Introduction
In almost all species studied, meiotic maturation is controlled by the maturation promoting factor (MPF), the key regulator of transition of G2/M in cell cycles. Although all species share an MPF complex that is required for oocytes meiotic maturation, some clear differences exist among the different species. This implies that the species-specific regulations are present even in the basic machinery for cell cycle regulation.
Significant progress has been made towards understanding the regulation of the meiotic cell cycle in Bufo arenarum oocytes although the pathway selected by progesterone, the physiological inducer of M-phase entry in this species, remains to be determined.
In immature oocytes of Xenopus (Gautier & Maller Reference Gautier and Maller1991; Minshull et al., Reference Minshull, Murray, Colman and Hunt1991), B. arenarum (Sánchez Toranzo et al., Reference Sánchez Toranzo, Bonilla, Zelarayán, Oterino and Bühler2006) and starfish (Kishimoto, Reference Kishimoto1998) there is an inactive complex (pre-MPF) that consists of cyclin B-bound Cdc2 phosphorylated on Thr-161 and on the inhibitory residues Thr-14/Tyr-15. In B. arenarum oocytes arrested in G2, the Thr14 and Tyr15 residues of pre-MPF are phosphorylated by the Myt1 kinase. To induce the MPF activation the inhibitory residues Thr-14/Tyr-15 must be dephosphorylated by the phosphatase Cdc25 (Duckworth et al., Reference Duckworth, Weaver and Rudeman2002).
The activity of the Cdc25 phosphatase is regulated by multiple phosphorylation and dephosphorylation events. Fully grown oocytes preincubated with Cdc25 inhibitors such as sodium metavanadate (NaVO3) are unable to activate MPF and cannot undergo meiotic resumption, highlighting the importance of this phosphatase in the process (Sánchez Toranzo et al., Reference Sánchez Toranzo, Bonilla, Zelarayán, Oterino and Bühler2006).
B. arenarum oocytes obtained during the reproductive period (spring–summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. In this species it is possible to obtain oocytes competent and incompetent to undergo spontaneous maturation according to the seasonal period in which animals are captured (Zelarayán et al., Reference Zelarayán, Oterino and Bühler1995). Interestingly, fully grown B. arenarum oocytes always respond to progesterone, independently of the time of the year in which they are obtained (Zelarayán et al., Reference Zelarayán, Oterino and Bühler1996).
In B. arenarum oocytes, the classical adenylyl cyclase/cAMP/protein kinase A (PKA) signalling pathway is considered to be the primary signalling cascade through which progesterone regulates meiosis reinitiation. We previously showed that maintaining high cAMP levels by stimulation of oocytes adenylyl cyclase (AC) with forskolin, by including the analogous membrane-permeable dbcAMP, or by the pharmacological blockade of phosphodiesterase (PDE) with theophyline, prevented meiotic reinitiation (Zelarayán et al., Reference Zelarayán, Oterino, Sánchez Toranzo and Bühler2000; Sánchez Toranzo et al., Reference Sánchez Toranzo, Bonilla, Zelarayán, Oterino and Bühler2006). These results suggest that cAMP-dependent protein kinase (PKA) is active in G2-arrested oocytes and has an important negative role in oocytes maturation. In agreement with this idea, injection of PKA inhibitors should cause maturation in the absence of progesterone whereas overactivation of PKA potently inhibits oocyte maturation (Kwon & Lee, Reference Kwon and Lee1991).
Therefore, a simple hypothesis may explain the cAMP and PKA inhibitory effects on MPF activity. High cAMP levels within the oocytes result in the phosphorylation of Cdc2 on Thr14 and Tyr15, rendering it inactive (Duckworth et al., Reference Duckworth, Weaver and Rudeman2002). A decrease in oocytes cAMP early in oocyte maturation leads to the dephosphorylation of Cdc2 on Thr14 and Tyr15, and the MPF complex becomes active so that oocytes can re-enter meiosis.
However, no evidence that MPF is a direct substrate of PKA has been reported, suggesting that intermediate steps are required. However, there is still no information on what the relevant PKA substrates in oocytes could be. The discrete sets of steps through which cAMP activates or inactivates MPF are still under investigation. It has been reported that PKA phosphorylates and inactivates Cdc25 in Xenopus oocytes (Duckworth et al., Reference Duckworth, Weaver and Rudeman2002).
The decrease in cAMP concentration and the subsequent inactivation of PKA are the first early events known to be induced by progesterone (Maller & Krebs, Reference Maller and Krebs1977; Huchon et al., Reference Huchon, Ozon and Demaille1981). Previous studies have shown that a transient increase in intracellular cAMP levels produced by inhibition of PDE activity or activation of adenyl cyclase (AC) is sufficient to inhibit GVBD induced by progesterone (Zelarayán et al., Reference Zelarayán, Oterino, Sánchez Toranzo and Bühler2000).
The identity of the protein phosphatase that catalyzes the dephosphorylation of the different inhibitory residues of Cdc25 is still uncertain. However, a role for high concentrations of PP2A in meiosis has long been suspected because MPF inactivation depends on an okadaic acid-sensitive phosphatase 2A.
In Xenopus oocytes in vitro, high concentrations of PP2A dephosphorylate the hyperphosphorylated form of Cdc25. Furthermore, okadaic acid (OA), a specific inhibitor of PP2A phosphatase, induces MPF auto-amplification (Goris et al., 1989; Felix et al., 1990; Izumi et al., Reference Izumi, Walker and Maller1992; Karaiskou et al., Reference Karaïskou, Jessus, Brassac and Ozon1999).
In addition, the inhibition of PP2A by okadaic acid (Rime et al., Reference Rime, Huchon, Jessus, Goris, Merlevede and Ozon1990; Picard et al., Reference Picard, Labbe, Barakat, Cavadore and Doree1991) induces meiotic resumption in the oocytes of many species, including both competent and incompetent mouse oocytes (Rime & Ozon, Reference Rime and Ozon1990; Rime et al., Reference Rime, Huchon, Jessus, Goris, Merlevede and Ozon1990).
Karaiskou et al. (1998), during the meiotic maturation of Xenopus, investigated the different molecular partners that are implicated in the network that allows Cdc2 activation in the mechanism of the MPF autocatalytic activation. This finding demonstrates that the initiation of the Cdc2/Cdc25 feedback loop, leading to an abrupt Cdc2 kinase activation, requires two sets of phosphorylation reactions on Cdc25: one is Cdc2 kinase-dependent and the second requires Plx1 activity and PP2A inhibition. Evidence for a crosstalk between cAMP–PKA and OA-sensitive protein phosphatases was presented (Schwartz & Schultz, Reference Schwartz and Schultz1991; Lu et al., Reference Lu, Smith, Chen, Yang, Han, Schatten and Sun2001); however, the direct physiological target of PP2A has not been unequivocally identified.
In the oocytes of some species, MAP kinase is activated as a consequence of the activation of a cascade that involves the kinase Mos, including MAP kinase kinase (MEK) or through the Ras–Raf–MEK pathway (Palmer & Nebreda 2000; Lu et al., Reference Lu, Smith, Chen, Yang, Han, Schatten and Sun2001; Bodart et al., Reference Bodart, Gutierrez, Nebreda, Buckner, Resau and Duesbery2002). MAPKs have thus been implicated in the cascade leading to MPF activation.
Active MAP kinase itself was found to be capable of inducing Xenopus oocytes maturation in the absence of hormone stimulation (Haccard et al., Reference Haccard, Lewellyn, Hartley, Erikson and Maller1995). Activated MAPKs may directly or indirectly regulate some proteins that control MPF activity, such as Cdc25 phosphatase or Myt1 kinase. However, in starfish, no MAP kinase activation is required for Cdc2 kinase activation and GVBD induction (Picard et al., Reference Picard, Galas, Peaucellier and Dorée1996) and, in rodents MAP kinase is only activated after GVBD and Cdc2 kinase activation (Verlhac et al., Reference Verlhac, Pennart, Maro, Cob and Clarke1993).
It is well established that in B. arenarum oocytes progesterone-induced maturation depends on the synthesis of new proteins (Zelarayán et al., Reference Zelarayán, Oterino and Bühler1996). Several studies in different species have demonstrated that Mos synthesis is required for hormone induced GVBD (Wasserman & Masui, Reference Wasserman and Masui1975; Takemoto & Horiuchi Reference Tatemoto and Horiuchi1995). In Xenopus, the protein kinase Mos is the only newly synthesized protein that has been shown to be necessary for progesterone-induced maturation (Sagata et al., 1988). Inhibition of protein synthesis in porcine oocytes blocks MAP kinase activation and GVBD. However, in starfish, neither protein synthesis nor MAP kinase activation are required for Cdc2 kinase activation and GVBD induction. Mos is a MAP kinase kinase kinase that leads to the activation of MAPK through the activation of MEK (MAPKK) (Nebreda et al., Reference Nebreda and Hunt1993; Posada et al., Reference Posada, Yew, Ahn, Van de Woude and Cooper1993; Shibuya & Ruderman, Reference Shibuya and Rudeman1993). Several studies have demonstrated that activation of MAP kinase is important or essential for MPF activation in Xenopus oocytes (Haccard et al., Reference Haccard, Jessus, Cayla, Goris, Merlevede and Ozon1990; Ferrell et al., Reference Ferrell, Wu, Gerhart and Martin1991), although Nebreda & Hunt (Reference Nebreda and Hunt1993), Posada et al. (Reference Posada, Yew, Ahn, Van de Woude and Cooper1993) and Shibuya & Ruderman (Reference Shibuya and Rudeman1993) showed that Mos leads to the activation of MAP kinase but not of Cdc2. However, in theory, the accumulation of a protein absolutely required for pre-MPF activation must depend on progesterone stimulation and be independent of Cdc2 activity.
The objectives of the present paper were to study the involvement and possible interactions of both cAMP–PKA and protein phosphatases in B. arenarum oocytes maturation and to determine if these pathways are independent or not of the MAPK cascade.
Materials and methods
Animals
Adult specimens of B. arenarum were collected in the northwestern area of Argentina from May to August (winter animals) and from September to December (summer animals) and kept at 15 °C until use.
Hormones and reagents
All hormones and reagents were purchased from Sigma. Progesterone was dissolved in ethanol and added directly to the culture medium to give a final concentration of 2.5 μM. Sodium metavanadate (NaVO3) was dissolved in ddH2O at 75 °C and various doses were added to the culture medium at a constant volume (5 μl). H-89 was dissolved in 4:6 ethanol/AR.
In vitro denuded oocytes culture
Experimental manipulation and culture were performed at room temperature (22–25 °C) in amphibian Ringer solution (AR) (6.6 g NaCl/l, 0.15 g CaCl2/l and 0.15 g KCl/l) containing penicillin G–sodium (30 mg/l) and streptomycin sulphate (50 mg/l), pH 7.4.
Denuded fully grown oocytes were obtained according to Lin and Schuetz (Reference Lin and Schuetz1985). Follicle cells were removed by gentle shaking (100 oscillations/min) (Zelarayán et al., Reference Zelarayán, Oterino and Bühler1995).
Randomized samples of 20 oocytes were distributed into separate wells containing 2 ml of AR; the reagents were added (5 μl) directly to the culture medium.
Oocyte maturation was assessed 24 h after hormone or reagent addition. Meiosis reinitiation was scored both by the presence of a transient white spot in the animal pole and by the absence of GV after dissection of the oocytes fixed in trichloroacetic acid.
Microinjections
Denuded fully grown immature oocytes were microinjected with 50 nl of mature cytoplasm, with 50 nl of immature cytoplasm, or with 50 nl of the GV content. Following injection, oocytes were placed in AR and scored for GVBD after incubation for 24 h.
Microinjection was performed with a sharpened glass micropipette (outer diameter 40–50 μm) attached to a micromanipulator.
Results
Effect of PKA inhibition on the maturation of fully grown oocytes
In order to evaluate whether in B. arenarum oocytes meiosis reinitiation is affected by the inhibition of PKA activity, we microinjected H-89, an inhibitor of the catalytic subunit of PKA, into the oocytes. Denuded oocytes incompetent to mature spontaneously were injected with 50 nl of different doses of H-89 (2.5–10 μM). GVBD was scored after 24 h of culture.
The result (Fig. 1) shows that the inhibition of PKA by treatment with H-89 is capable of inducing GVBD in a dose-dependent manner in the absence of progesterone. The maximum response (94%) was obtained with the dose of 10 μM of H-89.
Effect of H89 on oocytes maturation. Fully grown denuded oocytes incompetent to mature spontaneously were injected with 50 nl of different doses of H-89 (2.5–10 μM). GVBD was scored after 24 h of culture. Values are the mean ± SEM (n = 5). Each experiment was performed on a different animal.
Effect of Cdc25 activity inhibition during oocytes maturation induced by H-89
Phosphatase Cdc25 is involved in the Cdc2 Thr-14/Tyr-15 dephosphorylation of pre-MPF during oocytes maturation.
The participation of Cdc25 in the transduction pathway of maturation induced by H-89 was assayed using NaVO3, an inhibitor of Cdc25 activity.
Denuded fully grown oocytes incompetent to mature spontaneously were preincubated with NaVO3 (1 MM) for 60 min and then injected with 50 nl of different doses of H-89 (2.5–10 μM).
The results in Figure 2 indicate that NaVO3 effectively inhibited H-89-induced oocytes maturation, thus confirming that Cdc25 is involved in the PKA pathway of meiosis reinitiation.
Effect of Cdc25 activity inhibition during oocytes maturation induced by H-89. Denuded fully grown oocytes incompetent to mature spontaneously were preincubated with NaVO3 (1 MM) for 60 min and then injected with 50 nl of different doses of H-89 (2.5–10 μM). GVBD was scored after 24 h of culture. Values are the mean ± SEM (n = 4). Each experiment was performed on a different animal.
Effect of the inhibition of PP2A activity on GVBD of oocytes incompetent to mature spontaneously
To determine if PP2A is involved in the negative regulation of MPF, we used the specific inhibitor of this phosphatase, OA.
Denuded fully grown oocytes incompetent to mature spontaneously were injected with 50 nl of different doses of OA (0.5–2.5 μM). GVBD was scored after 24 h of culture.
The result (Fig. 3) shows that the inhibition of PP2A by treatment with OA is sufficient to release the meiosis block and allow GVBD in a dose-dependent manner. Continuous exposure of B. arenarum immature oocytes to PP2A inhibitor OA (1 μM) for 24 h significantly enhanced the percentage of oocytes that resumed meiosis (84% GVBD).
Effect of the inhibition of PP2A activity on the GVBD of oocytes incompetent to mature spontaneously. Denuded fully grown oocytes incompetent to mature spontaneously were injected with 50 nl of different doses of okadaic acid (0.5–2.5 μM). GVBD was scored after 24 h of culture. Values are the mean ± SEM (n = 3). Each experiment was performed on a different animal.
Effect of Cdc25 activity inhibition on MPF activation with OA
The direct physiological target of PP2A has not been unequivocally identified. It has been suggest that PP2A could be the phosphatase responsible for the dephosphorylation of the Cdc25 activator residues.
Our aim was to elucidate whether OA-induced meiotic resumption of incompetent oocytes was mediated through PP2A effect on Cdc25 activity.
Denuded fully grown incompetent oocytes were cultured for 60 min with NaVO3 (1 MM) and then injected with 50 nl of various doses of OA (0.5–2.5 μM).
The percentage of GVBD obtained (Fig. 4) showed that the inhibition of Cdc25 activity with NaVO3 reached values similar to those when oocytes were injected with OA alone, suggesting that OA induces GVBD by a transduction pathway in which Cdc25 phosphatase is not involved.
Effect of Cdc25 activity inhibition on MPF activation with OA. Denuded fully grown incompetent oocytes were cultured for 60 min with NaVO3 (1 MM) and then injected with 50 nl of various doses of OA (0.5–2.5 μM). GVBD was scored after 24 h of culture. Values are the mean ± SEM (n = 3). Each experiment was performed on a different animal.
Inhibition of MAPK activity on meiotic resumption in fully grown incompetent oocytes
We used an inhibitor of MEK, PD98059, as a pharmacological tool to study the involvement of MAPK in the induction of meiotic resumption of incompetent oocytes of B. arenarum. Different groups of fully grown incompetent oocytes were preincubated for 60 min in the presence of PD98059 at various doses (1–10 μM). Oocytes were subsequently exposed to progesterone (2.5 μM) for 24 h and GVBD was scored.
As shown in Figure 5, treatment with PD98059 consistently inhibited progesterone-induced meiotic maturation in a dose-dependent manner. These results indicate that MAPK activity was necessary for progesterone-induced maturation in denuded fully grown incompetent oocytes of this species.
Inhibition of MAPK activity on meiotic resumption in fully grown incompetent oocytes: fully grown incompetent oocytes were preincubated for 60 min in the presence of PD98059 at various doses (1–10 μM) and then exposed to progesterone (2.5 μM). GVBD was scored after 24 h of culture. Values are the mean ± SEM (n = 4). Each experiment was performed on a different animal.
Effect of MAPK inhibition on OA-induced maturation in fully grown ovarian oocytes
To determine whether the OA-induced meiotic resumption of incompetent oocytes was mediated through the MEK–MAPK pathway, fully grown denuded oocytes were preincubated for 60 min in the presence of PD98059 at various doses (1–10 μM) and then injected with 50 nl of OA (2.5 μM). Oocytes were cultured for 24 h before GVBD was scored.
The results in Figure 6 show that the inhibition of MAPK activity by PD98058 inhibited the meiosis reinitiation induced by OA in a dose-dependent manner.
Effect of MAPK inhibition on OA-induced maturation in fully grown ovarian oocytes. Fully grown denuded oocytes were preincubated for 60 min in the presence of PD98059 at various doses (1–10 μM) and then injected with 50 nl of OA (2.5 μM). Oocytes were cultured for 24 h before GVBD was scored. Values are the mean ± SEM (n = 3). Each experiment was performed on a different animal.
We also tested the effect of cycloheximide (10 μg/ml) on OA-induced meiotic resumption of incompetent oocytes because activation of MAP kinases requires protein synthesis. No oocytes underwent GVBD (0%), indirectly confirming the implication of MAP kinases in OA-induced meiotic resumption.
Together, these results indicate that OA induces meiotic resumption of incompetent oocytes through the activation of the MEK–MAPK pathway.
Effect of MAPK inhibition on the resumption of meiosis induced by PKA inhibition
To analyze the participation of MAPK in the GVBD induced by inhibition of PKA we pre-treated ovarian oocytes with PD98059 for 60 min and subsequently treated them with increasing concentrations of H-89, an inhibitor of PKA activity.
Figure 7 shows that the inhibition of MAPK with PD98059 did not significantly modify GVBD percentages induced by H-89. These results suggest that the MAPK pathway was not involved in the GVBD induced by the inhibition of PKA activity.
Effect of MAPK inhibition on the resumption of meiosis induced by PKA inhibition. Fully grown denuded oocytes were preincubated for 60 min in the presence of PD98059 (1–10 μM) and subsequently treated with increasing concentrations of H-89. Oocytes were cultured for 24 h before GVBD was scored. Values are the mean ± SEM (n = 3). Each experiment was performed on a different animal.
Discussion
Our results indicated that the inhibition of PKA by treatment with H-89 was capable of inducing GVBD in a dose-dependent manner by a pathway in which Cdc25 phosphatase but not the MAPK cascade is involved. In addition, the assays with 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.
It has been reported that, in B. arenarum oocytes, progesterone decreases cAMP intracellular levels by inhibiting adenylyl cyclase or cAMP-dependent PDE activity (Zelarayán et al., Reference Zelarayán, Oterino, Sánchez Toranzo and Bühler2000; Sánchez Toranzo et al., Reference Sánchez Toranzo, Bonilla, Zelarayán, Oterino and Bühler2006). This progesterone effect is believed to be sufficient to reduce the activity of the cAMP-dependent PKA, which prevents the activation of the maturation-promoting factor (MPF) in the oocytes. The involvement of PKA in oocytes maturation has been demonstrated in Fundulus heteroclitus (Cerdà et al., Reference Cerdà, Petrino, Landin and Lin1997) and C. batrachus (Haider & Baqri, Reference Haider and Baqri2002). In both species, the PKA inhibitor H-8 or H-89 stimulated GVBD, bypassing the MIS. In the Atlantic croaker, PKA inhibitors (Rp-cAMP and KT5720) did not stimulate GVBD in the absence of the MIS (20β-S) nor did they enhance the efficacy of the MIS (Pace & Thomas, Reference Pace and Thomas2005). In the present study we demonstrated that the injection of 50 nl of H-89 10 μM produced GVBD percentages similar to those obtained with treatment with progesterone.
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 agreement with this hypothesis, recent findings demonstrate that in Xenopus and mouse oocytes Cdc2 is a direct substrate of PKA (Qian et al., Reference Qian, Erikson, Taieb and Maller2001; Han et al., Reference Han, Chen, Paronetto and Conti2005; Han & Conti, Reference Han and Conti2006).
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.
During the past few years, important information has been acquired on how the natural G2 arrest is maintained in oocytes with Myt1 activity probably playing a critical role. In this sense, a link between MAPK and Cdc2–cyclin B activation has been identified, via the p90-induced phosphorylation and inactivation of the Cdc2 inhibitory kinase Myt1. Mishra & Joy, (Reference Mishra and Joy2006) have shown a positive involvement of MAPK in catfish (Heteropneustes fossilis) oocytes maturation.
A role of PP2A in oocytes maturation has long been suspected because MPF inactivation depends on an OA-sensitive phosphatase (Forester et al., Reference Forester, Maddox, Louis, Goris and Virshup2007). It is a generally accepted view that PP2A negatively regulates Cdc2-induced Cdc25 phosphorylation (Izumi et al., Reference Izumi, Walker and Maller1992; Clarke et al., Reference Clarke, Hoffmann, Draetta and Karsenti1993), and it has been suggested that PP2A acts downstream of cAMP-dependent protein kinase (PKA) yet prior to, or at the point of, MPF activation (Rime et al., Reference Rime, Huchon, Jessus, Goris, Merlevede and Ozon1990), but it is unclear whether PP2A is regulated early or late during oocytes maturation. In Xenopus oocytes it was proposed that PP2A, a major Ser/Thr phosphatase present, is a negative regulator of Cdc2 activation (Hermann et al., Reference Hermann, Cayla, Dumortier, Goris, Ozon and Merlevede1988; Lee et al., Reference Lee, Turk and Kirschner1994). However, in B. arenarum oocytes, inhibition of Cdc25 with NaVO3 did not inhibit OA-induced maturation, suggesting that the target of PP2A was not the Cdc25 phosphatase.
OA stimulates oocytes maturation in starfish, Xenopus and mammals (Goris et al., 1989; Rime & Ozon, Reference Rime and Ozon1990; Gavin et al., 1991; Schwartz & Schultz, Reference Schwartz and Schultz1991; Sun et al., Reference Sun, Yu, Chiang, Fu and Lu2002), an effect that has been attributed to MPF (Goris et al., 1989) or MAPK (Zernika-Goetz et al., Reference Zernicka-Goetz, Verlhac, Géraud and Kubiak1997; Sun et al., Reference Sun, Yu, Chiang, Fu and Lu2002) activation. MAP kinase activation has been reported to be essential in Xenopus oocytes GVBD (Ferrell & Machleder, Reference Ferrell and Machleder1998).
In order to confirm the implication of the MAPK pathway in the meiotic resumption of B. arenarum incompetent oocytes treated with progesterone, we used an inhibitor of MEK, PD98059, which selectively blocks the activity of MEK by inhibiting the activation of MAPK and subsequent phosphorylation of MAPK substrates. Our results showed that this inhibitor prevented activation of MAPKs and meiotic resumption, suggesting that MAP kinase activation is an early event in response to progesterone in this species.
It has recently been proposed (Palmer et al., Reference Palmer, Gavin and Nebreda1998) that kinase acting downstream of MAP kinase p90 inactivates the Cdc2 inhibitory kinase Myt1 by phosphorylation and thereby links MAP kinase activation to progesterone-induced MPF activation. However, Fisher et al., (Reference Fisher, Stirling Meyer, Malik and Helgason1999) showed that Xenopus oocytes can undergo GVBD in the absence of MAP kinase activity.
In B. arenarum oocytes we demonstrated that the inhibition of MAP kinase by PD 98059 prevented the activation of MPF induced by OA, suggesting that the activation of the MAP kinase cascade produced an inhibition of Myt1 and, in consequence, the activation of MPF without participation of the Cdc25 phosphatase.
In many species, including starfish, Spisula and mice, protein synthesis is not required for fully grown oocytes to undergo GVBD in response to the natural maturation-inducing signals. In B. arenarum, however, protein synthesis is required for GVBD. It is possible that this requirement is for Mos synthesis and thus MAP kinase activation, and no further cyclin B synthesis is required before GVBD.
It has been suggested that imbalanced coordination between protein kinases and protein phosphatases determines cellular responses. 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 regulate the activity of the Myt1; and (2) the inhibition of AMPc–PKA, which affects the activity of the Cdc25 phosphatase.
Acknowledgements
This work was supported by a grant from the Science Council of the National University of Tucumán (CIUNT), and the National Agency for Promotion of Science and Technology (FONCYT).
