The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF.

In unfertilized eggs from vertebrates, the cell cycle is arrested in metaphase of the second meiotic division (metaphase II) until fertilization or activation. Maintenance of the long-term meiotic metaphase arrest requires mechanisms preventing the destruction of the maturation promoting factor (MPF) and the migration of the chromosomes. In frog oocytes, arrest in metaphase II (M II) is achieved by cytostatic factor (CSF) that stabilizes MPF, a heterodimer formed of cdc2 kinase and cyclin. At the metaphase/anaphase transition, a rapid proteolysis of cyclin is associated with MPF inactivation. In Drosophila, oocytes are arrested in metaphase I (M I); however, only mechanical forces generated by the chiasmata seem to prevent chromosome separation. Thus, entirely different mechanisms may be involved in the meiotic arrests in various species. We report here that in mouse oocytes a CSF-like activity is involved in the M II arrest (as observed in hybrids composed of fragments of metaphase II-arrested oocytes and activated mitotic mouse oocytes) and that the high activity of MPF is maintained through a continuous equilibrium between cyclin B synthesis and degradation. In addition, the presence of an intact metaphase spindle is required for cyclin B degradation. Finally, MPF activity is preferentially associated with the spindle after bisection of the oocyte. Taken together, these observations suggest that the mechanism maintaining the metaphase arrest in mouse oocytes involves an equilibrium between cyclin synthesis and degradation, probably controlled by CSF, and which is also dependent upon the three-dimensional organization of the spindle.     

An M-phase-specific protein kinase of Xenopus oocytes: partial purification and possible mechanism of its periodic activation

The activity of a Ca2+- and cyclic nucleotide-independent protein kinase(s) which catalyzes hyperphosphorylation of a set of endogenous proteins, including a 95-kDa soluble phosphoprotein, is found to fluctuate in both the meiotic and mitotic cell cycles of Xenopus oocytes and activated eggs. The activity is high in M-phase and hardly detectable in interphase. The activity copurifies with a major histone kinase(s) throughout four purification steps: ammonium sulfate precipitation, DEAE-cellulose chromatography, high-performance liquid chromatography on TSK G3000, and CM-Sepharose chromatography. This suggests that a single enzyme shares activity against endogenous proteins and added histones. Changes in the activity of the M-phase-specific protein kinase(s) as assayed in vitro correlate with changes in the extent of protein phosphorylation in oocytes pulse-labeled with 32P-phosphate by microinjection during meiotic maturation and the early embryonic cell cycle. This suggests that the kinase(s) has a broad specificity and plays a key role in the increased protein phosphorylation which occurs at the transition to M-phase. Microinjection of the maturation-promoting factor (MPF) into immature oocytes triggers, after a 10-min lag period, the activation of the M-phase specific kinase(s), even in the absence of protein synthesis. In contrast MPF microinjection does not induce kinase activation in cycloheximide-treated oocytes arrested after completion of the first meiotic cell cycle or in activated eggs arrested in S-phase by incubation in cycloheximide. This suggests that immature oocytes contain an inactive kinase precursor (prokinase) which is synthesized at each of the following cell cycles. In the absence of MPF addition, the prokinase to kinase transition occurs "spontaneously" after a 2-hr lag period in high-speed supernatants prepared from prophase-arrested oocytes if low-molecular-weight metabolites are eliminated by gel filtration. Addition of ATP, but not of AMP-PNP (adenylyl-imidodiphosphate), prevents spontaneous kinase activation in gel-filtered extracts. We propose that MPF activates the M-phase-specific protein kinase in the intact cell by inactivating a factor which requires phosphorylation conditions to inhibit the prokinase to kinase transition.     

Cytostatic Activity Develops during Meiosis I in Oocytes of LT/Sv Mice

Oocytes of wild-type mice are ovulated as the secondary oocytes arrested at metaphase of the second meiotic division. Their fertilization or parthenogenetic activation triggers the completion of the second meiotic division followed by the first embryonic interphase. Oocytes of the LT/Sv strain of mice are ovulated either at the first meiotic metaphase (M I) as primary oocytes or in the second meiotic metaphase (M II) as secondary oocytes. We show here that duringin vitromaturation a high proportion of LT/Sv oocytes progresses normally only until metaphase I. In these oocytes MAP kinase activates shortly after histone H1 kinase (MPF) activation and germinal vesicle breakdown. However, MAP kinase activation is slightly earlier than in oocytes from wild-type F1 (CBA/H × C57Bl/10) mice. The first meiotic spindle of these oocytes forms similarly to wild-type oocytes. During aging, however, it increases in size and finally degenerates. In those oocytes which do not remain in metaphase I the extrusion of first polar bodies is highly delayed and starts about 15 h after germinal vesicle breakdown. Most of the oocytes enter interphase directly after first polar body extrusion. Fusion between metaphase I LT/Sv oocytes and wild-type mitotic one-cell embryos results in prolonged M-phase arrest of hybrids in a proportion similar to control LT/Sv oocytes and control hybrids made by fusion of two M I LT/Sv oocytes. This indicates that LT/Sv oocytes develop cytostatic factor during metaphase I. Eventually, anaphase occurs spontaneously and the hybrids extrude the polar body and form pronuclei in a proportion similar as in controls. In hybrids between LT/Sv metaphase I oocytes and wild-type metaphase II oocytes (which contain cytostatic factor) anaphase I proceeds at the time observed in control LT/Sv oocytes and hybrids between two M I LT/Sv oocytes, and is followed by the parthenogenetic activation and formation of interphase nuclei. Also the great majority of hybrids between M I and M II wild-type oocytes undergoes the anaphase but further arrests in a subsequent M-phase. These observations suggest that an internally triggered anaphase I occurs despite the presence of the cytostatic activity both in LT/Sv and wild-type M I oocytes. Anaphase I triggering mechanism must therefore either inactivate or override the CSF activity. The comparison between spontaneous and induced activation of metaphase I LT/Sv oocytes shows that mechanisms involved in anaphase I triggering are altered in these oocytes. Thus, the prolongation of metaphase I in LT/Sv oocytes seems to be determined by delayed anaphase I triggering and not provoked directly by the cytostatic activity.     

Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF.

MAP kinase is activated and phosphorylated during M phase of the Xenopus oocyte cell cycle, and induces the interphase-M phase transition of microtubule dynamics in vitro. We have carried out molecular cloning of Xenopus M phase MAP kinase and report its entire amino acid sequence. There is no marked change in the MAP kinase mRNA level during the cell cycle. Moreover, studies with an anti-MAP kinase antiserum indicate that MAP kinase activity may be regulated posttranslationally, most likely by phosphorylation. We show that MAP kinase can be activated by microinjection of MPF into immature oocytes or by adding MPF to cell-free extracts of interphase eggs. These results suggest that MAP kinase functions as an intermediate between MPF and the interphase-M phase transition of microtubule organization.     

Phosphorylated ERK5/BMK1 transiently accumulates within division spindles in mouse oocytes and preimplantation embryos

MAP kinases of the ERK family play important roles in oocyte maturation, fertilization, and early embryo development. The role of the signaling pathway involving ERK5 MAP kinase during meiotic and mitotic M-phase of the cell cycle is not well known. Here, we studied the localization of the phosphorylated, and thus potentially activated, form of ERK5 in mouse maturing oocytes and mitotically dividing early embryos. We show that phosphorylation/dephosphorylation, i.e. likely activation/inactivation of ERK5, correlates with M-phase progression. Phosphorylated form of ERK5 accumulates in division spindle of both meiotic and mitotic cells, and precisely co-localizes with spindle microtubules at metaphase. This localization changes drastically in the anaphase, when phospho-ERK5 completely disappears from microtubules and transits to the cytoplasmic granular, vesicle-like structures. In telophase oocytes it becomes incorporated into the midbody. Dynamic changes in the localization of phospho-ERK5 suggests that it may play an important role both in meiotic and mitotic division.     

Temporal regulation of embryonic M-phases

Temporal regulation of M-phases of the cell cycle requires precise molecular mechanisms that differ among different cells. This variable regulation is particularly clear during embryonic divisions. The first embryonic mitosis in the mouse lasts twice as long as the second one. In other species studied so far (C. elegans, Sphaerechinus granularis, Xenopus laevis), the first mitosis is also longer than the second, yet the prolongation is less pronounced than in the mouse. We have found recently that the mechanisms prolonging the first embryonic M-phase differ in the mouse and in Xenopus embryos. In the mouse, the metaphase of the first mitosis is specifically prolonged by the unknown mechanism acting similarly to the CSF present in oocytes arrested in the second meiotic division. In Xenopus, higher levels of cyclins B participate in the M-phase prolongation, however, without any cell cycle arrest. In Xenopus embryo cell-free extracts, the inactivation of the major M-phase factor, MPF, depends directly on dissociation of cyclin B from CDK1 subunit and not on cyclin B degradation as was thought before. In search for other mitotic proteins behaving in a similar way as cyclins B we made two complementary proteomic screens dedicated to identifying proteins ubiquitinated and degraded by the proteasome upon the first embryonic mitosis in Xenopus laevis. The first screen yielded 175 proteins. To validate our strategy we are verifying now which of them are really ubiquitinated. In the second one, we identified 9 novel proteins potentially degraded via the proteasome. Among them, TCTP (Translationally Controlled Tumor Protein), a 23-kDa protein, was shown to be partially degraded during mitosis (as well as during meiotic exit). We characterized the expression and the role of this protein in Xenopus, mouse and human somatic cells, Xenopus and mouse oocytes and embryos. TCTP is a mitotic spindle protein positively regulating cellular proliferation. Analysis of other candidates is in progress.     

Mos-induced p42 mitogen-activated protein kinase activation stabilizes M-phase in Xenopus egg extracts after cyclin destruction

Previously, we have shown that the addition of a constitutively-active mitogen-activated protein kinase kinase protein (MAPKK = MEK) to cycling Xenopus egg extracts activates the p42MAPK pathway, leading to a G2 or M-phase cell cycle arrest. The stage of the arrest depends on the timing of p42MAPK activation. If p42MAPK is activated prior to M-phase, or after exit from M-phase, the extract is arrested in G2. If p42MAPK is activated during entry into M-phase, the extract is arrested in M-phase. In this study, we show that the addition of recombinant Mos protein (which directly phosphorylates and activates MEK) to cycling egg extracts has the same effect as those described for MEK. The addition of Mos to the extract at the start of incubation leads to a G2 arrest with large interphase nuclei with intact nuclear envelopes. If Mos is added at later times, however, the activation of p42MAPK leads to an M-phase arrest with condensed chromosomes and mitotic arrays of microtubules. Moreover, the extent of M-phase specific phosphorylations is shown by the sustained presence of phosphoproteins that are detected by the monoclonal antibody MPM-2. Unexpectedly, in certain M-phase arrested extracts, histone H1 kinase activity levels reach a peak on entry into M-phase but then fall abruptly to interphase levels. When these extracts are analyzed by immunoblotting, Cyclin B2 is destroyed in those samples containing low maturation promoting factor activity (MPF, cyclin B/Cdc2), yet chromosomes remain condensed with associated mitotic arrays of microtubules and M-phase-specific phosphorylations are sustained. These results suggest that although MPF is required for entry into M-phase, once established, M-phase can be maintained by the p42MAPK pathway after the proteolysis of mitotic cyclins.     

Effects of spindle removal on MPF and MAP kinase activities in porcine matured oocytes

Intracellular localization of maturation/M-phase promoting factor (MPF) and mitogen activated protein (MAP) kinase in mature oocytes has been examined by immunocytochemical methods and the authors of these studies have reported that they are localized on spindles during M-phase. Although these reports showed the relative localization of MPF and MAPK on spindles, it has never been shown whether these kinases are present in the cytoplasm and, if they are present, how many parts of the kinases are localized on the metaphase spindle. In the present study, we made quantitative analyses of MPF and MAP kinase localized on oocyte spindles by kinase assays and immunoblotting after removal of the spindles from porcine mature oocytes. First, we certified their intracellular distribution by immunocytochemical methods and observed sharp signals of cyclin B1 on spindle poles and MAP kinase signals on the microtubule of metaphase spindles. In contrast to these results by immunostaining, the amounts of MPF and MAP kinase localized on spindles examined by immunoblotting and kinase assays were undetectable and less than 20%, respectively. These results indicate that the immunocytochemical technique is a powerful method for showing relative localization, but it is not suitable for quantitative analysis, and that the removal of metaphase spindles from mature oocytes does not have a severe negative impact on the subsequent MPF and MAP kinase activity and on the cell cycle progression in early embryo development.     

Protein phosphatases are involved in the in vivo activation of histone H1 kinase in mouse oocyte

Histone H1 kinase and protein phosphorylation have been studied in mouse oocyte. Histone H1 kinase activity increases when the oocyte enters M-phase at the time of GVBD and is paralleled with a burst of protein phosphorylation. This activity dramatically drops after parthenogenetic activation induced by puromycin. Okadic acid (OA), a potent inhibitor of protein phosphatases, induces GVBD when oocytes are arrested in the first meiotic prophase by dbc-AMP; the continuous presence of the phosphatase inhibitor, however, inhibits the polymerization of metaphase microtubules. Following activation of metaphase II-arrested mouse eggs by puromycin, OA can induce the breakdown of the nuclear envelope and the activation of histone H1 kinase. This indicates that in the absence of protein synthesis, and therefore of cyclin synthesis, inhibition of protein phosphatases may be sufficient to induce the entry into M-phase during the first cell cycle of the mouse parthenogenetic activated oocyte.     

Inactivation of p42 mitogen-activated protein kinase is required for exit from M-phase after cyclin destruction.

By using cycling Xenopus egg extracts, we have previously found that if mitogen-activated protein kinase (p42 MAPK) is activated on entry into mitosis (M-phase), the extract is arrested with condensed chromosomes and spindle microtubules. Here we show that these arrested extracts have high levels of M-phase promoting factor (MPF, Cyclin B/Cdc2) activity, stabilized levels of Cyclin B, and sustained M-phase-specific phosphorylations. We also examined the role of p42 MAPK in DNA damage checkpoint-arrested extracts that were induced to enter M-phase by the addition of Cdc25C protein. In these extracts, Cdc25C protein triggers the abrupt, premature activation of MPF and entry into M-phase. MPF activity then drops suddenly due to Cyclin B proteolysis, just as p42 MAPK is activated. Unexpectedly, however, M-phase is sustained, as judged by maintenance of M-phase-specific phosphorylations and condensed chromosomes. To determine if this M-phase arrest depended on p42 MAPK activation, we added PD98059 (PD), an inhibitor of p42 MAPK activation, to egg extracts with exogenous Cdc25. Both untreated and PD-treated extracts entered M-phase simultaneously, with a sharp peak of MPF activity. However, only PD-treated extracts subsequently exited from M-phase and entered interphase. In PD-treated extracts, p42 MAPK was not activated, and the transition to interphase was accompanied by the formation of decondensed nuclei and the disappearance of M-phase-specific phosphorylation of proteins. These results show that although entry into M-phase requires the activation of MPF, exit from M-phase even after cyclin destruction, is dependent on the inactivation of p42 MAPK.     

Nuclear responses to MPF activation and inactivation in Xenopus oocytes and early embryos

The cell cycle of most organisms is highlighted by characteristic changes in the appearance and activity of the nucleus. Structural changes in the nucleus are particularly evident when a cell begins to divide. At this time, the nuclear envelope is disassembled, the chromatin condenses into metaphase chromosomes, and the chromosomes associate with a newly formed spindle. Upon completion of cell division the nuclear envelope reassembles around the chromosomes as they form telophase nuclei, and subsequently interphase nuclei, in the daughter cells. The cytoplasmic control of nuclear behavior has been the theme of Yoshio Masui's research for much of his career. His pioneering demonstration that the cytoplasm of maturing amphibian oocytes causes the resumption of the meiotic cell cycle when it is injected into an immature oocyte provided unequivocal evidence that a cytoplasmic factor could initiate the transition from interphase to metaphase (M-phase) in intact cells. As described in several reviews in this and the previous issue of Biology of the Cell (see Beckhelling and Ford; Duesbery and Vande Woude; Maller), Masui initially called this activity maturation promoting factor (MPF), but when it was realized that it was a ubiquitous regulator of both mitotic and meiotic cell cycles, MPF came to stand for M-phase promoting factor. Biochemical evidence indicates that MPF activity is composed of a mitotic B-type cyclins and cyclin-dependent kinase 1. The increase in the protein kinase activity of cdk1 initiates the changes in the nucleus associated with oocyte maturation and with the entry into mitosis. This article will attempt to provide a brief summary of the responses of the nucleus to the activation of MPF. In addition, the effect of MPF inactivation on nuclear envelope assembly at the end of mitosis will be discussed. This article is written as a tribute to Yoshio Masui on his retirement from the University of Toronto, and as an expression of gratitude for his guidance while I was a student in his laboratory. I have felt very privileged to have known him as a mentor and a friend.     

Control of duration of the first two mitoses in a mouse embryo

The duration of M-phase is largely determined by the time necessary for the formation of a functional metaphase spindle and the correct alignment of all chromosomes on the metaphase plate. The spindle assembly checkpoint prevents the exit from M-phase before the proper alignment of all chromosomes on a metaphase plate in many cell types. In the present paper we show that the first mitotic M-phase of the mouse embryo lasts about 119 min, while the second embryonic M-phase lasts only about 70 min. Histone H1 kinase is activated rapidly during nuclear envelope breakdown in both mitoses. Its maximum, however, is followed by a plateau only during the first mitosis. In the second mitosis, the inactivation of histone H1 kinase activity follows its maximum directly. Histone H1 kinase is more stable in the cytoplasts obtained from mouse embryos during the first embryonic M-phase than during the second one. The stability of histone H1 kinase is greatly increased by the presence of the mitotic apparatus in both M-phases. The mitotic spindle assembly during the first and the second mitoses differs and the first metaphase spindle is stabilised during the period of maximum histone H1 kinase activity. These data show that an unknown developmentally regulated mechanism controls the duration of the two first mitoses in the mouse embryo.     

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.

During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified.     

Association of MPF, MAPK, and nuclear progression dynamics during activation of young and aged bovine oocytes

We have previously shown that bovine oocytes parthenogenetically activated after 40 hours (hr) of in vitro maturation proceed through the cell cycle faster than those after 20 hr of maturation. In the present study, we used this model of different speed of nuclear progression to investigate the correlation of two hallmarks of nuclear events, exit of metaphase arrest and pronuclear formation, with dynamics of MPF and MAPK. Bovine oocytes were matured in vitro for 20 hr (young) or 40 hr (aged) and activated in 7% ethanol followed by incubation in cycloheximide for 0, 0.5, 1, 3, 5, or 7 hr. Activity of MPF and MAPK was lower in aged than young oocytes. The responses to oocyte activation by both the two kinases and nuclear progression were faster in aged than in young oocytes. The activity of MPF declined to undetectable levels (P < 0.05) as early as 0.5 hr after activation in aged oocytes, while this did not happen in young oocytes until 3 hr after activation. The inactivation of MAPK occurred approximately 2 hr earlier in aged oocytes (5 hr post-activation) than in young oocytes (7 hr post-activation). Furthermore, the decline in MPF activity preceded that of MAPK in both young and aged oocytes by about 2 hr. The decrease in activity of MPF and MAPK corresponded with the exit from meiosis and pronuclei formation regardless of the speed of nuclear progression. Despite dramatic changes in activity of MPF and MAPK, the levels of Cdc2 and Erk2 proteins were unchanged (P > 0.05) during the first 7 hr of activation. These observations suggest that inactivation of MPF and MAPK are pre-requisite for the release from metaphase arrest and formation of pronuclei in bovine oocytes.     

Changes in MPF and MAPK activities in porcine oocytes activated by different methods

The effect of different oocyte activation methods on the dynamics of M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity in porcine oocytes were examined. Three activativation methods were tested: (1) electroporation (EP); (2) electroporation combined with butyrolactone I (BL), an inhibitor of cdc2 and cdk2 kinases; (3) electroporation followed by a treatment with cycloheximide (CHX), a protein synthesis blocker. The activity of cdc2 in MII oocytes was 0.067+/-0.011pmol/oocyte/min (mean+/-S.E.M.), which by 1h decreased in every treatment group (P<0.05) and stayed at low levels until 6h post-activation, approximately the time of pronuclear formation. The initial MAPK activity (0.123+/-0.017pmol/oocyte/min) also decreased 1h after each type of activation treatment (P<0.005). However, in the electroporation only group, activity reached its lowest level at 3h; thereafter, it started to recover and at later time points, MAPK activity did not differ from that in non-treated oocytes (P>0.1). In contrast, oocytes where electroporation was followed by protein kinase or protein synthesis inhibition had low MAPK activity by the time pronuclei were to be formed. Pronuclear formation in these groups (86.3+/-3.3% for EP+BL and 87.6+/-3.7% for EP+CHX) was higher compared to that found in the EP-only oocytes (69.4+/-3.3%; P<0.05). These findings demonstrated that electroporation alone efficiently triggered the inactivation of MPF but not that of MAPK. In order to achieve low MAPK activity to allow high frequency of pronuclear formation, electroporation should be followed by a treatment that inhibits protein synthesis or specific protein kinases. The combined activation methods provided stimuli that efficiently induced both MPF and MAPK inactivation and triggered pronuclear formation with high frequencies.     

Induction of a G2-Phase Arrest in Xenopus Egg Extracts by Activation of p42 Mitogen-activated Protein Kinase

Previous work has established that activation of Mos, Mek, and p42 mitogen-activated protein (MAP) kinase can trigger release from G₂-phase arrest in Xenopus oocytes and oocyte extracts and can cause Xenopus embryos and extracts to arrest in mitosis. Herein we have found that activation of the MAP kinase cascade can also bring about an interphase arrest in cycling extracts. Activation of the cascade early in the cycle was found to bring about the interphase arrest, which was characterized by an intact nuclear envelope, partially condensed chromatin, and interphase levels of H1 kinase activity, whereas activation of the cascade just before mitosis brought about the mitotic arrest, with a dissolved nuclear envelope, condensed chromatin, and high levels of H1 kinase activity. Early MAP kinase activation did not interfere significantly with DNA replication, cyclin synthesis, or association of cyclins with Cdc2, but it did prevent hyperphosphorylation of Cdc25 and Wee1 and activation of Cdc2/cyclin complexes. Thus, the extracts were arrested in a G₂-like state, unable to activate Cdc2/cyclin complexes. The MAP kinase-induced G₂ arrest appeared not to be related to the DNA replication checkpoint and not to be mediated through inhibition of Cdk2/cyclin E; evidently a novel mechanism underlies this arrest. Finally, we found that by delaying the inactivation of MAP kinase during release of a cytostatic factor-arrested extract from its arrest state, we could delay the subsequent entry into mitosis. This finding suggests that it is the persistence of activated MAP kinase after fertilization that allows the occurrence of a G₂-phase during the first mitotic cell cycle.     

Does the M-Phase Promoting Factor (MPF) Activate a Major Ca2+-and Cyclic Nucleotide-Independent Protein Kinase in Starfish Ocytes?

In starfish, the activity of a major Ca²⁺-and cyclic nuleotide-independent protein kinase has been shown to fluctuate in phase with that of MPF along meiotic and mitotic cell cycle (23, 25). Microinjection of α-naphthylphosphate (α-NP), a potent phosphatase inhibitor, increased considerably (from 15 to 546 picomoles/min/mg protein) the activity of this major cycling kinase in homogenates. Although this result supported the view that kinase phosphorylation might induce its own activation, this hypothesis was eliminated because injection of cytoplasm from hormone-stimulated enucleated oocytes, which contained the fully activated kinase but no MPF, failed to trigger kinase activation in recipient oocytes. In contrast, kinase activation was induced in recipient oocytes injected with either cytoplasm taken from nucleated maturing oocytes, which contained high MPF and kinase activities, or cytoplasm taken later from hormone-stimulated and ATP-γ-S-injected oocytes which contained high MPF but low kinase activites. These results indicate that inhibiting dephosphorylation of some regulatory protein activates the M-phase-specific protein kinase. The possibility that the M-phase or maturation-promoting factor (MPF) might be this regulatory protein is discussed.     

cdc25 is one of the MPM-2 antigens involved in the activation of maturation-promoting factor.

MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes recognized by the monoclonal antibody MPM-2, are phosphorylated during M-phase induction. Our previous studies suggested that certain MPM-2 antigens are involved in the appearance of maturation-promoting factor (MPF) activity. Because the central mitotic regulator cdc2 kinase has been shown to exhibit MPF activity, we explored the possibility that certain MPM-2 antigens are regulators of cdc2 kinase. We found that MPM-2 binding of its antigens would inhibit the autoamplification of cdc2 kinase in Xenopus oocytes and interfere with cyclin-activation of cdc2 kinase in Xenopus interphase egg extract. Immunodepletion of MPM-2 antigens from cyclin-induced M-phase egg extract caused the inactivation of cdc2 kinase, which was accompanied by an inhibitory phosphorylation of p34cdc2 on Thr 14 and Tyr 15, indicating that at least one MPM-2 antigen is a positive regulator of p34cdc2 dephosphorylation. We then showed that cdc25 from M-phase arrested egg extract is an MPM-2 antigen. These results suggest that phosphorylation of the epitope recognized by MPM-2 may be a crucial event in the activation of cdc25 and that the kinase(s) that phosphorylates this MPM-2 epitope may be an important regulator of cdc2 kinase activation.     

Phosphorylation changes associated with the early cell cycle in Xenopus eggs

Enucleated and nondividing amphibian eggs undergo cyclic changes in cell morphology and in the level of maturation promoting factor (MPF) with a period similar to the early cleavage cycle. We show here that there is a corresponding phosphorylation and dephosphorylation of specific proteins associated with this fundamental cell cycle. M-phase is associated with a general increase in phosphatase activity and specific phosphorylation of a small set of M-phase proteins, reflected in an increased stochiometry of phosphate and increased turnover. At the end of metaphase and correlated with a drop in MPF the phosphoproteins are rapidly lost. By microinjecting M-phase phosphoproteins into arrested interphase and metaphase eggs we could show that the specific M-phase phosphorylation was not due to specificity in phosphatase action. The ability to segregate synthesis from phosphorylation demonstrates that regulation is not on the level of synthesis of the M-phase proteins. Taken together these data suggest that regulation of kinase activity in M-phase in the face of general rapid phosphate turnover in the egg plays an important role in the regulation of the fundamental mitotic cycle.     

Maturation-promoting factor induces nuclear envelope breakdown in cycloheximide-arrested embryos of Xenopus laevis

We have studied the effect of maturation-promoting factor (MPF) on embryonic nuclei during the early cleavage stage of Xenopus laevis development. When protein synthesis is inhibited by cycloheximide during this stage, the embryonic cell cycle arrests in an artificially produced G2 phase-like state, after completion of one additional round of DNA synthesis. Approximately 100 nuclei can be arrested in a common cytoplasm if cytokinesis is first inhibited by cytochalasin B. Within 5 min after injection of MPF into such embryos, the nuclear envelope surrounding each nucleus disperses, as determined histologically or by immunofluorescent staining of the nuclear lamina with antilamin antiserum. The breakdown of the nuclear envelope occurs at levels of MPF comparable to or slightly lower than those required for oocyte maturation. Amplification of MPF activity, however, does not occur in the arrested egg as it does in the oocyte. These results suggest that MPF can act to advance interphase nuclei into the first events of mitosis and show that the nuclear lamina responds rapidly to MPF.