Inhibition of mitosis in fertilized sea urchin eggs by inhibition of the cyclic AMP-dependent protein kinase

Inhibition of cAMP-dependent protein kinase activity by microinjection of a specific physiologic protein inhibitor into sea urchin eggs inhibits the first cleavage after fertilization. Inhibition apparently occurs at some time prior to or during formation of the mitotic spindle. Measurement of the total protein kinase activity of sea urchin egg homogenates after fertilization showed that cAMP-dependent phosphorylation increases after fertilization and then declines prior to or at the time of the first cleavage. It is concluded that a cAMP-dependent phosphorylation plays a significant role in events leading to regulation of mitotic spindle assembly.     

A MAPK pathway is involved in the control of cortical granule reaction and mitosis during bovine fertilization

In order to understand the mechanism by which mitogen-activated protein kinase (MAPK) regulates fertilization, we examined the effect of the MAPK pathway inhibitor U0126 on polyspermy, cortical granule reaction and mitosis in bovine oocytes during and after fertilization. Oocytes were treated with 30 microM U0126 for 30 min prior to insemination, or from 15 to 27 hr following insemination. Western blotting with antibodies that detect active, phosphorylated MAPK revealed that MAPK activity was decreased in U0126 treated oocytes. Oocytes that were treated with U0126 before insemination displayed a significantly higher incidence of polyspermic penetration and incomplete cortical granule reaction than that observed in untreated oocytes (P < 0.05). Exposure of oocytes to 30microM U0126 15-27 hr after insemination induced aberrant microtubule assembly and cell division, often resulting in the formation of two or three daughter cells with altered shapes and sizes. These results suggest that an ERK-like cascade is part of a mechanism that controls cortical granule reaction and the formation of the mitotic spindle following sperm penetration in the bovine.     

Phosphorylation of a 225-kDa centrosomal component in mitotic CHO cells and sea urchin eggs

Components of centrosomes are those among cellular proteins that are phosphorylated at the transition from interphase to mitosis. Using an anti-phosphoprotein antibody (CHO3) directed against isolated mitotic CHO spindles, we identified a 225-kDa centrosomal phosphocomponent in mitotic CHO cells and in cleaving sea urchin eggs. The 225-kDa protein is tightly attached to the centrosome, which allowed us to separate it from other spindle-associated factors by high salt extraction. Phosphorylation of the 225-kDa protein occurred during mitosis. This was shown by isotope labeling on gels as well as by visualization of thiophosphorylated centrosomes with an anti-thiophosphoprotein antibody (M. Cyert, T. Scherson, and M. W. Kirschner, 1988, Dev. Biol. 129, 209) after preincubation with ATP-gamma-S in vivo and in vitro. Mitotic spindles isolated from CHO cells retained their ability to phosphorylate the centrosomal component, whereas sea urchin spindles did not, possibly due to loss or inactivation of protein kinase(s) during spindle isolation. The enzyme associated with isolated CHO spindles was extractable by high salt treatment and was capable of phosphorylating many spindle components, including the 225-kDa centrosomal protein of CHO cells and sea urchin embryos. Such high salt extracts contain protein kinases, including cell cycle control protein kinase p34cdc2, suggesting that the enzyme responsible for centrosomal phosphorylation could be p34cdc2 or other downstream mitotic kinases activated by the action of p34cdc2.     

Role of phospholipase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos

It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eggs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the early embryo. How sperm induces these calcium transients is still far from being understood. In sea urchin eggs, both InsP3 and ryanodine receptors contribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP3 after fertilization remains an enigma. In order to determine whether PLCgamma might be the origin of the peaks of InsP3 production that punctuate the first mitotic cell cycles of the fertilized sea urchin egg, we have amplified by RT-PCR several fragments of sea urchin PLCgamma containing the two SH2 domains. The sequence shares similarities with SH2 domains of PLCgamma from mammals. One fragment was subcloned into a bacterial expression plasmid and a GST-fusion protein was produced and purified. Antibodies raised to the GST fusion protein demonstrate the presence of PLCgamma protein in eggs. Microinjection of the fragment into embryos interferes with mitosis. A related construct made from bovine PLCgamma also delayed or prevented entry into mitosis and blocked or prolonged metaphase. The bovine construct also blocked the calcium transient at fertilization, in contrast to a tandem SH2 control construct which did not inhibit either fertilization or mitosis. Our data indicate that PLCgamma plays a key role during fertilization and early development.     

Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggs at fertilization.

We have evaluated the regulation of a 43-kDa MAP kinase in sea urchin eggs. Both MAP kinase and MEK (MAP kinase kinase) are phosphorylated and active in unfertilized eggs while both are dephosphorylated and inactivated after fertilization, although with distinct kinetics. Reactivation of MEK or the 43-kDa MAP kinase prior to or during the first cell division was not detected. Confocal immunolocalization microscopy revealed that phosphorylated (active) MAP kinase is present primarily in the nucleus of the unfertilized egg, with some of the phosphorylated form in the cytoplasm as well. Incubation of unfertilized eggs in the MEK inhibitor U0126 (0.5 microM) resulted in the inactivation of MEK and MAP kinase within 30 min. Incubation in low concentrations of U0126 (sufficient to inactivate MEK and MAP kinase) after fertilization had no effect on progression through the embryonic cell cycle. Microinjection of active mammalian MAP kinase phosphatase (MKP-3) resulted in inactivation of MAP kinase in unfertilized eggs, as did addition of MKP-3 to lysates of unfertilized eggs. Incubation of unfertilized eggs in the Ca(2+) ionophore A23187 led to inactivation of MEK and MAP kinase with the same kinetics as observed with sperm-induced egg activation. This suggests that calcium may be deactivating MEK and/or activating a MAP kinase-directed phosphatase. A cell-free system was used to evaluate the activation of phosphatase separately from MEK inactivation. Unfertilized egg lysates were treated with U0126 to inactivate MEK and then Ca(2+) was added. This resulted in increased MAP kinase phosphatase activity. Therefore, MAP kinase inactivation at fertilization in sea urchin eggs likely is the result of a combination of MEK inactivation and phosphatase activation that are directly or indirectly responsive to Ca(2+).     

Bub1 is activated by the protein kinase p90(Rsk) during Xenopus oocyte maturation

BACKGROUND: The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins. This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle. In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint. In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint. In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90(Rsk), which leads to inhibition of the APC. However, it is not known whether CSF-dependent metaphase arrest caused by p90(Rsk) involves components of the spindle assembly checkpoint. RESULTS: xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis. In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bub1 upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme. The activation of Bub1 can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK. In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bub1 remains in its low-activity, unshifted form. Injection of a constitutively active target of MAPK, the protein kinase p90(Rsk), restores the activation of Bub1 in the presence of U0126. Moreover, purified p90(Rsk) phosphorylates Bub1 in vitro and increases its protein kinase activity. CONCLUSIONS: Bub1, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner. Moreover, a single substrate of MAPK, p90(Rsk), is sufficient to activate Bub1 in vitro and in vivo. These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bub1, are downstream of p90(Rsk) and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90(Rsk).     

MAPK inactivation is required for the G2 to M-phase transition of the first mitotic cell cycle.

Down-regulation of MAP kinase (MAPK) is a universal consequence of fertilization in the animal kingdom, although its role is not known. Here we show that MAPK inactivation is essential for embryos, both vertebrate and invertebrate, to enter first mitosis. Suppressing down-regulation of MAPK at fertilization, for example by constitutively activating the upstream MAPK cascade, specifically suppresses cyclin B-cdc2 kinase activation and its consequence, entry into first mitosis. It thus appears that MAPK functions in meiotic maturation by preventing unfertilized eggs from proceeding into parthenogenetic development. The most general effect of artificially maintaining MAPK activity after fertilization is prevention of the G2 to M-phase transition in the first mitotic cell cycle, even though inappropriate reactivation of MAPK after fertilization may lead to metaphase arrest in vertebrates. Advancing the time of MAPK inactivation in fertilized eggs does not, however, speed up their entry into first mitosis. Thus, sustained activity of MAPK during part of the first mitotic cell cycle is not responsible for late entry of fertilized eggs into first mitosis.     

A calsequestrin-like protein in the endoplasmic reticulum of the sea urchin: localization and dynamics in the egg and first cell cycle embryo

Using an antiserum produced against a purified calsequestrin-like (CSL) protein from a microsomal fraction of sea urchin eggs, we performed light and electron microscopic immunocytochemical localizations on sea urchin eggs and embryos in the first cell cycle. The sea urchin CSL protein has been found to bind Ca++ similarly to calsequestrin, the well-characterized Ca++ storage protein in the sarcoplasmic reticulum of muscle cells. In semi-thin frozen sections of unfertilized eggs, immunofluorescent staining revealed a tubuloreticular network throughout the cytoplasm. Staining of isolated egg cortices with the CSL protein antiserum showed the presence of a submembranous polygonal, tubular network similar to ER network patterns seen in other cells and in egg cortices treated with the membrane staining dye DiIC16[3]. In frozen sections of embryos during interphase of the first cell cycle, a cytoplasmic network similar to that of the unfertilized egg was present. During mitosis, we observed a dramatic concentration of the antibody staining within the asters of the mitotic apparatus where ER is known to aggregate. Electron microscopic localization on unfertilized eggs using peroxidase-labeled secondary antibody demonstrated the presence of the CSL protein within the luminal compartment of ER-like tubules. Finally, in frozen sections of centrifugally stratified eggs, the immunofluorescent staining concentrated in the clear zone: a layer highly enriched in ER and thought to be the site of calcium release upon fertilization. This localization of a CSL protein within the ER of the egg provides evidence for the ability of this organelle to serve a Ca++ storage role in the regulation of intracellular Ca++ in nonmuscle cells in general, and in the regulation of fertilization and cell division in sea urchin eggs in particular.     

Mos mediates the mitotic activation of p42 MAPK in Xenopus egg extracts

The ERK1/ERK2 MAP kinases (MAPKs) are transiently activated during mitosis, and MAPK activation has been implicated in the spindle assembly checkpoint and in establishing the timing of an unperturbed mitosis. The MAPK activator MEK1 is required for mitotic activation of p42 MAPK in Xenopus egg extracts; however, the identity of the kinase that activates MEK1 is unknown. Here we have partially purified a Cdc2-cyclin B-induced MEK-activating protein kinase from mitotic Xenopus egg extracts and identified it as the Mos protooncoprotein, a MAP kinase kinase kinase present at low levels in mitotic egg extracts, early embryos, and somatic cells. Immunodepletion of Mos from interphase egg extracts was found to abolish Delta90 cyclin B-Cdc2-stimulated p42 MAPK activation. In contrast, immunodepletion of Raf-1 and B-Raf, two other MEK-activating kinases present in Xenopus egg extracts, had little effect on cyclin-stimulated p42 MAPK activation. Immunodepletion of Mos also abolished the transient activation of p42 MAPK in cycling egg extracts. Taken together, these data demonstrate that Mos is responsible for the mitotic activation of the p42 MAPK pathway in Xenopus egg extracts.     

Cell cycle specific variations in transport capacity of an isolated Ca2+-transport system

From sea urchin eggs as well as from mammalian cells a Ca2+-transporting system is described in its properties. One of its main components is the "mitotic" Ca2+-ATPase. If its activity is studied during the cell cycle of fertilized sea urchin eggs, fluctuations of the Ca2+-uptake capacity are found with a maximum in every cell cycle at mitosis. Additionally, only in the first cycle after fertilization, another activity increase occurs at the time of spermaster formation. This system, then, seems to qualify for one of the main regulators of the mitotic process.     

Fertilization and the first cleavage mitosis in insects

Fertilization in animals is now considered to be of the "sea urchin type"; that is, haploid male and female pronuclei completely fuse shortly after sperm entry into the egg, followed by the formation of a mitotic spindle to allow cleavage mitoses to proceed. However, two other patterns of fertilization and early embryonic mitosis in some animal species are known: an Ascaris type and a gonomeric type. The gonomeric type of fertilization in insects and other arthropods is not well known and is quite different from the sea urchin and Ascaris types. In the present article, the author examines the peculiar gonomeric fertilization, using mainly the silkworm as an example.     

Caulerpenyne blocks MBP kinase activation controlling mitosis in sea urchin eggs

In a previous study, we demonstrated that caulerpenyne (Cyn), a natural sesquiterpene having an antiproliferative potency, blocked the mitotic cycle of sea urchin embryos at metaphase and inhibited the phosphorylation of several proteins, but did not affect histone H1 kinase activation (Pesando et al, 1998, Eur. J. Cell Biol. 77, 19-26). Here, we show that concentrations of Cyn that blocked the first division of the sea urchin Paracentrotus lividus embryos in a metaphase-like stage (45 microM) also inhibited the stimulation of mitogen-activated protein kinase (MAPK) activity in vivo as measured in treated egg extracts using myelin basic protein (MBP) as a substrate (MBPK). However, Cyn had no effect on MBP phosphorylation when added in vitro to an untreated egg extract taken at the time of metaphase, suggesting that Cyn acts on an upstream activation process. PD 98059 (40 microM), a previously characterized specific synthetic inhibitor of MAPK/extracellular signal-regulated kinase-1 (MEK1), also blocked sea urchin eggs at metaphase in a way very similar to Cyn. Both molecules induced similar inhibitory effects on MBP kinase activation in vivo, but had no direct effect on MBP kinase activity in vitro, whereas they did not affect H1 kinase activation neither in vivo nor in vitro. As a comparison, butyrolactone 1 (100 microM), a known inhibitor of H1 kinase activity, did inhibit H1 kinase of sea urchin eggs in vivo and in vitro, and blocked the sea urchin embryo mitotic cycle much before metaphase. Immunoblots of mitotic extracts, treated with anti-active MAP-kinase antibody, showed that both Cyn and PD 98059 reduced the phosphorylation of p42 MAP kinase (Erk2) in vivo. Our overall results suggest that Cyn blocks the sea urchin embryo mitotic cycle at metaphase by inhibiting an upstream phosphorylation event in the MBPK activation pathway. They also show that H1 kinase and MBPK activation can be dissociated from each other in this model system.     

Microtubule assembly is required for the formation of the pronuclei,nuclear lamin acquisition,and DNA synthesis during mouse,but not sea urchin,fertillization

Microtubule assembly is required for the formation of the male and female pronuclei during mouse, but not sea urchin, fertilization. In mouse oocytes, 50 μM colcemid prevents the decondensation of the maternal meiotic chromosomes and of the incorporated sperm nucleus during in vitro fertilization. Nuclear lamins do not associate with either of the parental chromatin sets although peripherin, the PI nuclear peripheral antigen, appears on both. DN A synthesis docs not occur in these fertilized, colcemid-arrested oocytes. This effect is limited to the first hours after ovulation, since colcemid added 4–6 hours later no longer prevents pronuclear development, lamin acquisition, or DNA synthesis. Neither microtubule stabilization with 10 μM taxol nor microfilament inhibition with 10 μM cytochalasin D or 2.2 μg/ml lalrunculin A prevent these pronuclear events; these drugs will inhibit the apposition of the pronuclei at the egg center. In sea urchin eggs, colcemid or griseofulvin treatment doe? not result in the same effect and the male pronucleus forms with the attendant accumulation of the nuclear lamins. The differences in the requirement for microtubule assembly during pronucleus formation may be related to the cell cycle: In mice the sperm enters a meiotic cytoplasm, whereas in sea urchin eggs it enters an interphase cytoplasm. Refertilization of mitotic sea urchin eggs was performed to test the possibility that this phenomenon is related to whether the sperm enters a meiotic/mitotic cytoplasm or one at interphase; during refertilization at first mitosis, the incorporated sperm nucleus is unable to decondense and acquire lamins. These results indicate a requirement for microtubule assembly for the progression from meiosis to first interphase during mouse fertilization and suggest that the cytoskeleton is required for changes in nuclear architecture necessary during fertilization and the cell cycle.     

Involvement of Mos-MEK-MAPK pathway in cytostatic factor (CSF) arrest in eggs of the parthenogenetic insect, Athalia rosae

Extensive survey of meiotic metaphase II arrest during oocyte maturation in vertebrates revealed that the mitogen-activated protein kinase (MAPK) pathway regulated by the c-mos proto-oncogene product, Mos, has an essential role in cytostatic activity, termed cytostatic factor (CSF). In contrast, little is known in invertebrates in which meiotic arrest occurs in most cases at metaphase I (MI arrest). A parthenogenetic insect, the sawfly Athalia rosae, in which artificial egg activation is practicable, has advantages to investigate the mechanisms of MI arrest. Both the MAPK/extracellular signal-regulated protein kinase kinase (MEK) and MAPK were phosphorylated and maintained active in MI-arrested sawfly eggs, whereas they were dephosphorylated soon after egg activation. Treatment of MI-arrested eggs with U0126, an inhibitor of MEK, resulted in dephosphorylation of MAPK and MI arrest was resumed. The sawfly c-mos gene orthologue encoding a serine/threonine kinase was cloned and analyzed. It was expressed in nurse cells in the ovaries. To examine CSF activity of the sawfly Mos, synthesized glutathione S-transferase (GST)-fusion sawfly Mos protein was injected into MI-resumed eggs in which MEK and MAPK were dephosphorylated. Both MEK and MAPK were phosphorylated again upon injection. In these GST-fusion sawfly Mos-injected eggs subsequent mitotic (syncytial) divisions were blocked and embryonic development was ceased. These results demonstrated that the MEK-MAPK pathway was involved in maintaining CSF arrest in sawfly eggs and Mos functioned as its upstream regulatory molecule.     

The amino acid sequence, immunofluorescence and microinjection studies on the 15 kDa calcium-binding protein from sea urchin egg

The 15 kDa protein is the most abundant low molecular weight Ca2+-binding protein, different from calmodulin, in eggs of sea urchin, Hemicentrotus pulcherrimus. The data from the amino acid sequence demonstrated that the 15 kDa protein belonged to the troponin C superfamily. Based on immunofluorescent and immunomicroscopic observations, we showed that the 15 kDa protein localized in the nuclei of fertilized eggs and mitotic apparatus of dividing eggs. Microinjection of the antibody against 15 kDa protein into sea urchin blastomeres resulted in the arresting of cell division. These results suggest that the 15 kDa protein plays an important role in mitosis of sea urchin egg.     

Experimental separation of pronuclei in fertilized sea urchin eggs: chromosomes do not organize a spindle in the absence of centrosomes

We tested the ability of chromosomes in a mitotic cytoplasm to organize a bipolar spindle in the absence of centrosomes. Sea urchin eggs were treated with 5 X 10(-6) colcemid for 7-9 min before fertilization to block future microtubule assembly. Fertilization events were normal except that a sperm aster was not formed and the pronuclei remained up to 70 microns apart. After nuclear envelope breakdown, individual eggs were irradiated with 366-nm light to inactivate photochemically the colcemid. A functional haploid bipolar spindle was immediately assembled in association with the male chromosomes. In contrast to the male pronucleus, the female pronucleus in most of these eggs remained as a small nonbirefringent hyaline area throughout mitosis. High-voltage electron microscopy of serial semithick sections from individual eggs, previously followed in vivo, revealed that the female chromosomes were randomly distributed within the remnants of the nuclear envelope. No microtubules were found in these pronuclear areas even though the chromosomes were well-condensed and had prominent kinetochores with well-developed coronas. In the remaining eggs, a weakly birefringent monaster was assembled in the female pronuclear area. These observations demonstrate that chromosomes in a mitotic cytoplasm cannot organize a bipolar spindle in the absence of a spindle pole or even in the presence of a monaster. In fact, chromosomes do not even assemble kinetochore microtubules in the absence of a spindle pole, and kinetochore microtubules form only on kinetochores facing the pole when a monaster is present. This study also provides direct experimental proof for the longstanding paradigm that the sperm provides the centrosomes used in the development of the sea urchin zygote.     

The relationship between calcium, MAP kinase, and DNA synthesis in the sea urchin egg at fertilization

Fertilization releases the brake on the cell cycle and the egg completes meiosis and enters into S phase of the mitotic cell cycle. The MAP kinase pathway has been implicated in this process, but the precise role of MAP kinase in meiosis and the first mitotic cell cycle remains unknown and may differ according to species. Unlike the eggs of most animals, sea urchin eggs have completed meiosis prior to fertilization and are arrested at the pronuclear stage. Using both phosphorylation-state-specific antibodies and a MAP kinase activity assay, we observe that MAP kinase is phosphorylated and active in unfertilized sea urchin eggs and then dephosphorylated and inactivated by 15 min postinsemination. Further, Ca(2+) was both sufficient and necessary for this MAP kinase inactivation. Treatment of eggs with the Ca(2+) ionophore A23187 caused MAP kinase inactivation and triggered DNA synthesis. When the rise in intracellular Ca(2+) was inhibited by injection of a chelator, BAPTA or EGTA, the activity of MAP kinase remained high. Finally, inhibition of the MAP kinase signaling pathway by the specific MEK inhibitor PD98059 triggered DNA synthesis in unfertilized eggs. Thus, whenever MAP kinase activity is retained, DNA synthesis is inhibited while inactivation of MAP kinase correlates with initiation of DNA synthesis.     

Phosphorylation of the p220 subunit of eIF-4F by cAMP dependent protein kinase and protein kinase C in vitro

Changes in the extent of phosphorylation of the 25 kDa subunit of eIF-4F occur during several major biological events including mitosis and heat shock in mammalian cells and shortly after fertilization of sea urchin (Lytechinus pictus) eggs. In vitro phosphorylation studies using highly purified protein kinases demonstrated that the 220 kDa subunit of eIF-4F was phosphorylated by cAMP dependent protein kinase, protein kinase C and probably to a lesser extent by cGMP dependent protein kinase. In addition, eIF-4A was readily phosphorylated by cAMP and cGMP dependent protein kinases whereas p48 of eIF-4F was not. The effect of these phosphorylation events on eIF-4F function, its assembly or disassembly, susceptibility to viral initiated proteolysis or the ability of p25 to be phosphorylated at serine-53 remain to be investigated.     

Fertilization of sea urchin eggs is accompanied by 40 S ribosomal subunit phosphorylation

After fertilization of sea urchin (Arbacia punctulata) eggs, there is a single prominent alteration in the pattern of protein phosphorylation. In eggs preloaded with ³²PO₄, a 31,000 M_r protein (rp31) becomes labeled within 4 min of sperm addition. A new steady-state level of rp31 labeling is achieved by 11 min. The rate of protein synthesis in sea urchin zygotes also increases at 8–10 min after fertilization. Protein rp31 corresponds to mammalian ribosomal S6 because it cosediments with 40 S subunits on high salt-sucrose gradients, it is similar to the mammalian protein in M_r and charge, and it becomes phosphorylated during an increase in protein synthesis. The specific activity of phosphorylated rp31 (relative to rRNA) is similar between free 80 S monosomes and polysomes, indicating that rp31 phosphorylation is not sufficient for ribosomal activity. A phosphatase, highly specific for rp31, is present in extracts of eggs and very early embryos. This phosphatase becomes inactive at about the same time that the degree of labeling of rp31 increases in embryos. Evidently a control system that maintains a low level of rp31 phosphorylation is active in sea urchin eggs. Inactivation of this system shortly after fertilization leads to the accumulation of phosphorylated ribosomes.     

Localised MPF regulation in eggs

In this review we discuss the evidence that activation and inactivation of M-phase promoting factor (MPF), the universal mitotic activator, are regulated locally within the cell, and consider the mechanisms that might be responsible. Localised initiation of MPF activation has been demonstrated in Xenopus eggs and egg fragments by examination of the timing of surface contraction waves (SCWs), indicators of MPF activity, and confirmed by direct measurement of MPF in such fragments. Both the timing and the site of SCW initiation relate to the presence of nuclei and of associated centriole-nucleated microtubules. Localised MPF activation is likely to occur in the perinuclear cytoplasm as well as within the nucleus. Studies in a number of cell types show that the perinuclear/centrosomal region is the site of accumulation of MPF itself (the cyclin B-Cdc2 kinase complex) and of many of its molecular regulators. It also harbours calcium-regulating machinery, and in sea urchin eggs is the site of transient calcium release at the onset of mitosis. During mitosis MPF, regulatory molecules and calcium signalling components associate with spindle structures. Inactivation of MPF to end mitosis has been shown to be initiated locally at the mitoic spindle in Drosophila embryos. In sea urchin and frog eggs, calcium transients are required for both mitotic entry and exit and in mouse eggs, MPF inactivation requires both a calcium signal and an intact spindle. It thus appears that calcium signals coinciding with localised accumulation of MPF regulators are required first to set off and/or amplify the MPF activation process around the nucleus, and later to promote MPF inactivation via cyclin B destruction. Calcium release from sequestering machinery organised around nuclear and astral structures may act co-operatively with localised MPF regulatory molecules to trigger both mitotic entry and exit.