-6).

Analysis of endogenous Xe-wee1 during oocyte maturatirevealed that the protein product appeared at the onsemeiosis II (Fig. 2A). Using unfertilized eggs activated witionophore, we also found that the levels of Xe-wee1 did nchange during the first cell cycle but that the electrophoremobility increased during interphase and decreased in M-ph(Fig. 2B and Mueller et al., 1995a). During earlyembryogenesis, the Xe-wee1 protein was detected in embrat fairly constant levels until mid-gastrulation, however, it waundetectable after neurulation (Fig. 2C). The modest increat the medium cell blastula stage was not consistenobserved. Thus, Xe-wee1 is synthesized at the onset of meiII and is present at constant levels until gastrulation. Texpression pattern of Xe-wee1 differs significantly from thof the opposing phosphatase, Xe-cdc25, which is presenconstant levels from immature stage VI oocytes througastrulation (Hartley et al., 1996; Izumi et al., 1992).

In Xenopus, cdc2 is tyrosine phosphorylated at three poinduring early development: immature stage VI oocyteinterphase of the first cell cycle and after the mid-blastutransition (MBT; Ferrell et al., 1991). As the levels of cdc2do not vary during oocyte maturation or early embryogene(Hartley et al., 1996; Izumi et al., 1992), the synthesis of Xwee1 during oocyte maturation results in an increase in wee1/cdc25 ratio before the first mitotic interphase. Could thincrease in the wee1/cdc25 ratio mediate the cdc2 tyrosphosphorylation observed in the first cell cycle? We tested tpossibility by generating a dominant negative form of Xewee1; the lysine at position 242 was changed to an isoleucThe autophosphorylation activity of the mutated Xe-wee1 (Kwee1) was significantly reduced relative to the wild-type Xwee1 when the in vitro translated gene products were assain an immune complex kinase assay (data not shown). Tbiological activity of KM wee1 was assessed by monitoring theffect on oocyte maturation. Following the injection of RNAinto stage VI oocytes, the expression of wild-type and Kwee1 was verified by western analysis (Fig. 3A legend). contrast to wild-type Xe-wee1 which inhibited progesteroninduced GVBD (Fig. 1D and Fig. 3A), KM wee1 consistentl

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accelerated the rate of maturation (Fig. 3A), however, it did ninduce oocyte maturation in the absence of progesterone.

We examined the effect of KM-wee1 on the first embryoncycle by injecting stage VI oocytes with KM-wee1 RNA. Thexpression of KM-wee1 had no effect on the progression frmeiosis I to meiosis II (data not shown). The oocytes we

Fig. 1.Characterization of Xenopuswee1.(A) Xe-wee1 phosphorylates cyclinB/cdc2. In vitro translated Xe-wee1 wasimmunoprecipitated with Pre-immune(PI) or Immune (I) serum (Ab 725). Theimmune complexes were washed withkinase buffer and incubated with akinase-inactive cyclin B/cdc2 complex(prepared from baculovirus generouslyprovided by M. S. L. and H. P. W.) and[γ-32P]ATP. The phosphorylation ofcyclin B is mediated by the wild-typebaculovirus cdc2 which co-purifies withthe GST-cyclin B. The arrow indicates theposition of cdc2. (B) Xe-wee1phosphorylation of cdc2 occursexclusively on tyrosine. The band in A,corresponding to phosphorylated cdc2,was isolated and subjected to acidhydrolysis followed by 2Delectrophoresis. Circles indicate theposition of the amino acids markerstyrosine (tyr), threonine (thr) and serine(ser). C) Xe-wee1 inhibits the H1 kinaseactivity of cyclin B/cdc2 complexes. Invitro translated Xe- wee1 wasimmunoprecipitated with two anti-peptideantibodies (Ab 725 and 1532), with orwithout competing peptide (I or I/pep) orPre-immune serum (PI). Immunecomplexes were incubated with 5 ng ofactive cyclin B/cdc2 complexes (UBI) for15 minutes at room temperature, afterwhich time, histone H1, [γ-32P]ATP andPKA inhibitor peptide were added and thereaction continued for another 15minutes. Arrow indicates the position ofhistone H1. (D) Xe-wee1 inhibition ofoocyte maturation. Xe-wee1 RNA in vitrotranscribed in either the sense (S) orantisense (AS) direction was injected intoimmature stage VI oocytes and incubatedat 18°C for 16 hours. After this time,progesterone (10 µg/ml) was added andgerminal vesicle breakdown (GVBD) wasscored by the appearance of a white spotin the animal pole. (E) Characterizationof Xe-wee1 antibodies; the lower band represents Xe-wee1. Lysatimmunoblotting with antibody 725. Xe-wee1 was not detected in sNP-40, Brij 35, sarkosyl, octylglucoside; not shown). Lysates wereng Xe-wee1 RNA (lane 2); unfertilized eggs (lane 3); unfertilized e(lane 5); the supernatant from unfertilized egg lysate immunodepleprecipitate from unfertilized egg lysate incubated with Ab 725 (laneinterfere with the detection of the various phosphorylated forms ofmass in an in vitro transcription/translation reaction using a cDNA localization of Xe-wee1. Stage VI oocytes were injected with 40 ngsynthesis. Lysates were prepared from; germinal vesicles (nuc), woocytes (wce). Each lane contains material from approx. 1.5 oocyLanes 1-3, uninjected stage VI oocyte; lanes 4-6, stage VI oocyte

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matured in vitro with progesterone, then treated with ionophorThe activated eggs did not undergo cleavage since they werefertilized, however, the profile of H1 kinase activity was similato that of the first cell cycle in a fertilized embryo (Gerhart et a1984; Hartley et al., 1996). By western analysis, the level of KMwee1 was approx. 2 fold higher than endogenous levels (data

es from 1.5 oocytes or eggs were prepared in EB and subjected totage VI even after using a panel of detergent lysis conditions (Triton X-100, prepared from stage VI oocytes (lane 1); stage VI oocytes injected with 40ggs 30 minutes after treatment with ionophore (lane 4); unfertilized eggsted with Ab 725 (lane 6) or Ab 1532 (lane 7); unfertilized eggs (lane 8); the 9) or Ab 1532 (lane 10). Note that the upper cross reacting species does not

Xe-wee1. Antibody 725 also recognizes a protein of the same molecularencoding the Xe-wee1 isolated by Mueller et al. (not shown). (F) Subcellular Xe-wee1 RNA then left at 18°C for 16 hours to allow for adequatehich were manually isolated; the remaining cytoplasm (cyt) and wholetes. Note that the upper cross reacting species is exclusively cytoplasmic.injected with 40 ng Xe-wee1 RNA.

241Early embryonic cell cycles in Xenopus

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shown). These results were also observed when p13/sprecipitates were used to monitor the level of endogenous KM-wee1 expression (Fig. 3C). The same p13/suc1 precipiwas used to analyze the tyrosine phosphorylation state of cwith the anti-phosphotyrosine antibody 4G10. In control egcdc2 tyrosine phosphorylation was detected 30 minutes followentry into the first cell cycle and was absent by 80 minutes (3D, lower pair of panels). We observed that the expressionKM-wee1 significantly reduced the level of cdc2 tyrosinphosphorylation in the first cell cycle (Fig. 3D, upper pair panels). The absence of cdc2 tyrosine phosphorylation was

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Fig. 2.Characterization of endogenous Xe-wee1. (A) Westernanalysis of Xe-wee1 during oocyte maturation. Oocytes wereinduced to mature with progesterone and harvested at the indicattimes after GVBD. Approximately 1.5 oocytes were loaded per laand processed for western analysis with antibody 725. (B) Westeanalysis of Xe-wee1 during the first mitotic cell cycle. Unfertilizedeggs were dejellied and treated with calcium ionophore for 5minutes, lysates were prepared at the indicated times and subjecto immunoblotting with Ab 725. Each lane contains the lysate fromapproximately 1.5 eggs. (C) Western analysis of Xe-wee1 duringearly embryogenesis. Eggs were fertilized in vitro and lysates weprepared and processed for western analysis with Ab 725. Each contains approximately 1.5 oocytes, eggs or embryos. Arrows anbracket indicate the position of Xe-weel.

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due to lower levels of cdc2 protein in the precipitates (Fig. 3lower blot in each pair). We determined the effect of KM-weeon the length of the cell cycle and found that the cell cycle walways shortened by approx. 10 minutes (Fig. 3B). These dsuggest that Xe-wee1 contributes to the cdc2 tyrosiphosphorylation and the length of this cycle.

We were puzzled by the absence of cdc2 tyrosiphosphorylation in cycles 2-12, as the wee1/cdc25 ratio donot decrease after the first cycle. The levels of Xe-wee1 do decline (Fig. 2C) and the levels of Xe-cdc25 do not increaafter cycle 1 (Hartley et al., 1996; Izumi et al., 1992). While is possible that the levels of myt1 decrease after the first cywe tested for the presence of a wee1/myt1 inhibitor in cyc2-12. We lengthened the rapid embryonic cycles by injectip21/Cip, an inhibitor of cyclin E/cdk2 (El-Deiry et al., 1993)into one blastomere of a 2-cell embryo. A wee1/myt1 inhibitwould prevent cdc2 tyrosine phosphorylation when the rapcell cycles were lengthened. The effects of p21/Cip injectiare shown in Fig. 4A; the injected cells are larger, reflecting tfact that they have undergone fewer cell divisions. We analyzthe state of cdc2 tyrosine phosphorylation by immunoblottinp13/suc1 precipitates with an anti-phosphotyrosine antiboAs previously reported, we detected tyrosine phosphorylacdc2 in stage VI oocytes, during the interphase of the first ccycle, and in gastrula and neurula stage embryos (Fig. 4(Ferrell et al., 1991). We did not detect cdc2 tyrosinphosphorylation in unfertilized eggs, p21/Cip antisense injectembryos, uninjected early blastula stage embryos or embrarrested in M-phase with Mos protein (Fig. 4B). In contrast, wdid detect tyrosine phosphorylated cdc2 in embryos where oblastomere was injected with p21/Cip sense RNA (Fig. 4B, psense). Thus, cdc2 tyrosine phosphorylation was observed wthe rapid cell cycles of the embryos were lengthened wp21/Cip, arguing against the presence of a wee1/myt1 inhibin these cycles.

We postulated that there could be an additional componenthe first cell cycle, that extends the length of this cycle and resin cdc2 tyrosine phosphorylation. A cdk inhibitor such as p21/Cwould be an ideal candidate as it would inhibit DNA replicatiowhile the egg completes meiosis II. However, the Xenopusp21/p27 homologue is highly expressed at gastrulation and vlittle is found during cycles 1-12 (Shou and Dunphy, 1996Alternatively, it is possible that the Mos oncogene could functioto lengthen the first cell cycle; Mos inhibits DNA synthesis durinmeiosis (Furuno et al., 1994) and it is present during the first 30 minutes of the first cycle (Watanabe et al., 1991). To test role of Mos in the regulation of cell cycle length, we injected MoRNA into one cell embryos thereby arresting the embryos at M-phase of cycle 2 or 3 (Sagata et al., 1989). To restart the cycle, the Mos-arrested embryos were treated with calciuionophore which dramatically altered the cell shape and cauthe pigment to migrate to the animal hemisphere (Fig. 5A). Wmonitored the histone H1 kinase activity following activation; thexit from the M-phase arrest was identical in both the Moarrested embryo and unfertilized egg (Fig. 5B, 0-10 minuteStrikingly, the profile of H1 kinase activity following theactivation of the Mos-arrested embryo was also identical to tobserved in the unfertilized egg (Fig. 5B, 0-70 minutes), bodisplay an approx. 60 minute cell cycle. Furthermore, our resushow that the tyrosine phosphorylation of cdc2 which is normadetected during the first interphase (Fig. 5C, left panel) is a

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242 M. S. Murakami and G. F. Vande Woude

Fig. 3.A dominant negative KM-wee1 reduces cdc2tyrosine phosphorylation and shortens the first cellcycle. (A) KM wee1 enhances the rate of oocytematuration. Stage VI oocytes were injected with 40ng of KM wee1 or wild-type RNA, then incubatedfor 16 hours at 18°C. Lysates were prepared prior tothe addition of progesterone and the level ofexpression was determined by western analysis withAb 725, as shown on the right. Oocyte maturationwas initiated with the addition of progesterone at 10µg/ml and GVBD was scored according to theappearance of a white spot in the animal pole.GVBD was later confirmed by dissection followingfixation in 10% TCA. The KM wee1 mediatedacceleration in the rate of oocyte maturation wasobserved in seven separate experiments. KM – 61oocytes, Wild-type – 70 oocytes, Uninjected – 110oocytes. (B) KM wee1 shortens the length of thecell cycle. Stage VI oocytes were injected with 40ng KM-wee1 RNA or left uninjected. The oocyteswere matured in vitro with progesterone (10 µg/ml,16 hours at 18°C) and released from CSF/ meiosis IIarrest by treatment with calcium ionophore (0.5µg/ml in 1× MMR) for 5 minutes. Lysates wereprepared at 10-minute intervals and assayed forhistone H1 kinase activity. Activity was quantifiedon a Fuji Phosphoimager and presented in arbitraryunits. These results are representative of 5 separateexperiments. (C) Level of KM wee1 andendogenous Xe-wee1. The lysates described abovewere incubated overnight at 4°C with p13/suc1beads (Oncogene Science). The upper half of themembrane was reacted with antibody 725. Each lanecontains the p13/suc1 precipitate from the lysates of12 eggs. Note that the upper cross reacting speciesdetected by antibody 725 does not appear in thep13/suc1 precipitate. Similar levels of expressionwere detected using whole cell lysates (not shown).(D) KM wee1 reduces the level of cdc2 tyrosinephosphorylation. The lower half of the membranesdescribed above (Fig. 3C) were reacted with theanti-phosphotyrosine antibody 4G10 (gift from D.Morrison). The blots were stripped and reprobedwith an anti-cdc2 antibody (SC-54, lower blot ineach panel). Although the presence of endogenousXenopussuc1 could influence the amount of cdc2collected, the p13/suc1 beads have been reported toeffectively deplete cdc2 from extracts (Dunphy andNewport, 1989; Izumi and Maller, 1995).

243Early embryonic cell cycles in Xenopus

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Fig. 4. p21/Cip injection slows down the rapid embryonic cell cycleand results in tyrosine phosphorylation of cdc2. (A) Embryosinjected with p21/Cip. One blastomere of a two cell embryo wasinjected with 15 ng p21/Cip RNA (from B. Vogelstein) transcribed inthe sense or antisense direction. Embryos were left at roomtemperature for approx. 4.5 hours, then fixed in 0.1× MMR with 2%formaldehyde. Arrow indicates the position of the larger cells. (B)cdc2 tyrosine phosphorylation in embryos injected with p21/Cip.Lysates from 12 oocyte, egg or embryo equivalents were incubatedwith p13/suc1 beads and processed for immunoblotting withantibody 4G10. p13/suc1 precipitates were analyzed from stage VIoocytes (VI); unfertilized eggs arrested at meiosis II (mei II);unfertilized eggs 40 minutes after ionophore treatment (1stinterphase); embryos injected with p21/Cip sense RNA 4.5 hoursafter injection-stage 7.5 (p21 sense); equivalent embryos injectedwith p21/Cip antisense RNA (p21 antisense); equivalent uninjectedembryos (blastula); stage 11-12 embryos (gastrula); stage 15-16embryos (neurula) and embryos injected with MBP-Mos protein(MBP-Mos). This blot was stripped and reprobed for cdc2 (SC-54)(lower strip).

Fig. 5.Mos converts the 30-minute embryonic cell cycle into a 60-minute cell cycle. (A) Activation of Mos-arrested embryos withcalcium ionophore. One cell embryos were dejellied and injected withMos RNA (20 ng). Embryos arrested at the two or four cell stage weretreated with ionophore approx. 3.5 hours postfertilization (0.3 µM in0.3×MMR) for 5-10 minutes or until a change in cell shape wasevident. Similar ionophore treatment of the uninjected blastula had noobvious effect on the morphology or the time course of the cell cycleof uninjected embryos. Mos-arrested embryo at the two cell stage (leftpanel); 3 minutes (middle panel) and 6 minutes (right panel) afterincubation with ionophore. (B) Analysis of cell cycle following Mosarrest and ionophore activation. Lysates were prepared fromunfertilized eggs or Mos-arrested embryos at 10-minute intervalsfollowing ionophore activation. Histone H1 kinase assays wereperformed, quantified on a Fuji phosphoimager, and presented inarbitrary units. In other experiments, the histone H1 kinase activity ofthe Mos-injected embryos peaked at 60 minutes and declined by 80minutes, indicating that they were not completely arrested in the nextmitosis. These data are representative of 4 separate experiments.(C) Analysis of cdc2 tyrosine phosphorylation following Mos arrestand ionophore activation. Lysates were prepared from unfertilized eggs(left panel) and Mos-arrested embryos (right panel) at 0 and 40minutes following ionophore activation. Following incubation withp13/suc1 beads, the precipitates were processed for immunoblottingwith antibody 4G10. The blots were stripped and reprobed for cdc2(SC-54) (lower strips in each panel).

A

B

C

detected in the interphase following the activation of the Marrested embryo (Fig. 5C, right panel). These results demonsthat the 30 minutes embryonic cycle (with no detectable ctyrosine phosphorylation) can be converted into a 60 minute c(with cdc2 tyrosine phosphorylation) following the arrest wiMos and reactivation with ionophore.

To further assess the role of Mos in the regulation of ccycle length, we examined the consequence of constitutive Mexpression in the first cell cycle. We injected stage VI oocywith either the wild type (WT) or kinase mutant (KM) versionof the MBP-Mos fusion protein. Following maturation in vitrwith progesterone, we activated the eggs and monitoredlevel of endogenous Mos and exogenous MBP-Mos. While

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endogenous Mos was degraded after 20-minutes (Fig. 6A, upper panel), the MBPMos fusion proteins were not degradefollowing activation (Fig. 6A, middle andlower panels). We assessed the tyrosinphosphorylation state of MAP kinase(MAPK) which reflects the activation ofthis signaling cascade by Mos (Nebredand Hunt, 1993; Posada et al., 1993Shibuya and Ruderman, 1993; for reviewsee Kosako et al., 1994). In control eggthe pattern of MAPK tyrosinephosphorylation closely follows the levelsof endogenous Mos: both are present fothe first 20 minutes of the cell cycle (Fig6B, upper and middle pair of panels; Roet al., 1996). However, when the eggs weinjected with the wild-type MBP-Mosfusion protein, MAPK remained tyrosinephosphorylated during the entire timecourse (Fig. 6B, lower pair of panels)These data demonstrate that the MBP Mofusion proteins are not degraded followingactivation and that the wild-type MBP-Mos fusion protein remains activethroughout the entire time course.

The effect of constitutive wild-type Mosexpression was striking, while the kinasinactive MBP-Mos had no effect on thelength of the cell cycle, the wild typeMBP-Mos extended the length of the firscycle to 140 minutes (from 80 minutes(Fig. 6C). These results indicate that thelongation of the cell cycle is not simplydue to an influx of calcium or the recoveryfrom an M-phase arrest. Moreover, theffect of Mos must be mediated throughthe kinase, as the kinase inactive MBPMos has no effect (Fig. 6C).

We examined the mechanism by whicMos mediated this M-phase delay. Weassessed the effect of constitutive Moexpression on the accumulation of cyclinA1 and B1/2. Western analysis shows thawild type MBP-Mos did not markedly alterthe appearance of either cyclin A1 or B1/(Fig. 6D, 0-60 minutes), although itdramatically inhibited the degradation othese cyclins (Fig. 6D, 80-100 minutes)Therefore, the delayed activation of MPFdoes not appear to be mediated through tslower translation of either cyclin A1 orB1/2. We examined the effect of constitutiveMos expression on DNA replication.Previous studies have shown that Mos involved in the suppression of DNAreplication during meiosis (Furuno et al.1994). Furthermore, the inhibition ofreplication in the mitotic cycle by p21/Cip1 in Xenopusextracts (Jackson et al., 1995Strausfeld et al., 1994) blocks the

245Early embryonic cell cycles in Xenopus

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Fig. 6. A non-degradable Mos increases the length of the first cell cyc(A) Western analysis of endogenous Mos and exogenous MBP-Mos.VI oocytes were left uninjected or injected with either 9 ng wild type (MBP-Mos fusion protein or kinase mutant (KM) MBP-Mos fusion proOocytes were matured in vitro with progesterone, washed in 1× MMR thentreated with A23187 for 5 minutes to trigger activation. Lysates wereprepared at the indicated times and 1.5 oocyte equivalents were loadlane. Western analysis was performed with an anti-Mos antibody (SCNote that the endogenous Mos degrades normally in the presence oMBP-Mos proteins. (B) Analysis of MAP kinase tyrosine phosphorylaWhole cell lysates were prepared as described in A. 1.5 oocyte equivwere loaded per lane and analyzed by western analysis with the antiphosphotyrosine antibody 4G10. The blots were then stripped andreprobed with an anti-MAP kinase antibody (SC-154) (lower strips inpair). (C) MBP-Mos increases the length of the first cell cycle. Histonkinase assays were performed on the lysates described in A. Thephosphorylated histone was cut from the gel and the activity was quaby scintillation counting and presented as cpm/minute/mg of histone.(D) Analysis of cyclin synthesis in the presence of MBP-Mos. Whole lysates were prepared as described in A, then analyzed by western awith antibodies directed against cyclin A and cyclin B1 and B2 (gift frJ. Maller). Approximately 1.5 oocyte equivalents was loaded per lane(E) Analysis of DNA replication in the presence of MBP-Mos. Stage Voocytes were injected and matured in vitro as described in A. Eggs wactivated and labeled upon injection of 1 µCi [α-32P]dCTP supplementedwith 0.1 mM CaCl2. At the appropriate interval, lysates from 10 oocytewere prepared and processed for nucleic acid analysis as described Materials and Methods. (F) Analysis of cdc2 tyrosine phosphorylationLysates were prepared from oocytes as described in A, the lysates froocyte equivalents were incubated with 30 µl p13/suc 1 beads overnight 4ºC. The precipitates were washed and processed for western analyantibody 4G10.

subsequent entry into M-phase (Guadagno and Newp1996). However, we observed no differences between the wtype MBP-Mos injected, kinase mutant MBP-Mos injected ouninjected eggs with respect to the timing of the onset of DNreplication (Fig. 6E). These data indicate that Mos does nappear to be solely responsible for the inhibition of replicatioand the Mos mediated elongation of the cell cycle does appear to be mediated through the inhibition of S-phase.

Lastly, we examined the state of cdc2 tyrosinphosphorylation since neither cyclin synthesis nor DNreplication was markedly affected in the presence of witype MBP-Mos (Fig. 6D,E). While the initial appearance ocdc2 tyrosine phosphorylation appears to be unchangedthe presence of the wild type MBP-Mos fusion protein, thlevels and duration of tyrosine phosphorylated cdc2 increasignificantly (Fig. 6F, bottom panel). These differences wenot due to changes in the levels of cdc2 found in tp13/suc1 precipitates (data not shown) and the presencthe KM Xe-wee1 had no significant effect on the levels cdc2 tyrosine phosphorylation or cell cycle length (data nshown). Collectively, these data indicate that the activatiof the Mos/MAPK pathway in the mitotic cell cycle resultsin a longer cell cycle and the accumulation of tyrosinphosphorylated cdc2.

DISCUSSION

In Xenopus, the cycles which precede the MBT are minimcell cycles consisting of alternating S- and M-phas(Newport and Kirschner, 1982). The first cell cycle iunusual in that it appears to have gap phases, yet thereno transcriptional or growth requirements at this point development. In this cycle, the time required for thcompletion of meiosis II appears to account for the ‘gabefore S-phase, while the time required for the fusion of tsperm and egg nuclei may account for the ‘gap’ after S-pha(Gerhart et al., 1984; Gerhart, 1980; Kirschner et al., 198(Fig. 7). This cycle is also unique in that cdc2 is tyrosinphosphorylated in the interphase of this cycle but not in tmeiotic ‘interphase’ or the interphases of cycles 2-12. Wobserved that Xe-wee1 protein was absent in immature stVI oocytes and was synthesized at the onset of meiosisThis pattern of expression results in an increase of the raof wee1 to cdc25 before the interphase of the first cell cycindicating that Xe-wee1 might contribute to the cdctyrosine phosphorylation observed in this cycleInterestingly, these findings also indicate that Xe-wee1 is ninvolved in the generation or regulation of pre-MPF iimmature stage VI oocytes. We examined the role of Xwee1 in the first cell cycle using a dominant negative (KMwee1) which modestly decreased the level of cdc2 tyrosphosphorylation and the cell cycle length (Fig. 3). Whilthese results indicate that Xe-wee1 may, in part, contributo the length of this cycle, the pattern of Xe-wee1 expressdid not explain the lack of cdc2 tyrosine phosphorylationor the rapidity of the following eleven cycles. Thewee1/cdc25 ratio does not decrease during cycles 2-12fact, we found that the levels of Xe-wee1 declined aftgastrulation (Fig. 2C) when cdc2 tyrosine phosphorylatioand cell cycle length are greater.

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Fig. 7. Meiosis, the first cell cycle and the minimal embryonic cell cycle. Oocyte maturation, in response to progesterone, consists of meiosis I(reductive division) and entry into meiosis II. Unfertilized eggs are arrested at metaphase of meiosis II, which is maintained by cytostatic factor(CSF) (Sagata, 1997; Yew et al., 1993). This arrest can also be mediated by Mos (RNA or protein) (Sagata et al., 1989; Yew et al., 1992).Fertilization releases the arrest and is followed by the completion of meiosis II (anaphase, telophase and second polar body extrusion), whichrequires 20-30 minutes. The first round of DNA synthesis begins approx. 30 minutes after fertilization and takes place in two separate nuclei,which fuse after S-phase. Mitosis of the first cycle begins 60-70 minutes after fertilization and is completed by 80 minutes postfertilization.However, cytokinesis continues until 90 minutes postfertilization (Graham, 1966). The onset of the second round of DNA synthesis (cycle 2)begins 85 minutes postfertilization, concurrent with cytokinesis of cycle 1. Thus, the S-phase of the second cell cycle occurs during thecytokinesis of the first cycle, resulting in two rounds of DNA replication between fertilization and the first cytokinesis, (Gerhart, 1980; Graham,1966; Hartley et al., 1996; Miake-Lye et al., 1983).

phosphorylation in cycles 2-12? We tested for a wee1/minhibitor (or cdc25 activator) in cycles 2-12 by slowing dowthe rapid cycles (cycles 2-12) then monitoring the level of cdtyrosine phosphorylation. We hypothesized that the preseof a wee1/myt1 inhibitor would prevent the appearance tyrosine phosphorylated cdc2 when the cell cycles were slowHowever, the injection of p21/Cip into cleaving embryo(which resulted in slower cell divisions) resulted in thdetection of tyrosine phosphorylated cdc2 (Fig. 4). Theresults argue against the presence of a wee1/myt1 inhibitorcdc25 activator) in cycles 2-12.

We then considered the possibility that the first cell cycmight have an additional component (that is degraded beforesecond cycle), which extends the length of the cycle and resin cdc2 tyrosine phosphorylation. A Xenopushomologue ofp21/Cip would be an ideal candidate, however, the expressiothe Xenopusp21/p27 homologue is very low in eggs and earembryos (Shou and Dunphy, 1996). Although it is possible tother inhibitors may be specific to cycle 1, this pattern expression has not been described (Kumagai and Dunphy, 1Lee and Kirschner, 1996; Lee et al., 1994b; Su et al., 1995).tested the role of Mos in the first cell cycle because westanalysis showed that Mos was present during the first 20minutes of the first cycle (Fig. 6A and Roy et al., 1996; Watanaet al., 1991). The injection of Mos into fertilized eggs arrestthe embryos at M-phase of cycle 2 or 3. After the cell cycle wrestarted by ionophore treatment, the resultant cell cycle waminutes long, twice the usual length (Fig. 5). We also deteccdc2 tyrosine phosphorylation in these Mo

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activated/ionophore-activated embryos. It is unlikely that theeffects are the result of the calcium influx; experiments dowith calcium chelators indicate that calcium promotes H1 kinaactivation (Lindsay et al., 1995; Snow and Nuccitelli, 1993Another possibility was that our results were simply thconsequence of arresting and restarting the cell cycle. We furtcharacterized the role of Mos on cell cycle length by injectinthe eggs with a non-degradable form of Mos (MBP- Mos). Thconstitutive presence of the wild type MBP-Mos fusion protein the first mitotic cycle resulted in a significant delay in thactivation of MPF (approx. 140 minute cell cycle) (Fig. 6C). Thdelay of MPF activation was not attributable to slower cyclin or B translation and the onset of DNA replication was naffected (Fig. 6D and E). However, the levels and duration cdc2 tyrosine phosphorylation were significantly greater in tpresence of the MBP-Mos fusion protein (Fig. 6F). While theresults are similar to those of Pan et al. (1994) and Picard e(1996) who showed that the activation of MAPK in interphasresults in a G2 arrest, we observe a M-phase delay rather thanG2 arrest. Collectively, these results suggest that the activatof the MAPK pathway can lead to a longer embryonic cell cyc

The consequence of Mos/MAPK activation varies as thoocyte progresses through maturation to embryogenesis. Wfrom our lab and others has shown that Mos promotes entry imeiosis, prevents exit out of meiosis II (for review see Saga1996), and delays entry into mitosis (this paper). These varioeffects of Mos/MAPK appear to be contradictory, however, wnote that Mos mediated entry into meiosis requires several ho(Yew et al., 1992) and that the cast of cell cycle proteins chan

247Early embryonic cell cycles in Xenopus

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significantly during the course of meiotic maturation (i.e., cycE and Xe-wee1; Rempel et al., 1995; and Fig. 2). In addition,timing of Mos/MAPK activation is critical; CSF arrest requirethe activation of the Mos/MAPK pathway just before, but nduring M-phase (Abrieu et al., 1996), and Mos does not meda M-phase arrest in meiosis I (Yew et al., 1992). We propose the activation of the Mos/MAPK pathway during early interphasedelays the onset of M-phase, while the activation of Mos/MAPin late interphase results in CSF arrest (Abrieu et al., 1996).

At this point the downstream targets of Mos/MAPK aunknown. Both Mos and cdc25 are present in the meiointerphase yet there is no cdc2 tyrosine phosphorylat(Ohsumi et al., 1994), thus, it seems unlikely that Mos functioto inhibit cdc25. On the other hand, it is possible that Mos mfunction to activate Xe-wee1, as this kinase is not present umeiosis II (Fig. 2A). However, we have not been able to detactivation of Xe-wee1 by Mos, MEK or MAPK, and thdominant negative Xe-wee1 does not reverse the Mos mediM-phase delay (data not shown). We have been able to deboth the endogenous Mos and MBP-Mos fusion proteinp13/suc 1 precipitates (data not shown and Zhou et al., 19This observation is notable since recent experiments have shthat the immunodepletion of the Xenopussuc1 homologue (p9)at interphase prevents entry into M-phase and leads toaccumulation of tyrosine phosphorylated cdc2. Moreover, depletion of p9 during M-phase prevents the extracts fractivating the destruction of cyclin B (Patra and Dunphy, 199The effects of p9 depletion are similar to the effects of Mos; constitutive presence of Mos in the mitotic cycle delays activation of MPF with the accumulation of tyrosinphosphorylated cdc2 (Fig. 6C and F), and Mos inhibits degradation of cyclin in embryos and extracts (Abrieu et 1996; Jones and Smythe, 1996; Sagata et al., 1989 and Fig.One possible explanation of our data would be that the preseof Mos at the beginning of the first cycle affects sommodification that is similar to the depletion of p9.

Meiosis consists of two consecutive M-phases and resultan egg arrested at metaphase of meiosis II. Fertilization relethis arrest and marks the beginning of the first cycConsequently, the end of meiosis (polar body extrusion), decondensation of the sperm nucleus and the fusion of thepronuclei are events which are unique to the first cycle (Figand Graham, 1966). Furthermore, the S-phases of cycles occur during the cytokinesis of the preceding cycle (Fig. Significantly, the first S-phase cannot occur during meio(formation of second polar body) thus, there are two S-phabetween fertilization and the first cytokinesis (Fig. 7; Graha1966; Graham and Morgan, 1966; Miake-Lye et al., 1983; also Hartley et al., 1996, showing two S-phase peaks of cyE/cdk2 activity before cytokinesis). While these events justthe unusual length of the first cycle, we examined some ofcell cycle components which might regulate the length of tcycle. We found that the wee1/cdc25 ratio increased prior tofirst mitotic interphase. While this could explain the presenof cdc2 tyrosine phosphorylation in, and the length of, this ficycle, we believe that other components contribute to the lenof this cycle: the ratio of wee1/cdc25 remains the same afterfirst cycle, and we found no evidence of a wee1/myt 1 inhibiin cycles 2-12. We propose that the activation of tMos/MAPK pathway during earlyinterphase contributes to thegeneration of the atypical gap phases in the first cycle.

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We are grateful to H. Okayama for the human wee1 cDNA, M. Lee and H. Piwnica-Worms for the purified baculovirus cyclin B/cdccomplex, B. Vogelstein for the p21/Waf1/Cip vector, D. Morrison fothe 4G10 antibody, J. Maller for cyclin antibodies, and W. Dunphfor Xe-wee1 cDNA and antibody. We are indebted to I. Daar, MDasso, L. Lock, W. Matten, D. Morrison, M. Oskarsson, R. RempL. Roy, and T. Stukenberg for valuable advice, discussion and critireading of the manuscript, and we thank J. Newport for adviregarding the activation of arrested embryos. We also thank Ave Clfor expert manuscript preparation. Research sponsored by National Cancer Institute, DHHS, under contract with ABL.

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