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BOR - Papers in Press, published online ahead of print November 19, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020610
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BIOLOGY OF REPRODUCTION 70, 797–804 (2004)
DOI: 10.1095/biolreprod.103.020610
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Mitogen-Activated Protein Kinase Kinase Inhibitor Suppresses Cyclin B1 Synthesis and Reactivation of p34cdc2 Kinase, Which Improves Pronuclear Formation Rate in Matured Porcine Oocytes Activated by Ca2+ Ionophore1

Junya Ito, Masayuki Shimada2, and Takato Terada

Laboratory of Animal Reproduction, Graduate School of Biosphere Sciences, Hiroshima University, Higashi-Hiroshima, 739-8528, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To investigate the role of mitogen-activated protein (MAP) kinase kinase (MEK)/MAP kinase cascade on p34cdc2 kinase activity and cyclin B1 levels during parthenogenetic activation of porcine oocytes, MEK activity, MAP kinase activity, p34cdc2 kinase activity, and cyclin B1 levels were assayed in mature porcine oocytes after treatment with different concentrations of Ca2+ ionophore. A high concentration of Ca2+ ionophore (50 µM) rapidly reduced MEK activity in oocytes for up to 8 h of culture. MEK activity in the 10-µM treatment group was significantly higher. The low concentration treatment transiently decreased p34cdc2 kinase activity but did not affect MAP kinase activity and ultimately induced reactivation of p34cdc2 kinase via the synthesis of cyclin B1. On the other hand, treatments of a high concentration of Ca2+ ionophore or a low concentration of Ca2+ ionophore plus MEK inhibitor, U0126, linearly decreased MAP kinase activity following the decrease of p34cdc2 kinase activity; most of these oocytes formed pronuclei. These results suggest that decreasing MAP kinase activity is essential to maintaining low p34cdc2 kinase activity resulting from the degradation of cyclin B via a Ca2+-dependent pathway; lower activities of both MAP kinase and p34cdc2 kinase induce normal meiotic completion and pronuclear formation of parthenogenetically activated porcine oocytes.

calcium, developmental biology, kinases, meiosis, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
After sperm penetration, the first detectable event in mature oocytes is a rise in the intracellular calcium; the phenomenon has been observed in many species, including pigs [1, 2]. The transient rise of intracellular calcium has been shown to lead to cortical granule exocytosis, subsequent zona pellucida modifications [3], resumption of meiosis, and then pronuclear formation [4, 5]. An increase of intracellular calcium concentration has also been shown to stimulate degradation of cyclin B, a regulatory subunit of maturation-promoting factor (MPF) [6, 7]. Since the activation of p34cdc2 kinase, which is a catalytic subunit of MPF, depends on binding with cyclin B1 [8], it is considered that the degradation of cyclin B1 triggers the decrease of p34cdc2 kinase activity following pronuclear formation.

Additionally, Moos et al. [9] demonstrated that in mouse oocytes, mitogen-activated protein (MAP) kinase was inactivated during pronuclear formation. MAP kinase is phosphorylated and activated after germinal vesicle breakdown, and high activity is maintained at the metaphase II (MII) stage [10, 11]. The high level of MAP kinase activity has been reported to promote the synthesis of cyclin B, inducing the increase of p34cdc2 kinase activity, which results in a transient decrease at the anaphase I and telophase I stages [12]. It has been reported that MAP kinase is downstream of MAP kinase kinase (MEK), and that the treatment of matured oocytes with the MEK inhibitor, U0126, fails to cause arrest at the MII stage [13]. The inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, cyclin-dependent protein kinase-2 (cdk2), and c-Jun N-terminal kinases (JNK) [14]. Thus, MAP kinase is involved in the maintenance of the high activity of p34cdc2 kinase via cyclin B synthesis at the MII stage, suggesting that a decrease of its activity is also required for pronuclear formation. However, the role of decreasing MAP kinase activity on inactivation of p34cdc2 kinase and pronuclear formation in porcine matured oocytes has until now been unclear.

Release of oocytes from the MII stage can also be induced by artificial stimulants, such as electrical pulse [1, 15], ethanol [16], or calcium ionophore [17–19]. In mouse oocytes, these treatments resulted in pronuclear formation similar to fertilization [9]. However, in bovine and pig oocytes, it has been well known that a treatment by calcium ionophore A23187 alone is not sufficient to form pronuclei, and most of these oocytes progress to the meiotic metaphase III (MIII) stage, which has a meiotic spindle with a second polar body [4, 17, 20–22]. Also, in our previous study [21] when oocytes were treated with 10 µM Ca2+ ionophore, over 50% progressed to the MIII stage, and a high activity of MAP kinase was observed. Therefore, the investigation of the role of MAP kinase in the MII stage of porcine oocytes seems to be required for successful artificial activation.

In this study, we focused on the role of the MEK/MAP kinase cascade on p34cdc2 kinase activity and cyclin B1 levels during parthenogenetic activation of porcine oocytes. MAP kinase activity, p34cdc2 kinase activity, and cyclin B1 levels were analyzed in mature porcine oocytes after treatment by different concentrations of Ca2+ ionophore. Moreover, it was examined that a combinational treatment of a low concentration of Ca2+ ionophore (10 µM) and U0126 affected kinase activities, cyclin B1 levels, and pronuclear formation in mature porcine oocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and Culture of Porcine Oocyte Complexes

Porcine oocyte complexes (COCs) were isolated as described previously [23]. Porcine ovaries were collected from 5- to 7-month-old prepubertal gilts at a local slaughterhouse. The surface of healthy antral follicles measuring from 3 to 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. 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. COCs were cultured for 48 h in 100-µl drops of basic medium supplemented with 0.6 µg/ml porcine FSH (Sigma, St. Louis, MO) and 1.3 µg/ml equine LH (Sigma; about 20 oocytes/drop), and covered with mineral oil (Sigma) at 39°C in a humidified atmosphere of 5% CO2 in air. The proportion of oocytes matured at the MII stage in our culture system was 85% ± 4.7%. The basic medium was modified NCSU37 [24] supplemented with 10% (v/v) fetal calf serum (Gibco BRL, Invitrogen, Carlsbad, CA); 7 mM Turine (Sigma); 10% (v/v) essential amino acid (Gibco); and 5% (v/v) nonessential amino acid (Gibco).

Calcium Ionophore Treatment

Calcium ionophore treatment was performed according to the method described by Funahashi et al. [25] with some modifications. After 48 h culture, COCs were denuded mechanically by pipetting with flame-draw pipette tips that had inner diameters slightly larger than the oocyte diameter. Cumulus-free oocytes were washed three times in the basic medium. The oocytes were treated with 10 or 50 µM calcium ionophore A23187 in the basic medium for 5 min at 39°C and washed with basic medium to quench the action of the ionophore. Oocytes were exposed to Ca2+ ionophore three times at 5-min intervals as described above. The oocytes then were washed at least three times, and each group of 20 oocytes was cultured in 100-µl drops of the basic medium covered with mineral oil for 12 h at 39°C in a humidified atmosphere of 5% CO2 in air.

Assessment of Nuclear Maturation

After incubation, the oocytes were mounted on slides, fixed with acetic acid/ethanol (1:3) for 48 h, and stained with aceto-lacmoid before being examined under a phase-contrast microscope (400x) to evaluate their chromatin configuration. The nuclear status of oocytes was classified into categories according to our previous report [21]: A,T-II (anaphase, telophase-II), characterized by separating chromosomes attached to a slightly elongated spindle or extraction of the second polar body (Fig. 1A); MIII, characterized by a metaphase spindle with the release of a second polar body (Fig. 1B); PN, characterized by one or two pronuclei; CC, condensed chromosome; or Deg, degenerate oocytes.



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  		FIG. 1. Nuclear status in porcine oocytes activated by 10 µM Ca2+ ionophore. A) A,T-II; separating chromosomes and extraction of the first polar body. B) MIII; metaphase with the release of two polar bodies. PB, polar body; C, chromosome. Original magnification x400

Extract Preparation for In Vitro Kinase Assay

Oocytes were lysed according to the technique used by Shimada et al. [26]. In brief, oocytes were washed several times in PBS and transferred 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% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 mg/ml Leupaptin, and 1 mM PMSF (Sigma). All chemicals except PMSF were purchased from New England Biolabs (Beverly, MA). After suspension of the oocytes, the samples were frozen in liquid nitrogen and 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. The oocyte extracts were frozen and stored at -80°C until just before use.

Western Blot Analysis of Cyclin B1

Western blot analysis was based on the procedures reported by Shimada and Terada [27]. Twenty oocytes were put into plastic tubes containing 5 µl Laemmli sample buffer. After denaturing by boiling at about 98°C for 5 min, 4 µl of protein sample was separated by SDS-PAGE on 12.5% polyacrylamide gel (Amersham Biosciences, Uppsala, Sweden), then transferred onto PVDF membrane (Amersham) using the PhastTransfer system (Amersham). The membrane was blocked using blocking buffer (3% [w/v] nonfat dry milk [Amersham] in T-PBS), then incubated with mouse monoclonal anticyclin B1 antibody (Upstate, Charlottesville, VA) at 1:250 dilution overnight at 4°C in blocking buffer. After three washes in T-PBS, the membranes were treated with horseradish-peroxidase-labeled anti-mouse immunoglobulin G (IgG; 1:1000, Amersham) in 10% (v/v) SuperBlock blocking buffer (Pierce, Rockford, IL) in T-PBS for 1.5 h at room temperature. After five washes of 5 min each with T-PBS, peroxidase activity was visualized using the ECL Plus Western blotting detection system (Amersham) according to the manufacturer's instructions. The intensity of the bands was analyzed using a Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD).

In Vitro p34cdc2 Kinase Assay

The p34cdc2 kinase assay was performed according to the method described by Ito et al. [23]. Five microliters of oocyte extract (containing 10 oocytes) was mixed with 45 µl kinase assay buffer A composed of 25 mM Hepes buffer (pH 7.5; MBL, Nagoya, Japan), 10 mM MgCl2 (MBL), 10% (v/v) mouse vimentin peptide solution (SLYSSPGGAYC; MBL), and 0.1 mM ATP (Sigma). The mixture was incubated for 30 min at 30°C. The reaction was terminated by the addition of 200 µl PBS containing 50 mM EGTA (MBL). The phosphorylation of mouse vimentin peptides was detected using an ELISA MESACUP cdc2 kinase assay kit (MBL, code no. 5234). Data were expressed in terms of the strength of p34cdc2 kinase activity in oocytes matured for 48 h.

In Vitro MAP Kinase Assay

A p44/42 MAP kinase assay kit (Cell Signaling Technology, Beverly, MA) was used for measuring MAP kinase activity. The methods used for the MAP kinase assay were based on those reported by Shimada and Terada [27]. Five microliters of oocyte extract (containing 20 oocytes) was mixed with 25 µl kinase assay buffer B, 25 mM Tris (pH 7.5), 5 mM b-glycerophosphate, 2 mM dithiothreitol, 0.1 mM MgCl2 with 0.1 mM ATP (Sigma), and 2 µg Elk 1 fusion protein (Cell Signaling Technology), and the mixture was incubated for 30 min at 30°C. Chemicals except for ATP were purchased from New England Biolabs. The reaction was terminated by the addition of 10 µl 4x Laemmli sample buffer; the samples were boiled at 100°C for 5 min and then subjected to 12.5% SDS-PAGE. The phosphorylation of Elk 1 fusion protein was detected by immunoblot analysis and chemiluminescence detection using antiphospho-specific Elk 1 antibody. The data were expressed in terms (means ± SEM) of the fold strength of MAP kinase activity in oocytes matured for 48 h.

In Vitro MEK Assay

The methods for MEK assay were based on instructions provided by the manufacturer (Cell Signaling Technology). Briefly, 5 µl oocyte extract (containing 30 oocytes) was mixed with 25 µl kinase assay buffer B, with 0.1 mM ATP and 2 µg inactive p42 MAP kinase (Cell Signaling Technology) as a substrate. The mixture was incubated for 40 min at 30°C. The reaction was terminated by the addition of 10 µl 4x Laemmli sample buffer, and after boiling for 5 min, the samples were subjected to 12.5% SDS-PAGE. Phosphorylation of inactive p42 MAP kinase was detected by antiphospho p42 MAP kinase antibody by immunoblotting and ECL detection. Data were expressed as fold strength of the control, which represented activity of oocytes matured for 48 h.

Experimental Design

The effects of different concentrations of calcium ionophore A23187 on MEK, MAP kinase, and p34cdc2 kinase activity in mature porcine oocytes were examined. Cumulus-free matured oocytes were treated with 10 or 50 µM calcium ionophore. After ionophore treatment, the oocytes were washed several times and were cultured for up to 12 h at 39°C in a humidified atmosphere of 5% CO2 in air. After 4-, 8-, and 12-h culture, MAP kinase activity, p34cdc2 kinase activity, and cyclin B1 level and nuclear status were evaluated in oocytes. MEK assay was evaluated in oocytes cultured for 2, 4, and 8 h after ionophore treatment. Calcium ionophore A23187 (Sigma) was dissolved in dimethyl sulfoxide (DMSO, Sigma) at a concentration of 2 mM, and this A23187/DMSO solution was stored at -20°C until just before use.

Second, we investigated the time-dependent changes of MAP kinase, p34cdc2 kinase activity, and cyclin B1 levels in porcine oocytes cultured in the presence of 10 µM U0126 after 10-µM calcium ionophore treatment. Specifically, we examined the effect of the MEK inhibitor, U0126 (1,4-Diamino-2,3-diacyano-1,4-bis [2-aminophenylthio])butadiene, Sigma U-120) on MEK activity in oocytes cultured for 2 h after treatment with or without 10 µM Ca2+ ionophore. U0126 was dissolved in DMSO (Sigma) at 1 x 10-2 M stock and kept at -80°C until just before use. The final concentration was obtained by dilution with basic medium. For inactivation of MAP kinase in porcine oocytes, 10 µM is the most effective concentration of U0126 (refer to Shimada et al. [28]); this concentration does not inhibit other kinases, such as PKC, Cdk2, and JNK [14]. After 48-h culture, oocytes treated with 10 µM of both U0126 and Ca2+ ionophore were assayed for MAP kinase; p34cdc2 kinase was assayed at 4, 8, or 12 h. At 12 h, nuclear status was also evaluated.

It was investigated whether inactivation of MAP kinase alone could induce a decrease of p34cdc2 kinase activity and pronuclear formation in mature porcine oocytes. After 48-h culture and denudation, oocytes were washed in basic medium several times and cultured in basic medium supplemented with 10 µM U0126 for up to 24 h. For 8-, 16-, and 24-h culture, these oocytes were collected and used for assay of MAP kinase and p34cdc2 kinase activity. The rest of the oocytes cultured for 12 h were evaluated for nuclear status.

Statistical Analysis

Statistical analysis of the data from three or four replicates was carried out for the sake of comparison by analysis of variance (ANOVA) and Fisher protected least significant difference test using the STATVIEW (Abacus Concepts, Inc., Berkeley, CA) program. All percentage data were subjected to arcsine transformation before statistical analysis. Differences of P < 0.05 were considered significant. Values are means ± SEM of three replicates.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dose Effect of Ca2+ Ionophore on MEK/MAPK, MPF Activity, and Pronuclear Formation in Porcine Matured Oocyte

We investigated the dose effects of Ca2+ ionophore on mature porcine oocytes. MEK activities are shown in Figure 2. After 2-h culture following the treatment with 50 µM Ca2+ ionophore, the activity was rapidly reduced, and the significantly lower activity was maintained until 8 h of culture. At 2-h culture, the level of MEK activity in oocytes treated with 10 µM was comparable with that of control oocytes; however, the level in the 50-µM treatment group had significantly decreased (P < 0.05). At 4- and 8-h culture, the activity in the 50-µM treatment group was also significantly lower than that of the control and 10-µM treatment groups.



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  		FIG. 2. Time-dependent changes in MEK activity in porcine oocytes after 10- or 50-µM Ca2+ ionophore treatment. a-b,c-e,f-hDifferent superscripts show significant difference among treatments at the same culture period (P < 0.05). MEK activity data were expressed in terms of the fold strength of the activity in oocytes matured for 48 h

Time-dependent changes of MAP kinase, p34cdc2 kinase activity, and cyclin B1 levels in porcine oocytes treated with 10 or 50-µM Ca2+ ionophore are shown in Figures 3, 4, and 5, respectively. At 8-h culture, there was a significant difference in MAP kinase activity between the 10-µM and 50-µM treatment groups; at 12 h, the activity in the 10-µM treatment group showed a high level similar to the control. However, this activity in the 50-µM Ca2+ ionophore group was significantly lower than that in the 10-µM treatment group or control between 4-h and 12-h culture (P < 0.05). The activity of p34cdc2 kinase in oocytes treated with either 10 or 50-µM calcium ionophore decreased quickly until 8 h, and these activities at 8 h were significantly lower than that of the control. The low p34cdc2 kinase activity was sustained in oocytes treated with 50-µM calcium ionophore for up to 12-h culture, but had been rapidly reactivated by this time in oocytes treated with 10-µM calcium ionophore. The level of cyclin B1 was significantly reduced at 8 h in both 10- and 50-µM Ca2+ ionophore treatments (P < 0.05). The value in the 50-µM treatment group was kept at a low level until 12 h. However, the cyclin B1 level in the 10-µM treatment group had increased by 12 h, and was significantly higher than that in the 50-µM group.



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  		FIG. 3. Time-dependent changes in MAP kinase activity in porcine oocytes after 10- or 50-µM Ca2+ ionophore treatment. a-b,c-e,f-gDifferent superscripts show significant difference among treatments at the same culture period (P < 0.05). MAP kinase data were expressed in terms of the fold strength of the kinase activity in oocytes matured for 48 h



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  		FIG. 4. Time-dependent changes in p34cdc2 kinase activity in porcine oocytes after 10- or 50-µM Ca2+ ionophore treatment. a-b,c-d,e-fDifferent superscripts show significant difference among treatments at the same culture period (P < 0.05). The p34cdc2 kinase activity data were expressed in terms of the fold strength of the activity in oocytes matured for 48 h



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  		FIG. 5. Time-dependent changes in cyclin B1 level in porcine oocytes after 10- or 50-µM Ca2+ ionophore treatment. a-b,c-d,e-gDifferent superscripts show significant difference among treatments at the same culture period (P < 0.05). Cyclin B1 level data were expressed in terms of the fold strength of the level in oocytes matured for 48 h

As shown in Table 1, the majority of activated oocytes (72% ± 4.6%) in the 10-µM treatment group had progressed to the MIII stage after 12-h culture; this proportion was significantly higher than that of oocytes treated with 50-µM Ca2+ ionophore (11% ± 4.1%). On the other hand, the proportion of oocytes forming pronuclei was significantly higher in the 50-µM Ca2+ ionophore treatment group (78% ± 2.6%) than in the 10-µM Ca2+ ionophore treatment group (20% ± 9.4%) or control (0%).


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  		TABLE 1. Nuclear stage of porcine oocytes treated with different concentrations of Ca2+ ionophore after in vitro maturation for 48 h and subsequent culture for 12 h.a

Combination of 10 µM Ca2+ Ionophore with 10 µM U0126 Confirmed, Using Morphological and Biochemical Approaches, that MAP Kinase Activity Is an Essential Part of Cytostatic Factor

The effect of U0126 on MEK activity in pig oocytes with or without Ca2+ ionophore treatment is shown in Figure 6. Oocytes without U0126 after 10-µM Ca2+ ionophore treatment had a high activity of MEK, and this activity was not significantly different from that of control (nontreated) oocytes. On the other hand, the activity in oocytes treated with U0126 was significantly lower than that of control (nontreated) oocytes. Combinational treatment with U0126 and 10 µM Ca2+ ionophore decreased MEK activity to a level significantly lower than that of other treatments.



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  		FIG. 6. Effect of U0126 on MEK activity in oocytes cultured for 2 h after 10-µM Ca2+ ionophore treatment. a-cDifferent superscripts show significant difference. Control was nontreated oocytes after maturation

It was investigated whether addition of U0126 after 10-µM Ca2+ ionophore treatment inhibited reactivation of p34cdc2 kinase. MAP kinase activity in oocytes cultured with 10 µM U0126 after 10-µM Ca2+ ionophore treatment (combinational treatment) was dramatically decreased at 4 h and remained at a significantly lower level than that in the control for up to 12 h (Fig. 7A). Activity of p34cdc2 kinase in the combinational treatment group dramatically decreased at 4 h and was maintained at a low level until 12 h (Fig. 7B). The activity of the combinational treatment group at 4, 8, and 12 h was significantly lower than that of the control. Cyclin B1 levels in the combinational treatment group linearly decreased up to 12 h (Fig. 7C). At all culture periods, these values were significantly lower than those of the control. The nuclear stage of the combinational treatment group is shown in Table 1. Most oocytes reaching MII were activated (94%), and all of the activated oocytes had progressed to the pronuclear stage at 12 h.



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  		FIG. 7. Time-dependent changes in MAP kinase activity (A), p34cdc2 kinase activity (B), and cyclin B1 level (C) in porcine oocytes cultured in the medium supplemented with U0126 after 10-µM Ca2+ ionophore treatment. *Significant difference from control. MAP kinase, p34cdc2 kinase activity, and cyclin Bl level data were expressed in terms of the fold strength of each kinase activity and level in oocytes for 48 h

U0126 Treatment Alone Induces Inactivation of MAPK, but not Inactivation of p34cdc2 Kinase or Pronuclear Formation

We investigated whether only inhibition of MAP kinase by the treatment with U0126 induced pronuclear formation. Time-dependent changes of MAP kinase and p34cdc2 kinase activity in oocytes treated with U0126 are shown in Figure 8, A and B. MAP kinase activity of oocytes treated with U0126 dramatically decreased at 8 h (P < 0.05), and thereafter gradually until 24 h. However, the activity of the control group remained at a high level until 24 h, and the kinase activity at 8, 16, and 24 h was significantly higher than those of the U0126 treatment group.



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  		FIG. 8. Effect of U0126 on MAP kinase activity (A) and p34cdc2 kinase activity (B) in porcine matured oocytes. *Significant difference from control. MAP kinase and p34cdc2 kinase activity data were expressed in terms of the fold strength of each activity in oocytes for 48 h

On the other hand, p34cdc2 kinase activities of both the U0126 treatment group and the control group were maintained at high levels until 12 h. However, at 16 h, the activity in the U0126 treatment group was significantly lower than in the control. The nuclear stages of oocytes cultured for 12 h in the presence of U0126 are shown in Table 2. There was no significant difference in the percentage of MII-stage oocytes between the U0126 treatment group and the control. However, at 24 h after treatment, some oocytes treated with U0126 evidenced condensed chromatin.


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  		TABLE 2. Nuclear stage of porcine matured oocytes cultured in the medium supplemented with U0126 for 12 h


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In Xenopus oocytes activated by Ca2+ ionophore, a rise in the intracellular calcium level triggered the destruction of cyclin B via ubiquitin proteolysis, which induced inactivation of p34cdc2 kinase and pronuclear formation [29]. In the present study, we also observed that treatment with either 10 or 50 µM calcium ionophore significantly decreased p34cdc2 kinase activity and cyclin B1 levels until 8-h culture. These results suggest that a dose as low as 10 µM of Ca2+ ionophore can induce cyclin B degradation, which in turn results in the decrease of p34cdc2 kinase activity and release from the MII stage. At 12 h of culture after 10-µM Ca2+ ionophore treatment, a high proportion of these oocytes failed to form pronuclei but progressed to metaphase III. This observation is consistent with the report [17] in cattle oocytes that treatments with less than 10 µM Ca2+ ionophore induced progression to MIII. Suppression of p34cdc2 kinase by the cdk inhibitor, butyrolactone I, induced pronuclear formation in pig and bovine oocytes [30]. At the 12-h culture period, reactivation of p34cdc2 kinase and an increase in the cyclin B level were observed in these oocytes, which showed a high activity of MAP kinase. In Xenopus oocytes, it has been reported that the activation of MPF for progression from the A,T-I to the MII stage requires the binding of p34cdc2 kinase with de novo synthesized cyclin B, which is strictly dependent on the c-mos/MAP kinase/p90rsk pathway [31–33]. From these reports and the present results, it is estimated that the failure to decrease MAP kinase activity in oocytes activated by 10 µM Ca2+ ionophore allows reactivation of p34cdc2 kinase through the increase of the cyclin B1 level.

In starfish oocytes, MAP kinase activity is regulated by MEK, and the inactivation of the MEK/MAP kinase pathway has been shown to be essential for completion of meiosis and progression to S-phase after fertilization [34, 35]. Our present results showed that until 12 h, MEK activity in oocytes treated with 10 µM Ca2+ ionophore was maintained at a higher level than in the 50-µM Ca2+ ionophore group. On the other hand, U0126 rapidly induced inactivation of MEK and MAP kinase in oocytes treated with or without the Ca2+ ionophore. Therefore, we also examined whether combinational treatment of 10 µM Ca2+ ionophore and U0126 affected the decrease of MAP kinase activity and reactivation of p34cdc2 kinase. In these oocytes, which have low MAP kinase activity, neither reactivation of p34cdc2 kinase nor elevation of cyclin B1 were observed, and all of these oocytes progressed to the pronuclear stage. In Xenopus oocytes, newly synthesized Mos protein induced activation of MAP kinase via phosphorylation of MEK, which played an important role in meiotic progression and arrest at the MII stage [36]. Therefore, it seemed that the degradation of Mos during oocyte activation accelerated the decrease of MAP kinase activity. In mouse oocytes, inhibition of the Mos-dependent MAP kinase cascade by ablation of Mos mRNA suppresses accumulation of newly translated cyclin B1, which is required for p34cdc2 kinase activity [37]. These findings, together with those of the present study, suggest that a high concentration of Ca2+ ionophore (50 µM) might affect destruction of Mos. This is why p34cdc2 kinase and MAP kinase were inactivated by the high concentration of Ca2+ ionophore treatment. When oocytes were treated with a low concentration (10 µM), additional treatment with U0126 was required to induce inactivation of MEK, which results in the decrease of both p34cdc2 kinase and MAP kinase activity and an improved rate of pronuclear formation. Thus, since MAP kinase might have the potential to upregulate cyclin B synthesis after release from the MII stage, treatment with 10 µM Ca2+ ionophore resumed the arrest at the MII stage but allowed oocytes to progress to the MIII stage. This is the first report that lowered MAP kinase activity, which is required for the maintenance of low MPF activity and induces normal meiotic completion and pronuclear formation.

Recently, Phillips et al. [13] reported that 50-µM U0126 treatment effectively induced the inactivation of both MAP kinase and p34cdc2 kinase, resulting in an induction of the pronuclear formation of mouse oocytes. When oocytes were cultured in the presence of 10 µM U0126 without calcium ionophore treatment, no decline in p34cdc2 kinase was observed, in spite of rapid inactivation of MEK and MAP kinase, resulting in arrest at the MII stage and failure to progress to the pronuclear stage (Fig. 4, A and B; Table 2). This result differs from that reported by Phillips et al. in their mouse study [13]. In our previous study, a higher concentration (20 µM) of U0126 suppressed not only MAP kinase, but also p34cdc2 kinase activity during maturation in porcine oocytes [27]. Moreover, high concentration of the drug suppresses other kinases such as cdk2, PKC, p38, and JNK [14]. Therefore, the disagreement of results between pig and mouse oocytes may have been due to the different concentrations of U0126. Judging from the results in this study, it is suggested that, at least in pig oocytes, the inactivation of MAP kinase does not itself decrease p34cdc2 kinase activity, but is required for the maintenance of p34cdc2 kinase activity at the low levels achieved by activation stimuli.

In summary, we showed that a low-concentration treatment of calcium ionophore transiently decreased p34cdc2 kinase activity, but did not affect MAP kinase activity, and allowed reactivation of p34cdc2 kinase via elevation of the cyclin B1 level, resulting in the progression of oocytes to the MIII stage. On the other hand, in oocytes treated with a high concentration of calcium ionophore or a low concentration of calcium ionophore plus U0126, MAP kinase activity linearly decreased following the decrease of p34cdc2 kinase activity, and most of these oocytes formed pronuclei. These results suggest that decreasing the MAP kinase activity was essential for the maintenance of the low p34cdc2 kinase activity induced by the degradation of cyclin B via the Ca2+-dependent pathway, and that the decreased activity of both MAP kinase and MPF induced normal meiotic completion and pronuclear formation of parthenogenetically activated porcine oocytes.


   ACKNOWLEDGMENTS
 
We thank the staff of the Meat Inspection Office in Hiroshima City for supplying the porcine ovaries.


   FOOTNOTES
 
1 Supported in part by Grant-in-Aid for Scientific Research (No. 14760179 to M.S.) of the Japan Society for the Promotion of Science (JSPS) and a Research Fellowship (No. 08254 to J.I.) of JSPS for Young Scientists. Back

2 Correspondence: Masayuki Shimada, Laboratory of Animal Reproduction, Graduate School of Biosphere Sciences, Hiroshima University, Higashi-Hiroshima, 739-8528, Japan. FAX: 81 824 24 7988; mashimad{at}hiroshima-u.ac.jp Back

Received: 24 June 2003.

First decision: 14 July 2003.

Accepted: 6 November 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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