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Biology of Reproduction 65, 442-448 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Inhibition of Phosphatidylinositol 3-Kinase or Mitogen-Activated Protein Kinase Kinase Leads to Suppression of p34cdc2 Kinase Activity and Meiotic Progression Beyond the Meiosis I Stage in Porcine Oocytes Surrounded with Cumulus Cells

Masayuki Shimadaa, Wen-Xian Zenga, and Takato Teradaa

a Faculty of Applied Biological Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8528, Japan

ABSTRACT

In this study, the effects of U0126 that inhibits the activity of mitogen-activated protein (MAP) kinase kinase (MEK), and LY294002, which is a phosphatidylinositol (PI) 3-kinase inhibitor, on meiotic progression beyond the metaphase I (MI) stage in porcine oocytes were examined. Cumulus-oocyte complexes (COCs) were cultured for 22 h with 50 µM LY294002 or 10 µM U0126 following cultivation for the initial 22 h. MAP kinase activity in oocytes cultured with LY294002 or U0126 was significantly lower than that in control oocytes cultured for up to 44 h. U0126 and LY294002 significantly decreased p34cdc2 kinase activity and the proportion of oocytes reaching the MII stage compared to those in control oocytes. Oocytes denuded after COCs had been cultured for 22 h were cultured further for 22 h with U0126 or LY294002. In the denuded oocytes, U0126 suppressed MAP kinase activity, p34cdc2 kinase activity, and meiotic progression to the MII stage; however, LY294002 did not significantly affect the activity of these kinases and meiotic progression. These results suggest that increasing MAP kinase activity in oocytes via the PI 3-kinase signaling pathway in cumulus cells is involved in the stimulation of maturation promoting factor, leading to meiotic progression beyond the MI to MII stage in porcine oocytes.

cumulus cells, gamete biology, kinases, meiosis, signal transduction

INTRODUCTION

Oocyte maturation depends on the activation of maturation promoting factor (MPF) that is composed of p34cdc2 kinase and cyclin B [1, 2]. Activated MPF induces germinal vesicle breakdown (GVBD) in amphibian oocytes [3, 4], mouse oocytes [5], porcine oocytes [6], and bovine oocytes [7]. After GVBD in porcine oocytes, a further increase of MPF activity is required for meiotic progression beyond the metaphase I (MI) stage [8]. Increasing MPF activity stimulates the ubiquitin proteolysis pathway that induces degradation of cyclin B, which triggers a decrease in MPF activity, leading to meiotic progression beyond the MI stage [9, 10]. Consistent with the increase of MPF activity after GVBD, mitogen-activated protein kinase (MAP kinase) in porcine oocytes is also activated [11]. Several researchers have shown that during meiotic resumption and progression, MAP kinase activity is essential for the conversion of pre-MPF (inactive form) into active MPF in Xenopus oocytes [12–14]; however, little information exists describing the role of MAP kinase activity during meiotic progression beyond the MI stage in porcine oocytes.

In our previous study [15, 16], the activation of MAP kinase and MPF in porcine oocytes surrounded by cumulus cells was inhibited by 50 µM LY294002 that selectively inhibited phosphatidylinositol (PI) 3-kinase activity [17], resulting in suppression of the meiotic progression from the MI to the MII stage. However, in the denuded oocytes cultured with LY294002 for 48 h MAP kinase activity, p34cdc2 kinase activity, and proportion of oocytes that had reached the MII stage were at a similar level to those of oocytes cultured without LY294002. These results suggest that PI 3-kinase in cumulus cells controls MAP kinase and MPF activity after GVBD in porcine oocytes. However, it remains to be determined whether PI 3-kinase in cumulus cells regulates the activation of MPF through a MAP kinase-dependent pathway.

In this report, we investigated the mechanisms by which PI 3-kinase in cumulus cells activate MPF and the relationship between MAP kinase and MPF during meiotic progression after GVBD in porcine oocytes. After 22 h of cultivation of porcine cumulus-oocyte complexes (COCs), some COCs were denuded. Then the remaining COCs and the denuded oocytes (DOs) were cultured further in the presence of LY294002 or U0126 that inhibits activation of MAP kinase by blocking the activity of MAP kinase kinase (MEK) [18], and the MAP kinase activity, the p34cdc2 kinase activity, and the phosphorylation form of p34cdc2 kinase in the cultured oocytes were analyzed.

MATERIALS AND METHODS

Isolation and Culture of Porcine COCs

Porcine ovaries were collected from 5- to 7-mo-old prepubertal gilts at a local slaughterhouse and transported within 1.5 h to the laboratory in 0.85% (w/v) NaCl containing 0.1 mg/ml kanamycin (Meiji Seika, Tokyo, Japan) at about 30°C. The surfaces of intact healthy antral follicles of 3–8 mm in diameter were cut with a razor blade, and oocytes were collected by scraping the inner surface of the follicle walls using a surgical blade. The oocytes collected were placed in prewarmed PBS (pH 7.4) supplemented with 0.1% (w/v) polyvinylpyrrolidone (PVP) (Sigma Chemical Co., St. Louis, MO) and 0.1 mg/ml kanamycin. Oocytes having evenly granulated cytoplasm with at least four layers of unexpanded cumulus oophorus cells were selected under a stereomicroscope and washed three times with maturation medium. The basic maturation medium was modified NCSU37 [19] containing 10% (v/v) fetal calf serum (FCS; Gibco BRL, Grand Island, NY), 0.6 µg/ml porcine FSH (Sigma), 1.3 µg/ml equine LH (Sigma), 7 mM taurine (Sigma), 2% (v/v) essential amino acids (ICN 16-011-49; Gibco), and 1% (v/v) nonessential amino acids (ICN 16-810-49; Gibco). The COCs were cultured for various time periods in 100-µl drops of different maturation medium (about 20 oocytes/drop) covered with mineral oil (Sigma) at 39°C in a humidified atmosphere of 5% CO2 in air.

Assessment of Nuclear Maturation

After incubation, the oocytes were freed from cumulus cells, then mounted on slides, fixed with acetic acid:ethanol (1:3, v/v) for 48 h, and stained with aceto-lacmoid before examination under a phase-contrast microscope (400x) for evaluation of their chromatin configuration.

Extract Preparation

Oocytes were washed several times in PBS and put into plastic tubes containing 5 µl cell lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% [v/v] Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM PMSF [Sigma]). All drugs used except for PMSF were purchased from New England Biolabs (Beverly, MA). After the oocytes were suspended, the samples were frozen in liquid nitrogen, and the frozen samples were then sonicated using an ultrasonic disruptor (UD-200; Tomy, Tokyo, Japan) fitted with Cup Horn (CH-0633; Tomy) three times for 25 sec each at 1°C. Cell extracts were frozen and stored at -80°C just before use.

In Vitro MAP Kinase Assay

A p44/42 MAP kinase assay kit (New England BioLabs) was used for measuring MAP kinase activity. The method for determination of MAP kinase activity was based on the report of Anas et al. [20]. Briefly, 5 µl of oocyte extract (containing 20 oocytes) was mixed with 25 µl of kinase assay buffer A (25 mM Tris [pH 7.5], 5 mM ß-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM MgCl2), 0.1 mM ATP (Sigma), and 2 µg Elk 1 fusion protein. The mixture was incubated for 30 min at 30°C. All the drugs used except ATP were purchased from New England BioLabs. The reactions were terminated by the addition of 10 µl of 4x Laemmli sample buffer, and after boiling for 5 min the samples were subjected to 12.5% SDS-PAGE. Phosphorylation of the Elk 1 fusion protein was detected by immunoblotting and chemiluminescence using anti-phospho-specific Elk 1 antibody. Each independent experiment was repeated four times.

In Vitro p34cdc2 Kinase Assay

The p34cdc2 kinase assay was performed using a Mesacup cdc2 kinase assay kit (code no. 5234; MBL, Nagoya, Japan), according to the method described by Shoujo et al. [21]. They showed that the correlation coefficient between p34cdc2 kinase activity as determined by using the Mesacup cdc2 kinase assay kit and histone H1 kinase activity as measured by radioactive method were as high as 0.9961.

Briefly, 5 µl of oocyte extract (containing 10 oocytes) was mixed with 45 µl of kinase assay buffer B composed of 25 mM Hepes buffer (pH 7.5) (MBL), 10 mM MgCl2 (MBL), 10% (v/v) MV peptide solution (SLYSSPGGAYC) (MBL), and 0.1 mM ATP (Sigma), and the mixture was incubated for 30 min at 30°C. The reaction was terminated using 200 µl PBS containing 50 mM EGTA (MBL). Phosphorylation of MV peptides was detected using an ELISA analysis (Mesacup cdc2 kinase assay kit [code no. 5234; MBL]). Values were expressed as the fold strength of p34cdc2 kinase in oocytes just after collection from their follicles, defined as 1. Each independent experiment was repeated four times.

Detection of p34cdc2 Kinase by Immunoblotting Analysis

Oocyte extracts (containing 60 oocytes) were diluted twofold with 2x Laemmli sample buffer [22]. After denaturing by boiling for 5 min, the protein samples were separated by SDS-PAGE on 12.5% polyacrylamide gel (Pharmacia Biotech, Uppsala, Sweden), then transferred onto polyvinylidene difluoride (PVDF) membrane (Amersham, Arlington Heights, IL) using the PhastTransfer system (Pharmacia Biotech). The membrane was blocked using SuperBlock blocking buffer (Pierce, Rockford, IL), then incubated with anti-cdc2 monoclonal antibody (cat. no. sc-54; Santa Cruz, CA) at 1:500 dilution overnight at 4°C in 10% (v/v) SuperBlock blocking buffer in 0.1% (v/v) Tween 20-PBS (T-PBS). After three washes in T-PBS, the membranes were treated with horseradish-peroxidase-labeled anti-mouse IgG (1:7000; Amersham) in 10% (v/v) SuperBlock blocking buffer in T-PBS for 1 h at room temperature. After three washes of 10 min each with T-PBS, peroxidase activity was visualized using the ECL Plus Western blotting detection system (Amersham), according to the manufacturer's instructions. Each experiment was repeated three times.

Experimental Design

In experimental 1, the dose effects of U0126 (Promega, Madison, WI) on MAP kinase activity in matured porcine oocytes were examined. After the COCs were cultured for 44 h in the basic maturation medium, COCs were mechanically denuded by pipetting in 0.1% (w/v) hyaluronidase (Sigma)/PBS with flame-draw pipette tips, whose inner diameters were slightly larger than the diameters of the oocytes. After the DOs were cultured further in the basic medium supplemented with 0, 1, 5, 10, and 20 µM U0126 for 6 h, MAP kinase activity was examined. U0126 was dissolved in dimethyl sulfoxide (DMSO; Sigma) at 20 mM, and stored at -20°C. Each final concentration was obtained by dilution in the basic medium.

In experiment 2, the effect of U0126 on p34cdc2 kinase activity during GVBD in porcine DOs was tested. COCs were mechanically denuded by pipetting in 0.02% (w/v) EDTA (Sigma)/PBS with flame-draw pipette tips. After the DOs were cultured in the maturation medium with 10 or 20 µM U0126 for up to 24 h, their p34cdc2 kinase activity was assayed.

Experiment 3 was undertaken to evaluate the effects of 50 µM LY294002 (Sigma) and 10 µM U0126 on meiotic progression, MAP kinase activity, and p34cdc2 kinase activity during meiotic progression beyond the MI stage in oocytes enclosed in cumulus cells or DOs. Previously [15], 5 µM LY294002 did not significantly affect the proportion of oocytes reaching the MII stage, but the addition of 50 µM LY294002 significantly suppressed meiotic progression to the MII stage. Thus, the concentration of 50 µM LY294002 was employed in this experiment. After the COCs had been cultured for 22 h in the basic maturation medium, some COCs were denuded mechanically with flame-draw pipette tips. The DOs and the remaining COCs were cultured further for 6, 10, 14, 18, and 22 h in the basic maturation medium supplemented with 50 µM LY294002 or 10 µM U0126, respectively. As a control, inhibitor-free medium was prepared by adding 0.098% (v/v) DMSO to the basic maturation medium. This concentration of DMSO had no adverse effect on porcine oocyte maturation [15].

Statistical Analysis

Statistical analyses of all data from three or four replicates for comparison were carried out using analysis of one-way ANOVA followed by Duncan multiple-range test using Statview (Abacus Concepts, Inc., Berkeley, CA). All percentage data were subjected to arc-sine transformation before statistical analysis.

RESULTS

Experiment 1: Dose Effects of U0126 on MAP Kinase Activity in Matured Oocyte

In order to determine the effective concentrations of U0126 that inhibit MAP kinase activity, oocytes cultured for 44 h in which MAP kinase activity might be high were cultured further with 1, 5, 10, and 20 µM U0126 for 6 h. Immunoblottings of phosphorylated Elk 1 fusion protein were used as a measure of MAP kinase activity. The activity is expressed relative to a positive control, 5 ng active MAP kinase, defined as 100%. At further cultivation in basic medium (control), a high level of MAP kinase activity was observed (Fig. 1). However, U0126 significantly decreased MAP kinase activity with increasing concentrations of the drug (Fig. 1). Each MAP kinase activity in the oocytes treated with 10 or 20 µM U0126 was significantly lower as compared to those of other groups (Fig. 1). After a 6-h treatment with U0126, the majority of oocytes (73–78%) in all treatment groups were arrested at the MII stage (data not shown).



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  		FIG. 1. Dose effects of U0126 (MEK inhibitor) on MAP kinase activity in mature porcine oocytes. {dagger}Relative amounts of MAP kinase activity that were determined using scanning densitometry (data are expressed as fold strength of positive control, 5 ng active MAP kinase as defined 100%). a–dNo common superscripts were significant (P < 0.01)

Experiment 2: Dose Effects of U0126 on p34cdc2 Kinase Activity in DOs During Meiotic Resumption

Because it was possible that MAP kinase in cumulus cells was associated with GVBD of oocytes [23], the effects of 10 or 20 µM U0126 on p34cdc2 kinase activity were examined using DOs that were cultured with 10 or 20 µM U0126 for 24 h. After the cultivation of denuded oocytes, p34cdc2 kinase activity was determined. p34cdc2 kinase activity was not suppressed by the addition of 10 µM U0126 into medium at any time point (Fig. 2), but after a 16-h cultivation of DOs, 20 µM U0126 significantly decreased this kinase activity up to 24 h. These results show that 10 µM U0126 does not have any effect on p34cdc2 kinase but on MAP kinase activity. Accordingly, the concentration of U0126 was adjusted to 10 µM in each maturation medium in the other experiments.



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  		FIG. 2. Effects of U0126 on p34cdc2 kinase activity in porcine DOs. *Significant difference in comparison with treatment with U0126 at the same time point (P < 0.01). {dagger}Data are expressed as fold strength of p34cdc2 kinase activity in oocytes just after collection from their follicles as defined 1

Experiment 3: Meiotic Progression in Porcine Oocytes Cultured with LY294002 or U0126 after the First 22 hof Cultivation

COCs or DOs were cultured for 6 or 22 h in the presence of 50 µM LY294002 or 10 µM U0126 following cultivation of COCs for the initial 22 h. As shown in Table 1, at 22 + 6 h of cultivation, LY294002 and U0126 had no significant effect on meiotic progression. However, after a further 22-h (22 + 22 h) cultivation of COCs, the addition of U0126 and LY294002 significantly increased the proportion of oocytes arrested at the GV and MI stages and significantly decreased the proportion of oocytes reaching the MII stage as compared to the control oocytes. In DOs, at 22 + 22 h of cultivation, U0126 significantly suppressed meiotic progression to the MII stage as compared to those treated with LY294002 and without this drug, although no significant difference in nuclear status between DOs cultured with LY294002 and in the basic medium was observed. When DOs were cultured for 22 + 22 h with U0126, 55% of oocytes did not undergo either chromosome separation or first polar body emission but had a chromatin cluster in the ooplasm.


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  		TABLE 1. Meitoic progression in porcine COCs or DOs that were cultured with LY294002 or U0126 further for 6 h or 22 h following 22 h cultivation of COCs in the basic medium

Mitogen-Activated Protein Kinase Activity in Porcine Oocytes Cultured with LY294002 or U0126 after the First 22 h of Cultivation

The MAP kinase activity in porcine oocytes surrounded with cumulus cells cultured in the basic maturation medium (control) was very low at 22 + 2 h but increased at 22 + 6 h of maturation (Fig. 3), when most of the oocytes had reached the MI stage (71%). At the same time point, however, MAP kinase in oocytes cultured in the presence of LY294002 and U0126 was not significantly activated as compared to that in oocytes cultured in the basic maturation medium for 22 + 2 h (Fig. 3). At 22 + 6 h, significant differences in MAP kinase activity existed between oocytes cultured with LY294002 or U0126 and those cultured without any drug (Fig. 3). The kinase activity in oocytes cultured with LY294002 or U0126 was significantly lower than that in oocytes cultured in the basic maturation medium for up to cultivation of 44 h (Fig. 3).



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  		FIG. 3. Effects of LY294002 or U0126 on MAP kinase activity in porcine oocytes. (COC): After COCs were cultured for 22 h in the basic medium, the COCs were cultured further for 22 h with 50 µM LY294002 or 10 µM U0126; (DO): after COCs were cultured for 22 h in the basic medium, the COCs were denuded and then the DOs were cultured further for 22 h with 50 µM LY294002 or 10 µM U0126. *Significant difference for each treatment at the same time point (P < 0.01). a–cNo common superscripts were significant (P < 0.01). {dagger}{dagger}Relative amounts of MAP kinase activity that were determined using scanning densitometry (data are expressed as fold strength of positive control, 5 ng active MAP kinase as defined 100%)

When COCs were denuded after 22 h of cultivation in the basic medium and then cultured further with LY294002 or U0126, MAP kinase activity in DOs not treated with any drug was significantly higher compared to those exposed to LY294002 or U0126 at the 22 + 6 h or 22 + 10 h time points (Fig. 3). The MAP kinase activity in DOs cultured with LY294002 then gradually rose, and at 22 + 14 h of cultivation, significant differences in MAP kinase activity between DOs cultured with and without LY294002 disappeared (Fig. 3). However, significant differences in MAP kinase activity were observed between DOs cultured with U0126 and those cultured without this drug from 22 + 6 h to 22 + 22 h cultivation (Fig. 3).

p34cdc2 Kinase Activity in Porcine Oocytes Cultured with LY294002 or U0126 after the First 22 h of Cultivation

During first up to 20 hr cultivation of COCs in the basic maturation medium, the low level of p34cdc2 kinase activity in oocytes was maintained, but it was dramatically increased after the 22 + 2 h cultivation (Fig. 4). This kinase activity reached a peak at 22 + 14 h of cultivation (Fig. 4). However, p34cdc2 kinase activity in oocytes incubated for 2, 6, and 10 h with 50 µM LY294002 or 10 µM U0126 after the first 22 h of cultivation was comparable to those in oocytes cultured in basic medium (control) (Fig. 4). After 14 h (22 + 14 h) of cultivation with LY294002 or U0126, oocytes showed significantly lower p34cdc2 kinase activity compared to that of oocytes cultured without any drug until 22 + 22 h of cultivation (Fig. 4).



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  		FIG. 4. Effects of LY294002 or U0126 on p34cdc2 kinase activity in porcine oocytes. (COC): After COCs were cultured for 22 h in the basic medium, the COCs were cultured further for 22 h with 50 µM LY294002 or 10 µM U0126; (DO): after COCs were cultured for 22 h in the basic medium, the COCs were denuded and then the DOs were cultured further for 22 h with 50 µM LY294002 or 10 µM U0126. *Significant difference for each treatment at the same time point (P < 0.01). {dagger}Data are expressed as fold strength of p34cdc2 kinase in oocytes just after collection from their follicles, defined as 1

When COCs were denuded mechanically after 22 h cultivation in the basic medium and cultured further for 22 h with or without drug exposure, p34cdc2 kinase activity in DOs cultured with LY294002 was comparable to that of oocytes cultured without this drug for 22 + 22 h (Fig. 4). In sharp contrast, after 22 + 14 h of cultivation in the presence of U0126, p34cdc2 kinase activity in DOs was maintained to be significantly lower compared to that of oocytes cultured in the absence of this drug (Fig. 4).

Immunoblotting analysis of p34cdc2 kinase using anti-cdc2 monoclonal antibody (cat. no. sc-54; Santa Cruz) showed marked differences between p34cdc2 kinase bands in oocytes, when COCs cultured further for 22 h with either 50 µM LY294002 or 10 µM U0126 following cultivation of COCs for the initial 22 h. Only a single band situated on SDS-PAGE at the 34-kDa region (lower band: L) was detected in oocytes cultured for 44 h without any drug (Fig. 5). In the extract of the oocytes cultured with LY294002 or U0126, another lower migration form (upper band: U) was also observed (Fig. 5).



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  		FIG. 5. Immunoblotting analysis of the changes of p34cdc2 kinase in porcine oocytes. 1) Porcine COCs were cultured for 44 h. 2) After COCs had been cultured for 22 h, COCs were cultured further for 22 h with 50 µM LY294002. 3) After COCs were cultured for 22 h, COCs were cultured further for 22 h in the presence of 10 µM U0126

DISCUSSION

Mitogen-activated protein kinase is activated during meiotic progression in Xenopus oocytes [12, 24–27], mouse oocytes [28, 29], porcine oocytes [11], bovine oocytes [30], and goat oocytes [31]. In Xenopus oocytes, the role of MAP kinase during meiotic progression is known to activate MPF [12–14]. On the other hand, using c-mos-deficient mice (Mos-/-), Verlhac et al. [32], Choi et al. [33], and Araki et al. [34] demonstrated that MPF was activated in its oocytes with the level of MAP kinase activity below the detection limit, resulting in the induction of GVBD as in the oocytes of Mos+/+ mice (wild type) that showed high MAP kinase activity during GVBD. This suggests that MPF activation is independent of MAP kinase activities in the mouse. Thus, in mammalian oocytes, the association of MAP kinase with MPF remains unclear.

In this report, it was determined that when COCs or DOs were cultured with 10 µM MEK inhibitor, U0126, that did not inhibit other kinases including PKC, Cdk2, Cdk4, JNK, and p38 MAP kinase [18] as well as p34cdc2 kinase in this study, MAP kinase activity was suppressed, leading to an inhibition of p34cdc2 kinase activity and meiotic progression beyond the MI stage in porcine oocytes. This shows that MAP kinase pathway mediates MPF activation during meiotic progression beyond the MI stage in porcine oocytes.

Our previous reports [16] showed that the MAP kinase activating pathway in porcine oocytes was regulated by PI 3-kinase in cumulus cells. In the present results, when porcine COCs were cultured with LY294002 after the first 22 h of cultivation, low p34cdc2 kinase activity was detected in the oocytes, and p34cdc2 kinase exhibited a phosphorylated form on SDS-PAGE followed by immunoblot analysis as in the case of treatment with U0126. However, p34cdc2 kinase and MAP kinase activities in DOs during meiotic progression beyond the MI stage were not significantly affected by the addition of LY294002. Judging from these findings, it is estimated that PI 3-kinase in cumulus cells induces MAP kinase activity, and the activated MAP kinase is associated with an increasing p34cdc2 kinase activity after the MI stage in porcine oocytes.

We also demonstrated that PI 3-kinase in cumulus cells attached to the oocyte played a crucial role in the regulation of gap junctional communications through the phosphorylation of connexin-43 [16, 35]. The gap junctional communications between cumulus cells and oocytes were seen to be disrupted at the point of first polar body emission in rats and pigs [36–38]. These cumulus cells have been reported to produce the suppressive factor for meiotic progression that is transferred to the oocytes through numerous gap junctions in pigs [39]. Thus, we favor the hypothesis that the activation of MAP kinase by the closing of the gap junctional communication due to PI 3-kinase activation in cumulus cells drives the increase of p34cdc2 kinase activity, resulting in meiotic progression beyond the MI to MII stage in porcine.

The activation of MAP kinase has been observed during meiotic progression after GVBD and activation of MPF in porcine oocytes [11, 16]. In this study, 10 µM U0126 that completely suppressed MAP kinase activity did not significantly affect p34cdc2 kinase activity in DOs cultured for 24 h as compared to that of oocytes cultured without this drug. However, during meiotic progression beyond the MI stage, this kinase inhibitor decreased both MAP kinase and p34cdc2 kinase activity. Thus, MAP kinase is not required to establish GVBD and activation of MPF but is needed for increasing MPF activity during the MI stage in porcine oocytes. In Xenopus oocytes pre-MPF (inactive form) is accumulated at the GV stage, and MAP kinase activity is essential for the conversion of pre-MPF into active MPF during GVBD [12–14]. Whereas in porcine oocytes pre-MPF was not observed at the GV stage but detected after the MI stage [40], and in the present study the addition of U0126 leaded to an increase of pre-MPF and an inhibition of p34cdc2 kinase activity during the MI stage in porcine oocytes. On the basis of these reports and present results, the evidence that in porcine oocytes MAP kinase increases some hours before GVBD and MPF activation suggests that the mechanisms of MPF activation during GVBD is differ from that in oocytes during the MI stage. Further study is necessary for better understanding of differences between both mechanisms governing the activation of MPF in porcine oocytes during GVBD and the MI stage.

In summary, the addition of LY294002 or U0126 to maturation medium suppressed MPF activity, meiotic progression beyond the MI stage, and MAP kinase activity in oocytes surrounded by cumulus cells. These results showed that increasing MAP kinase activity in oocytes was essential for the increase of MPF activity, leading to meiotic progression beyond the MI to MII stage in porcine oocytes.

FOOTNOTES

First decision: 22 January 2001.

1 Correspondence. FAX: 81 824 24 7988; tterada{at}hiroshima-u.ac.jp Back

Accepted: March 20, 2001.

Received: December 8, 2000.

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