Human Speedy: a novel cell cycle regulator that enhances proliferation through activation of Cdk2

The decision for a cell to self-replicate requires passage from G1 to S phase of the cell cycle and initiation of another round of DNA replication. This commitment is a critical one that is tightly regulated by many parallel pathways. Significantly, these pathways converge to result in activation of the cyclin-dependent kinase, cdk2. It is, therefore, important to understand all the mechanisms regulating cdk2 to determine the molecular basis of cell progression. Here we report the identification and characterization of a novel cell cycle gene, designated Speedy (Spy1). Spy1 is 40% homologous to the Xenopus cell cycle gene, X-Spy1. Similar to its Xenopus counterpart, human Speedy is able to induce oocyte maturation, suggesting similar biological characteristics. Spy1 mRNA is expressed in several human tissues and immortalized cell lines and is only expressed during the G1/S phase of the cell cycle. Overexpression of Spy1 protein demonstrates that Spy1 is nuclear and results in enhanced cell proliferation. In addition, flow cytometry profiles of these cells demonstrate a reduction in G1 population. Changes in cell cycle regulation can be attributed to the ability of Spy1 to bind to and prematurely activate cdk2 independent of cyclin binding. We demonstrate that Spy1-enhanced cell proliferation is dependent on cdk2 activation. Furthermore, abrogation of Spy1 expression, through the use of siRNA, demonstrates that Spy1 is an essential component of cell proliferation pathways. Hence, human Speedy is a novel cell cycle protein capable of promoting cell proliferation through the premature activation of cdk2 at the G1/S phase transition.     

Expression of cell-cycle regulators during Xenopus oogenesis

In full-grown Xenopus oocytes, cell-cycle regulators and an inactive form of maturation/M phase promoting factor (pre-MPF) are stored ready to bring about a specific cell cycle for oocyte maturation. We examined the expression pattern of these cell-cycle regulators as well as pre-MPF formation during oogenesis. Cdc2 and Cyclin B2 were already present in stage I oocytes and pre-MPF formation was also detected in stage I oocytes. Some negative regulators of MPF, Myt1 and Chk1, were synthesized early in oogenesis. In contrast, positive regulators of MPF, MEK, MAPK and Cdc25C, were mainly synthesized late in oogenesis. Northern blotting analysis suggested that the synthesis of these cell-cycle regulators was controlled by translation.     

Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase.

In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21Cip in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21Cip can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21Cip are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles.     

Inhibition of mos-induced oocyte maturation by protein kinase A

The relationship between the mos protooncogene protein and cAMP- dependent protein kinase (PKA) during the maturation of Xenopus oocytes was investigated. Microinjection of the PKA catalytic subunit (PKAc) into Xenopus oocytes inhibited oocyte maturation induced by the mos product but did not markedly affect the autophosphorylation activity of injected mos protein. By contrast, PKAc did not inhibit maturation promoting factor (MPF) activation or germinal vesicle breakdown (GVBD) that was initiated by injecting crude MPF preparations. In addition, inhibiting endogenous PKA activity by microinjecting the PKA regulatory subunit (PKAr) induced oocyte maturation that was dependent upon the presence of the endogenous mos product. Moreover, PKAr potentiated mos protein-induced MPF activation in the absence of progesterone and protein synthesis. These data are consistent with the hypothesis that progesterone-induced release from G2/M is regulated via PKAc and that PKAc negatively regulates a downstream target that is positively regulated by mos.     

A role for the MEK-MAPK pathway in okadaic acid-induced meiotic resumption of incompetent growing mouse oocytes

Fully grown competent mouse oocytes spontaneously resume meiosis in vitro when released from their follicular environment, in contrast to growing incompetent oocytes, which remain blocked in prophase I. The cell cycle regulators, maturation promoting factor (MPF; [p34(cdc2)/cyclin B kinase]) and mitogen-activated protein (MAP) kinases (p42(MAPK) and p44(MAPK)), are implicated in meiotic competence acquisition. Incompetent oocytes contain levels of p42(MAPK), p44(MAPK), and cyclin B proteins that are comparable to those in competent oocytes, but their level of p34(cdc2) is markedly lower. Okadaic acid (OA), an inhibitor of phosphatases 1 and 2A, induces meiotic resumption of incompetent oocytes. The kinetics and the percentage of germinal vesicle breakdown depends on whether or not oocytes have been cultured before OA treatment. We show that the fast kinetics and the high percentage of germinal vesicle breakdown induced by OA following 2 days in culture is neither the result of an accumulation of p34(cdc2) protein, nor to the activation of MPF in incompetent oocytes, but rather by the premature activation of MAP kinases. Indeed, a specific inhibitor of MAPK kinase (MEK) activity, PD98059, inhibits activation of MAP kinases and meiotic resumption. Altogether, these results indicate that the MEK-MAPK pathway is implicated in OA-induced meiotic resumption of incompetent mouse oocytes, and that the MEK-MAPK pathway can induce meiotic resumption in the absence of MPF activation.     

tpr-met oncogene product induces maturation-producing factor activation in Xenopus oocytes.

tpr-met, a tyrosine kinase oncogene, is the activated form of the met proto-oncogene that encodes the receptor for hepatocyte growth factor/scatter factor. The tpr-met product (p65tpr-met) was tested for its ability to induce meiotic maturation in Xenopus oocytes. While src and abl tyrosine kinase oncogene products have previously been shown to be inactive in this assay, p65tpr-met efficiently induced maturation-promoting factor (MPF) activation and germinal vesicle breakdown (GVBD) together with the associated increase in ribosomal S6 subunit phosphorylation. tpr-met-mediated MPF activation and GVBD was dependent on the endogenous c-mosxe, while the increase in S6 protein phosphorylation was not significantly affected by the loss of mos function. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine inhibits tpr-met-mediated GVBD at concentrations that prevent insulin- but not progesterone-induced oocyte maturation. Moreover, maturation triggered by tpr-met is also inhibited by cyclic AMP-dependent protein kinase. This is the first demonstration that a tyrosine kinase oncogene product, p65tpr-met, can induce meiotic maturation in Xenopus oocytes and activate MPF through a mos-dependent pathway, possibly the insulin or insulinlike growth factor 1 pathway.     

A characterization of cytostatic factor activity from Xenopus eggs and c-mos-transformed cells

In Xenopus oocytes, the mos proto-oncogene product is required during meiosis I for the activation of maturation promoting factor (MPF) and the subsequent breakdown of the germinal vesicle (GVBD). In addition, the mos product has been shown to be a candidate "initiator" of meiotic maturation and is an active component of cytostatic factor (CSF), an activity responsible for metaphase II arrest. Here we demonstrate that pp39mos is required throughout oocyte maturation. We found that in progesterone stimulated oocytes, depletion of mos RNA immediately before GVBD terminally decreased MPF. Likewise, oocytes depleted of mos RNA and induced to mature with crude MPF proceeded through GVBD but lacked the MPF activity required to arrest mature oocytes at metaphase II. Thus, during maturation the mos product is required, directly or indirectly, to sustain MPF activity. On the other hand, mouse NIH/3T3 cells transformed by the constitutive expression of pp39mosxc possessed CSF activity but lacked constitutive levels of MPF or its associated histone H1 kinase activity. Moreover, cytosols prepared from transformed NIH/3T3 cells or Xenopus eggs had similar levels of CSF activity, but pp39mos levels were greater than 40-fold higher in the transformed cell extract. These analyses show that maintenance of CSF during interphase does not result in the maintenance of MPF.     

A new role for Mos in Xenopus oocyte maturation: targeting Myt1 independently of MAPK

The resumption of meiosis in Xenopus arrested oocytes is triggered by progesterone, which leads to polyadenylation and translation of Mos mRNA, then activation of MAPK pathway. While Mos protein kinase has been reported to be essential for re-entry into meiosis in Xenopus, arrested oocytes can undergo germinal vesicle breakdown (GVBD) independently of MAPK activation, leading us to question what the Mos target might be if Mos is still required. We now demonstrate that Mos is indeed necessary, although is independent of the MAPK cascade, for conversion of inactive pre-MPF into active MPF. We have found that Myt1 is likely to be the Mos target in this process, as Mos interacts with Myt1 in oocyte extracts and Mos triggers Myt1 phosphorylation on some sites in vivo, even in the absence of MAPK activation. We propose that Mos is involved, not only in the MAPK cascade pathway, but also in a mechanism that directly activates MPF in Xenopus oocytes.     

Inhibition of poly(A) polymerase requires p34cdc2/cyclin B phosphorylation of multiple consensus and non-consensus sites.

We showed previously that p34(cdc2)/cyclin B (MPF) hyperphosphorylates poly(A) polymerase (PAP) during M-phase of the cell cycle, causing repression of its enzymatic activity. Mutation of three cyclin-dependent kinase (cdk) consensus sites in the PAP C-terminal regulatory domain prevented complete phosphorylation and MPF-mediated repression. Here we show that PAP also contains four nearby non-consensus cdk sites that are phosphorylated by MPF. Remarkably, full phosphorylation of all these cdk sites was required for repression of PAP activity, and partial phosphorylation had no detectable effect. The consensus sites were phosphorylated in vitro at a 10-fold lower concentration of MPF than the non-consensus sites. Consistent with this, during meiotic maturation of Xenopus oocytes, consensus sites were phosphorylated prior to the non-consensus sites at metaphase of meiosis I, and remained so throughout maturation, while the non-consensus sites did not become fully phosphorylated until after 12 h of metaphase II arrest. We propose that PAP's multiple cdk sites, and their differential sensitivity to MPF, provide a mechanism to link repression specifically to late M-phase. We discuss the possibility that this reflects a general means to control the timing of cdk-dependent regulatory events during the cell cycle.     

Spy1 interacts with p27Kip1 to allow G1/S progression

Progression through the G1/S transition commits cells to synthesize DNA. Cyclin dependent kinase 2 (CDK2) is the major kinase that allows progression through G1/S phase and subsequent replication events. p27 is a CDK inhibitor (CKI) that binds to CDK2 to prevent premature activation of this kinase. Speedy (Spy1), a novel cell cycle regulatory protein, has been found to prematurely activate CDK2 when microinjected into Xenopus oocytes and when expressed in mammalian cells. To determine the mechanism underlying Spy1-induced proliferation in mammalian cell cycle regulation, we used human Spy1 as bait in a yeast two-hybrid screen to identify interacting proteins. One of the proteins isolated was p27; this novel interaction was confirmed both in vitro, using bacterially expressed and in vitro translated proteins, and in vivo, through the examination of endogenous and transfected proteins in mammalian cells. We demonstrate that Spy1 expression can overcome a p27-induced cell cycle arrest to allow for DNA synthesis and CDK2 histone H1 kinase activity. In addition, we utilized p27-null cells to demonstrate that the proliferative effect of Spy1 depends on the presence of endogenous p27. Our data suggest that Spy1 associates with p27 to promote cell cycle progression through the G1/S transition.     

c-Mos and cyclin B/cdc2 connections during Xenopus oocyte maturation.

Fully-grown G2 arrested Xenopus oocytes can be induced to enter and progress into meiotic cell cycle by progesterone stimulation. This process is termed oocyte maturation. An early response to progesterone is the synthesis of the onco-protein c-Mos, defined as the candidate initiator of Xenopus oocyte maturation, which triggers the MAPK cascade, MPF activation and promotes CSF activity. Here we review our current knowledge on the synthesis, activation and functions of c-Mos in connection with MPF activation during maturation. We also discuss our recent results concerning the dispensability of cyclin B degradation in meiosis I-meiosis II transition and the stabilization of c-Mos through its direct phosphorylation by cyclin B/cdc2.     

Protein kinase A acts at multiple points to inhibit Xenopus oocyte maturation.

In Xenopus oocytes, initiation of maturation is dependent on reduction of cyclic AMP-dependent protein kinase (PKA) activity and the synthesis of the mos proto-oncogene product. Mos is required during meiosis I for the activation of both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Here we show that injection of the catalytic subunit of PKA (PKAc) prevented progesterone-induced synthesis of endogenous Mos as well as downstream MPF and MAPK activation. However, PKAc did not prevent injected soluble Mos product from activating MAPK. While MAPK is activated during Mos-PKAc coinjection, attendant MPF activation is blocked. Additionally, PKAc caused a potent block in the electrophoretic mobility shift of cdc25 that is associated with phosphatase activation. This inhibition of cdc25 activity was not reversed by progesterone, Mos, or MPF. We conclude that PKAc acts as a negative regulator at several points in meiotic maturation by preventing both Mos translation and MPF activation.     

A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1.

M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34(cdc2). In G2-arrested Xenopus oocytes, there is a stock of p34(cdc2)/cyclin B complexes (pre-MPF) which is maintained in an inactive state by p34(cdc2) phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34(cdc2) inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2-->M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. Here we show that the C-terminal regulatory domain of Myt1 specifically binds to p90(rsk), a protein kinase that can be phosphorylated and activated by MAPK. p90(rsk) in turn phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34(cdc2)/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK-p90(rsk) cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. We found that Myt1 preferentially associates with hyperphosphorylated p90(rsk), and complexes can be detected in immunoprecipitates from mature oocytes. Our results suggest that during oocyte maturation MAPK activates p90(rsk) and that p90(rsk) in turn down-regulates Myt1, leading to the activation of p34(cdc2)/cyclin B.     

Mos induces the in vitro activation of mitogen-activated protein kinases in lysates of frog oocytes and mammalian somatic cells.

Mitogen-activated protein kinases (MAPKs) are rapidly and transiently activated when both quiescent Go-arrested cells and G2-arrested oocytes are stimulated to reenter the cell cycle. We previously developed a cell-free system from lysates of quiescent Xenopus oocytes that responds to oncogenic H-ras protein by activating a MAPK, p42MAPK. Here, we show that the oncogenic protein kinase mos is also a potent activator of p42MAPK in these lysates. Mos also induces p42MAPK activation in lysates of activated eggs taken at a time when neither mos nor p42MAPK is normally active, showing that the mos-responsive MAPK activation pathway persists beyond the stage where mos normally functions. Similarly, lysates of somatic cells (rabbit reticulocytes) also retain a mos-inducible MAPK activation pathway. The mos-induced activation of MAPKs in all three lysates leads to phosphorylation of the pp90rsk proteins, downstream targets of the MAPK signaling pathway in vivo. The in vitro activation of MAPKs by mos in cell-free systems derived from oocytes and somatic cells suggests that mos contributes to oncogenic transformation by inappropriately inducing the activation of MAPKs.     

Induction of maturation-promoting factor during Xenopus oocyte maturation uncouples Ca(2+) store depletion from store-operated Ca(2+) entry.

During oocyte maturation, eggs acquire the ability to generate specialized Ca(2+) signals in response to sperm entry. Such Ca(2+) signals are crucial for egg activation and the initiation of embryonic development. We examined the regulation during Xenopus oocyte maturation of store-operated Ca(2+) entry (SOCE), an important Ca(2+) influx pathway in oocytes and other nonexcitable cells. We have previously shown that SOCE inactivates during Xenopus oocyte meiosis. SOCE inactivation may be important in preventing premature egg activation. In this study, we investigated the correlation between SOCE inactivation and the Mos-mitogen-activated protein kinase (MAPK)-maturation-promoting factor (MPF) kinase cascade, which drives Xenopus oocyte maturation. SOCE inactivation at germinal vesicle breakdown coincides with an increase in the levels of MAPK and MPF. By differentially inducing Mos, MAPK, and MPF, we demonstrate that the activation of MPF is necessary for SOCE inactivation during oocyte maturation. In contrast, sustained high levels of Mos kinase and the MAPK cascade have no effect on SOCE activation. We further show that preactivated SOCE is not inactivated by MPF, suggesting that MPF does not block Ca(2+) influx through SOCE channels, but rather inhibits coupling between store depletion and SOCE activation.     

Cdc2-cyclin B triggers H3 kinase activation of Aurora-A in Xenopus oocytes

Xenopus oocytes are arrested in meiotic prophase I and resume meiotic divisions in response to progesterone. Progesterone triggers activation of M-phase promoting factor (MPF) or Cdc2-cyclin B complex and neosynthesis of Mos kinase, responsible for MAPK activation. Both Cdc2 and MAPK activities are required for the success of meiotic maturation. However, the signaling pathway induced by progesterone and leading to MPF activation is poorly understood, and most of the targets of both Cdc2 and MAPK in the oocyte remain to be determined. Aurora-A is a Ser/Thr kinase involved in separation of centrosomes and in spindle assembly during mitosis. It has been proposed that in Xenopus oocytes Aurora-A could be an early component of the progesterone-transduction pathway, acting through the regulation of Mos synthesis upstream Cdc2 activation. We addressed here the question of Aurora-A regulation during meiotic maturation by using new in vitro and in vivo experimental approaches. We demonstrate that Cdc2 kinase activity is necessary and sufficient to trigger both Aurora-A phosphorylation and kinase activation in Xenopus oocyte. In contrast, these events are independent of the Mos/MAPK pathway. Aurora-A is phosphorylated in vivo at least on three residues that regulate differentially its kinase activity. Therefore, Aurora-A is under the control of Cdc2 in the Xenopus oocyte and could be involved in meiotic spindle establishment.     

O-linked N-acetylglucosaminyltransferase inhibition prevents G2/M transition in Xenopus laevis oocytes

Full-grown Xenopus oocytes are arrested at the prophase of the first meiotic division in a G(2)-like state. Progesterone triggers meiotic resumption also called the G(2)/M transition. This event is characterized by germinal vesicle breakdown (GVBD) and by a burst in phosphorylation level that reflects activation of M-phase-promoting factor (MPF) and MAPK pathways. Besides phosphorylation and ubiquitin pathways, increasing evidence has suggested that the cytosolic and nucleus-specific O-GlcNAc glycosylation also contributes to cell cycle regulation. To investigate the relationship between O-GlcNAc and cell cycle, Xenopus oocyte, in which most of the M-phase regulators have been discovered, was used. Alloxan, an O-GlcNAc transferase inhibitor, blocked G(2)/M transition in a concentration-dependent manner. Alloxan prevented GVBD and both MPF and MAPK activations, either triggered by progesterone or by egg cytoplasm injection. The addition of detoxifying enzymes (SOD and catalase) did not rescue GVBD, indicating that the alloxan effect did not occur through reactive oxygen species production. These results were strengthened by the use of a benzoxazolinone derivative (XI), a new O-GlcNAc transferase inhibitor. Conversely, injection of O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate, an O-GlcNAcase inhibitor, accelerated the maturation process. Glutamine:fructose-6-phosphate amidotransferase inhibitors, azaserine and 6-diazo-5-oxonorleucine, failed to prevent GVBD. Such a strategy appeared to be inefficient; indeed, UDP-GlcNAc assays in mature and immature oocytes revealed a constant pool of the nucleotide sugar. Finally, we observed that cyclin B2, the MPF regulatory subunit, was associated with an unknown O-GlcNAc partner. The present work underlines a crucial role for O-GlcNAc in G(2)/M transition and strongly suggests that its function is required for cell cycle regulation.     

Function of the Mos/MAPK pathway during oocyte maturation in the Japanese brown frog Rana japonica

Fully grown immature oocytes acquire the ability to be fertilized with sperm after meiotic maturation, which is finally accomplished by the formation and activation of the maturation-promoting factor (MPF). MPF is the complex of Cdc2 and cyclin B, and its function in promoting metaphase is common among species. The Mos/mitogen-activated protein kinase (MAPK) pathway is also commonly activated during vertebrate oocyte maturation, but its function seems to be different among species. We investigated the function of the Mos/MAPK pathway during oocyte maturation of the frog Rana japonica. Although MAPK was activated in accordance with MPF activation during oocyte maturation, MPF activation and germinal vesicle breakdown (GVBD) was not initiated when the Mos/MAPK pathway was activated in immature oocytes by the injection of c-mos mRNA. Inhibition of Mos synthesis by c-mos antisense RNA and inactivation of MAPK by CL100 phosphatase did not prevent progesterone-induced MPF activation and GVBD. However, continuous MAPK activation and MAPK inhibition through oocyte maturation accelerated and delayed MPF activation, respectively. Furthermore, Mos induced a low level of cyclin B protein synthesis in immature oocytes without the aid of MAPK. These results suggest that the general function of the Mos/MAPK pathway, which is not essential for MPF activation and GVBD in Rana oocytes, is to enhance cyclin B translation by Mos itself and to stabilize cyclin B protein by MAPK during oocyte maturation.     

c-mos and cdc2 cooperate in the translational activation of fibroblast growth factor receptor-1 during Xenopus oocyte maturation

During oocyte maturation in Xenopus, previously quiescent maternal mRNAs are translationally activated at specific times. We hypothesized that the translational recruitment of individual messages is triggered by particular cellular events and investigated the potential for known effectors of the meiotic cell cycle to activate the translation of the FGF receptor-1 (XFGFR) maternal mRNA. We found that both c-mos and cdc2 activate the translation of XFGFR. However, although oocytes matured by injection of recombinant cdc2/cyclin B translate normal levels of XFGFR protein, c-mos depletion reduces the level of XFGFR protein induced by cdc2/cyclin B injection. In oocytes blocked for cdc2 activity, injection of mos RNA induced low levels of XFGFR protein, independent of MAPK activity. Through the use of injected reporter RNAs, we show that the XFGFR 3' untranslated region inhibitory element is completely derepressed by cdc2 alone. In addition, we identified a new inhibitory element through which both mos and cdc2 activate translation. We found that cdc2 derepresses translation in the absence of polyadenylation, whereas mos requires poly(A) extension to activate XFGFR translation. Our results demonstrate that mos and cdc2, in addition to functioning as key regulators of the meiotic cell cycle, cooperate in the translational activation of a specific maternal mRNA during oocyte maturation.     

Participation of MAPK, PKA and PP2A in the regulation of MPF activity in Bufo arenarum oocytes

The objectives of the present paper were to study the involvement and possible interactions of both cAMP-PKA and protein phosphatases in Bufo arenarum oocyte maturation and to determine if these pathways are independent or not of the MAP kinase (MAPK) cascade. Our results indicated that the inhibition of PKA by treatment with H-89, an inhibitor of the catalytic subunit of PKA, was capable of inducing GVBD in a dose-dependent manner by a pathway in which Cdc25 phosphatase but not the MAPK cascade is involved. The injection of 50 nl of H-89 10 μM produced GVBD percentages similar to those obtained with treatment with progesterone. In addition, the assays with okadaic acid (OA), a PP2A inhibitor, significantly enhanced the percentage of oocytes that resumed meiosis by a signal transducing pathway in which the activation of the MEK-MAPK pathway is necessary, but in which Cdc25 phosphatase was not involved. Treatment with H-89, was able to overcome the inhibitory effect of PKA on GVBD; however, the inhibition of Cdc25 activity with NaVO3 was able to overcome the induction of GVBD by H-89. Although the connections between PKA and other signalling molecules that regulate oocytes maturation are still unclear, our results suggest that phosphatase Cdc25 may be the direct substrate of PKA. In Xenopus oocytes it was proposed that PP2A, a major Ser/Thr phosphatase present, is a negative regulator of Cdc2 activation. However, in Bufo arenarum oocytes, inhibition of Cdc25 with NaVO? did not inhibit OA-induced maturation, suggesting that the target of PP2A was not the Cdc25 phosphatase. MAPK activation has been reported to be essential in Xenopus oocytes GVBD. In B. arenarum oocytes we demonstrated that the inhibition of MAPK by PD 98059 prevented the activation of MPF induced by OA, suggesting that the activation of the MAPK cascade produced an inhibition of Myt1 and, in consequence, the activation of MPF without participation of the Cdc25 phosphatase. Our results suggest that in incompetent oocytes of B. arenarum two signal transduction pathways may be involved in the control of MPF activation: (1) the inhibition of phosphatase 2A that through the MEK-MAPK pathway regulates the activity of the Myt1; and (2) the inhibition of AMPc-PKA, which affects the activity of the Cdc25 phosphatase.