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BOR - Papers in Press, published online ahead of print July 30, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.018036
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BIOLOGY OF REPRODUCTION 69, 1675–1682 (2003)
DOI: 10.1095/biolreprod.103.018036
© 2003 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Effect of Protein Kinase C Activator on Mitogen-Activated Protein Kinase and p34cdc2 Kinase Activity During Parthenogenetic Activation of Porcine Oocytes by Calcium Ionophore1

Junya Ito, Masayuki Shimada, and Takato Terada2

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
 
The objective of this study was to elucidate the role of a [Ca2+]i rise and protein kinase C (PKC) activation on decreases of p34cdc2 kinase and mitogen-activated protein (MAP) kinase activity during parthenogenetic activation of porcine oocytes. In oocytes treated with 50 µM Ca2+ ionophore, degradations of both p34cdc2 kinase and MAP kinase activity were observed and half of these oocytes formed pronuclei. However, a supplement of PKC inhibitor, calphostin C, after 50 µM Ca2+ ionophore treatment, was sufficient to inhibit the inactivation of MAP kinase and pronuclear formation in the oocytes. These results showed that PKC played an important role in Ca2+-induced oocyte activation. On the other hand, 10 µM Ca2+ ionophore treatment could not affect the MAP kinase activity but induced a transient decrease of p34cdc2 kinase activity, which resulted in recovery of p34cdc2 kinase activity and progression to meiotic metaphase III stage. To investigate the effects of PKC activator on oocytes treated with 10 µM Ca2+ ionophore, matured oocytes were cultured with phorbol 12-myriatate 13-acetate (PMA), after 10 µM Ca2+ ionophore treatment. The additional treatment suppressed the recovery of p34cdc2 kinase activity and rapidly induced a decrease of MAP kinase activity, and these low activities were maintained until 12-h cultivation. As a result, a significantly higher percentage of these oocytes (67%) had pronuclei at 12-h cultivation. Moreover, PMA treatment without Ca2+ ionophore treatment effectively led to a decrease of MAP kinase activity in a dose-dependent manner but not p34cdc2 kinase activity in matured porcine oocytes. In conclusion, the parthenogenetic activation of porcine oocytes was mediated by the inactivation of p34cdc2 kinase via a calcium-dependent pathway and thereafter by the inactivation of MAP kinase via a PKC-dependent pathway.

calcium, gamete biology, kinases, meiosis, ovum


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Matured mammalian oocytes remained arrested at meiotic metaphase II (MII) until fertilization or parthenogenetic activation. The meiotic arrest at the MII stage was maintained by a high activity of maturation-promoting factor (MPF), a heterodimer of p34cdc2 kinase and cyclin B in Xenopus [1–4], mouse [5], and porcine [6–8] oocytes. It was well known that mitogen-activated protein kinase (MAP) was also required for arrest at MII stage [9]. Stimulation by spermatozoa will induce sequential intracellular calcium elevation in oocytes, which induces decreases of both MPF and MAP kinase activity in matured Xenopus oocytes [10, 11]. The inactivation of both MPF and MAP kinase at fertilization is respectively considered to be associated with the extrusion of the second polar body and pronuclear formation [12–14].

It has been well established that an intracellular calcium level artificially elevated by the Ca2+ ionophore A23187 [15], ethanol [13], or electric pulse [16], promotes parthenogenetic activation in mouse oocytes. The [Ca2+]i elevation by Ca2+ ionophore treatment was observed also in porcine and bovine oocytes; however, this drug alone could not effectively produce parthenogenetic activation in those species [17–19]. It was reported that treatment with either the protein synthesis inhibitor cycloheximide or the protein phosphorylation inhibitor, 6-dimethylaminopurine (6-DMAP), induced inactivation of MPF and MAP kinase and then led to meiotic progression of matured bovine oocytes [20–22]. Liu and Yang [21] found that a combined treatment with Ca2+ ionophore and 6-DMAP also leads to the effective stimulation of pronuclear formation; however, this treatment could not provoke the extrusion of the second polar body in bovine oocytes. It was also shown that the treatment of matured oocytes with cycloheximide caused a strong inhibition and/or delay of DNA synthesis in cattle [23]. These results suggest that the nonspecific, broad-spectrum protein synthesis and/or phosphorylation inhibitors have detrimental effects on subsequent zygotic development, although the treatment with these drugs increased the rate of pronuclear formation.

Protein kinase C (PKC) activity was well recognized to increase dramatically as a result of a [Ca2+]i elevation after sperm penetration or artificial activation in Xenopus [24, 25] and mouse [26] oocytes. Recently in swine, Fan et al. [27] demonstrated that PKC was activated after sperm penetration, which induced cortical granule exocytosis. It was also shown in mice that the treatment with an artificial PKC activator, phorbol 12-myriatate 13-acetate (PMA), induced pronuclear formation at a high level of efficiency [28]. These experimental observations suggest that PKC plays important roles in egg activation in pronuclear formation in oocytes. Moreover, Lu et al. [29] reported that the treatment with PMA caused MAP kinase dephosphorylation in matured mouse oocytes. However, it is still unclear how [Ca2+]i and PKC activate porcine oocytes. Therefore, the investigations of the role of [Ca2+]i and PKC for the loss of MPF and MAP kinase activity are required for a successful parthenogenetic activation of porcine and bovine oocytes. The objective of this study was to elucidate the role of a [Ca2+]i rise and PKC activation in the decrease of p34cdc2 kinase and MAP kinase activity during parthenogenetic activation of porcine oocytes. When matured oocytes were treated with 10 µM Ca2+ ionophore, the treatment could not affect the MAP kinase activity and induced a transient decrease of p34cdc2 kinase activity in these oocytes, which resulted in recovery of the activity and progression to meiotic metaphase III (MIII) stage. The additional treatment with PMA induced the decrease both of p34cdc2 kinase and MAP kinase. On the other hand, PMA treatment without Ca2+ ionophore treatment effectively led to a decrease of MAP kinase activity in a dose-dependent manner but not a decrease of p34cdc2 kinase activity. In conclusion, the activation of porcine oocytes was mediated by the inactivation of p34cdc2 kinase via a Ca2+-dependent pathway, and thereafter by the inactivation of MAP kinase via a PKC-dependent pathway.


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

Porcine ovaries were collected from 5- to 7-mo-old prepubertal gilts at a local slaughterhouse and transported to the laboratory in 0.85% (w/v) NaCl containing 0.1 mg/ml kanamycin (Meiji Seika, Tokyo, Japan) at 30°C within 1.5 h. The surfaces 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. The oocytes that were collected were placed in prewarmed phosphate-buffered saline (pH 7.4) supplemented with 0.1% (w/v) polyvinyl-pyrrolidone (Sigma Chemical Co., St. Louis, MO). 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. Porcine cumulus oocyte complexes (COCs) were cultured for 48 h in 100-µl drops of basic medium supplemented with 0.6 µg/ml porcine FSH (pFSH) (Sigma), 1.3 µg/ml equine LH (eLH) (Sigma) (about 20 oocytes/drop), and covered with mineral oil (Sigma) at 39°C in a humidified atmosphere of 5% CO2 in air. The basic medium was modified NCSU37 [30] supplemented with 10% (v/v) fetal calf serum (Gibco BRL, Grand Island, NY), 7 mM Taurine (Sigma), 10% (v/v) essential amino acid (Gibco), and 5% (v/v) nonessential amino acid (Gibco).

Calcium Ionophore Treatment

Calcium ionophore treatment was according to the method described by Funahashi et al. [31] with some modification. After 48-h cultivation, COCs were denuded mechanically by pipetting with flame-draw pipette tips which 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 (Sigma) in the basic medium for 5 min at 39°C and washed with the basic medium to quench the action of the ionophore. Oocytes were exposed to Ca2+ ionophore three times at 5-min intervals as described above. Then the oocytes were washed at least three times, and each group of 15 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 Status

After the incubation, the oocytes were mounted on slides, fixed with acetic acid/ethanol (1:3) for 48 h, and stained with acetolacmoid before being examined under a phase-contrast microscope (400x) to evaluate their chromatin configuration. Nuclear status of oocytes as classified according to the report of Liu et al. [18] into categories: A,T-II, characterized by separating chromosomes attached to a slightly elongated spindle or the extrusion of the second polar body; MIII, characterized by metaphase spindle with the release of a second polar body; PN, characterized with one or two pronuclei.

Extract Preparation for In Vitro Kinase Assay

Oocytes were lysed according to the technique used by Shimada and Terada [32]. 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 oocytes extracts were frozen and stored at -80°C until just before use.

In Vitro p34cdc2 Kinase Assay

The p34cdc2 kinase assay was performed according to the method described by Ito et al. [33]. 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 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 (New England Biolab) was used for measuring MAP kinase activity. The methods used for the MAP kinase assay were based on those reported by Shimada et al. [34]. 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, and the mixture was incubated for 30 min at 30°C. Chemicals except for ATP were purchased from New England Biolab. The reaction was terminated by the addition of 10 µl 4x Laemmli sample buffer; and 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 of the fold strength of MAP kinase activity in oocytes matured for 48 h.

Experimental Design

In experiment 1, we determined the effective concentration of calphostin C for PKC inhibition in oocytes activated by Ca2+ ionophore. COCs cultivated for 48 h were denuded and washed several times in the basic medium. The denuded oocytes were treated with 50 µM Ca2+ ionophore, a concentration considered to be effective for the activation of porcine oocytes [19], and were further cultured for 12 h in the medium supplemented with 1, 10, and 100 µM of calphostin C (Sigma). These oocytes were collected and subjected to MAP kinase assay. Thereafter, to explore the relationship between both kinase activities and pronuclear formation, p34cdc2 kinase and MAP kinase activity at 4, 8, and 12 h and nuclear status at 12 h were evaluated in oocytes that were treated with 50 µM Ca2+ ionophore with or without 10 µM calphostin C (see Fig. 6).



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  		FIG. 6. Schematic representation of experimental design and mechanism of oocyte activation. Treatment with 50 µM Ca2+ ionophore induces the degradations of both p34cdc2 kinase and MAP kinase; the inactivation of MAP kinase was inhibited by a supplement of PKC inhibitor, calphostin C (Experiment 1). PMA treatment without Ca2+ ionophore treatment effectively led to a decrease of MAP kinase activity but not p34cdc2 kinase activity (Experiment 2). Treatment with 10 µM Ca2+ ionophore could not affect the MAP kinase activity but induced a transient decrease of p34cdc2 kinase activity. The additional treatment with PMA suppressed the recovery of p34cdc2 kinase activity and rapidly induced a decrease of MAP kinase activity (Experiment 3). These results suggest that the activation of porcine oocytes was mediated by the inactivation of p34cdc2 kinase via a calcium-dependent pathway and thereafter by the inactivation of MAP kinase via a PKC-dependent pathway

The aim of experiment 2 was to investigate the PMA concentration that effectively induced the inactivation of MAP kinase and pronuclear formation in porcine oocytes. Matured oocytes were denuded and further cultured for 12 h in the medium supplemented with 1, 10, or 100 µM PMA (Sigma). After cultivation of oocytes, nuclear status and MAP kinase activity were analyzed. Second, we determined whether calphostin C led to the abrogation of PKC activity stimulated by an effective concentration of PMA. Matured oocytes were cultured for 12 h in the medium supplemented with 10 µM PMA and different concentrations of calphostin C. These oocytes cultured for 4, 8, and 12 h were collected and used for assay of p34cdc2 kinase and MAP kinase activity, and the rest of oocytes cultured for 12 h were assessed for evaluation of nuclear status (see Fig. 6).

In experiment 3, we tested the effectiveness of treatment with Ca2+ ionophore combined with PMA on inducing a decrease in the activities of both p34cdc2 kinase and MAP kinase and the consequent pronuclear formation in matured porcine oocytes. The matured oocytes were denuded and treated with 10 µM Ca2+ ionophore. After the treatment, oocytes were further cultured for 12 h in medium supplemented with or without 10 µM PMA. These oocytes were subjected to assessment of p34cdc2 kinase, MAP kinase, and nuclear status (see Fig. 6).

Statistical Analysis

Statistical analysis of the data from three or four replicates was carried out for the sake of comparison by 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1. Effective Concentration of Calphostin C to Inhibit Pronuclear Formation and Its Effect on MPF and MAP Kinase Inactivation in Oocytes Treated with Calcium Ionophore

The percentage of oocytes that had pronuclei after 10 or 50 µM Ca2+ ionophore treatment is shown in Table 1. Control oocytes did not display pronuclei, and the treatment with 10 µM Ca2+ ionophore induced a low rate of pronuclear formation (12%). On the other hand, the treatment with 50 µM Ca2+ ionophore significantly induced a higher proportion (50%) of pronuclear formation than that of 10 µM Ca2+ ionophore treatment and control. The treatment with 50 µM Ca2+ ionophore significantly decreased MAP kinase activity in oocytes at 12 h cultivation. The decrease of MAP kinase in the oocytes treated with 50 µM Ca2+ ionophore was overcome by additional calphostin C in a dose-dependent manner at concentrations up to 10 µM (Fig. 1). The maximum activity of MAP kinase at 10 µM Calphostin C was comparable with that in oocytes without Ca2+ ionophore treatment. Accordingly, the final concentration of calphostin C was adjusted to 10 µM in each medium in following experiments.


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  		TABLE 1. Effect of 10 µM calphostin C on nuclear status in oocytes cultured for 12 h after 50 µM Ca2+ ionophore treatment



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  		FIG. 1. MAP kinase activity in oocytes treated with different concentrations of calphostin C for 12 h after 50 µM Ca2+ ionophore treatment. a–cDifferent superscripts show significant differences (P < 0.05). 1Data were expressed in terms of the fold strength of MAP kinase activity in oocytes matured for 48 h. *After 48 h maturation, COCs were denuded and further cultured in the basic medium for 12 h

In the oocytes treated with 50 µM Ca2+ ionophore alone, MAP kinase activity was significantly decreased in a time-dependent manner and became markedly low level at 12 h (Fig. 2A, P < 0.05). In oocytes treated with both 50 µM Ca2+ ionophore and 10 µM calphostin C, the activity was significantly higher, compared with that of oocytes treated with only Ca2+ ionophore treatment at every culture period (P < 0.05). Oocytes treated with 10 µM calphostin C without Ca2+ ionophore treatment maintained significantly higher activity of MAP kinase at 4 and 8 h of cultivation, but the activity at 12 h of cultivation significantly decreased to the level at the onset of culture (P < 0.05).



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  		FIG. 2. MAP kinase activity in porcine oocytes treated with different concentrations of PMA for 12 h after maturation. a–cDifferent superscripts show significant differences (P < 0.05). 1Data were expressed in terms of the fold strength of MAP kinase activity in oocytes matured for 48 h. *After 48 h maturation, COCs were denuded and further cultured in the basic medium for 12 h

The p34cdc2 kinase activity in control oocytes remained at a high level until 12 h of cultivation, whereas the activity in oocytes treated with 50 µM Ca2+ ionophore was decreased in an approximately linear response over 8 h of cultivation. When oocytes treated with Ca2+ ionophore were cultured in the medium with 10 µM calphostin C, p34cdc2 kinase activity had declined rapidly at 4 h; however, the activity at 8 and 12 h culture period was significantly higher than that in oocytes treated with Ca2+ ionophore and then cultured without calphostin C (P < 0.05). Oocytes cultured in the medium with 10 µM calphostin C alone for 4-h cultivation had a level of p34cdc2 kinase activity similar to that in oocytes at the beginning of culture.

All control oocytes did not display pronuclear formation, and the slight percentage of oocytes treated with calphostin C alone formed pronuclei (16%). Half of the oocytes treated with 50 µM Ca2+ionophore alone formed a pronucleus. However, when oocytes were treated with calphostin C after Ca2+ ionophore treatment, the percentage of pronuclear formation (29%) was significantly decreased (Table 1).

Experiment 2. MAP Kinase Activity and Pronuclear Formation in Matured Porcine Oocytes Treated with PMA and/or Calphostin C

The addition of PMA to the culture medium led to a decrease of MAP kinase activity in a dose-dependent manner, whereas there was no significant difference of MAP kinase activity between oocytes treated with 10 and 100 µM PMA (Fig. 3). PMA-treated oocytes cultured in the medium without calphostin C showed a significantly low level of MAP kinase activity, compared with control oocytes (Fig. 4). The addition of 1 µM calphostin C into the medium with 10 µM PMA also displayed a low level of MAP kinase activity similar to that of oocytes treated with only PMA, but the addition of 10 µM calphostin C to the PMA-containing medium overcame the low level of MAP kinase activity induced by PMA, and the activity was at a similar level, compared with that of control. In the case of the addition of 100 µM calphostin C, MAP kinase activity was lower than that in the oocytes cultured in the medium with 10 µM PMA and 10 µM calphostin C. Furthermore, after 12 h of cultivation in the medium with PMA alone, the proportion of oocytes possessing pronuclei was only 16%, despite a low level of MAP kinase activity (Table 2).



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  		FIG. 3. Time-dependent changes in MAP kinase activity (A) and p34cdc2 kinase (B) in oocytes cultured with calphostin C after 50 µM Ca2+ ionophore treatment. A–IDifferent superscripts show significant differences between treatments at the same culture period (P < 0.05). 1Data were expressed in terms of the fold strength of kinase activity in oocytes matured for 48 h



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  		FIG. 4. MAP kinase activity in oocytes that were treated with 10 µM PMA and then cultured with different concentrations of calphostin C for 12 h. a–dDifferent superscripts show significant differences (P < 0.05). 1Data were expressed in terms of the fold strength of MAP kinase activity in oocytes matured for 48 h. *After 48 h maturation, COCs were denuded and further cultured in the basic medium for 12 h


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  		TABLE 2. Nuclear status of oocytes cultured for 12 h in the medium supplemented with 10 µM PMA

Experiment 3. The Effectiveness of Combined Treatment with Ca2+ Ionophore and PMA on Pronuclear Formation in Matured Porcine Oocytes

MAP kinase in control oocytes and oocytes treated with 10 µM Ca2+ ionophore alone gradually declined during 12 h of cultivation (Fig. 5A). However, when oocytes treated with 10 µM Ca2+ ionophore were cultured in the medium with 10 µM PMA, the activities of MAP kinase were rapidly decreased. In oocytes treated with 10 µM PMA alone, the activities declined and showed a similar level to that in the oocytes cultured in 10 µM PMA after 10 µM Ca2+ ionophore treatment.



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  		FIG. 5. Time-dependent changes in MAP kinase activity (A) and p34cdc2 kinase (B) in oocytes cultured with PMA for 12 h after 10 µM Ca2+ ionophore treatment. A–IDifferent superscripts show significant differences between treatments at the same culture period (P < 0.05). 1Data were expressed in terms of the fold strength of kinase activity in oocytes matured for 48 h

The p34cdc2 kinase activity in PMA-treated oocytes was kept at a level similar to that of the control until 12 h of cultivation (Fig. 5B). However, those activities in oocytes cultured in PMA-containing medium after 10 µM Ca2+ ionophore treatment were dramatically decreased at 4 h and showed lower levels at 8 and 12 h. On the other hand, p34cdc2 kinase activity in oocytes treated only with 10 µM Ca2+ ionophore was dramatically decreased until 8 h of cultivation, but the activity was recovered at 12 h.

The percentage of pronuclear formation in oocytes treated with 10 µM Ca2+ ionophore alone was 12%, and 43% of the oocytes progressed to the MIII stage (Table 3). A significantly higher percentage of oocytes (67%) treated with PMA after 10 µM Ca2+ ionophore treatment displayed pronuclear formation.


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  		TABLE 3. Effect of 10 µM PMA on nuclear status in oocytes cultured for 12 h after 10 µM Ca2+ ionophore treatment


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In many species, sequential intracellular calcium elevation has been observed in oocytes after penetration by sperm [15, 35, 36]. Moreover, during fertilization in Xenopus [37], mouse [26], and pig [27] oocytes, some PKC isoforms (cPKC, nPKC, and aPKC) were detectable, and the activation of PKC was induced by GTP-binding protein (G protein) via phospholipase C (PLC) [38–40]. Both calcium elevation and PKC activation have been shown to stimulate oocyte activation; however, it is still unclear how [Ca2+]i and PKC activate eggs including those of swine.

The present study revealed that the treatment of matured porcine oocytes with PKC activator, PMA, induces the inactivation of MAP kinase. It was also shown in pig oocytes that injection of the G protein stimulator, guanosine-5'-O-(3'thiophosphate), into matured oocytes caused parthenogenetic activation [41]. Several studies support the theory that activation of G protein-induced productions of diacylglecerol (DG) and inositol phosphate 3 via PLC pathway result in the activation of PKC in vertebrate oocytes [39, 42, 43]. Additionally, PMA has been reported to directly induce the activation of PKC as does DG [44]. From these reports, it seems that PKC activation is related to production of DG via G protein and PLC, which is needed for porcine oocyte activation. In the present study, it was also found that calcium elevation by 50 µM Ca2+ ionophore alone could facilitate a decrease of MAP kinase activity in pig oocytes and that the degradation of this kinase was blocked by the PKC inhibitor, calphostin C. From these results, because it is estimated that the activation of PKC in pig oocytes was dependent on a rise of [Ca2+]i and DG, the PKC involved in the activation of pig oocytes may be cPKC, which has binding sites of [Ca2+]i and DG [44]. Thus, it is hypothesized that cPKC, which is activated by [Ca2+]i and DG, induces a decrease of MAP kinase activity during the activation of porcine oocytes.

Several researchers have found that treatment with a PKC activator induces pronuclear formation in mice [24, 28, 29]; however, in pig, the treatment with the drug cannot trigger the resumption of oocyte meiosis from MII stage and pronuclear formation [45]. In the present study, a majority of oocytes treated with 10 µM PMA, which rapidly reduced MAP kinase activity but not p34cdc2 kinase activity, did not display pronuclear formation. Although in mouse oocytes, inhibition of MAP kinase kinase (MEK) induced inactivation both of p34cdc2 kinase and MAP kinase, resulting in parthenogenetic activation [46], we showed that treatment of matured porcine oocytes with MEK inhibitor, u0126, mediated a low activity of MAP kinase, but none of these oocytes formed pronuclei (unpublished data). Thus, a decrease of MAP kinase activity alone was insufficient for the activation of porcine oocytes. However, the additional treatment with 10 µM PMA after 10 µM Ca2+ ionophore treatment sufficiently induced inactivation of p34cdc2 kinase and pronuclear formation. In oocytes treated with 50 µM Ca2+ ionophore, the inactivation of p34cdc2 kinase was not suppressed by additional treatment of calphostin C. This result was not consistent with the report of Colonna et al. [47], who demonstrated that a disappearance of MPF activity was inhibited by PKC inhibitor in mouse oocytes. These results showed that the inactivation of p34cdc2 kinase in matured porcine oocytes is evoked solely by [Ca2+]i elevation via a PKC-independent pathway unlike mouse oocytes. Thus, to our knowledge, this is the first report that oocyte activation in swine is mediated by the inactivation of p34cdc2 kinase via a Ca2+-dependent pathway and the inactivation of MAP kinase via a PKC-dependent pathway (Fig. 6).

Concerning the inactivation of p34cdc2 kinase in MII oocytes, a rise of calcium ion during fertilization in Xenopus oocytes has been reported to lead to calmodulin-dependent protein kinase II activation, which stimulates the destruction of cyclin B via the ubiquitin pathway, resulting in the inactivation of p34cdc2 kinase [11]. In mouse oocytes, MAP kinase has been found to participate in the synthesis of cyclin B, which is involved in the activation of p34cdc2 kinase during MI/MII transition [13]. We also showed that during meiotic progression from MI to MII stage, MAP kinase activity was involved in the further activation of p34cdc2 kinase [33]. In the present study, when the oocytes were treated with 10 µM Ca2+ ionophore alone, a transient decrease of p34cdc2 kinase activity and high level of MAP kinase activity were observed and these oocytes could not undergo pronuclear formation but progressed to MIII. Moreover, in our previous study (unpublished data), when oocytes were treated with 10 µM Ca2+ ionophore and then cultured with U0126 (MEK specific inhibitor), MAP kinase activity, reactivation of p34cdc2 kinase, and increase of cyclin B1 level at 12 h were significantly suppressed, resulting in the induction of pronuclear formation. From these results, because high activity of MAP kinase may be responsible for the reactivation of p34cdc2 kinase via cyclin B1 synthesis, the treatments that reduce both p34cdc2 kinase and MAP kinase activity are essential for a successful parthenogenetic activation in porcine oocytes.

In this study, about 30% of oocytes that were cultured with 10 µM calphostin C without Ca2+ ionophore treatment had resumed meiosis from MII stage. Coskun and Lin [48] reported that the addition of calphostin C inhibited meiotic progression to MII stage in porcine oocytes. Viveiros et al [49] demonstrated that in mouse oocytes, PKC delta located in meiotic spindle and the suppression of PKC activity promoted entry into anaphase. Moreover, microinjection of PKC zeta into Xenopus oocytes induced the activation of p34cdc2 kinase [50]. These observations suggested that inhibition of PKC activity was associated with a decrease of p34cdc2 kinase activity and with the progression from metaphase to anaphase. In the present study, the addition of calphostin C induced a decrease of p34cdc2 kinase activity, compared with control. PKC delta belongs to nPKC family, which is DG dependent, but [Ca2+]i independent [51]. PKC zeta is one member of a PKC family that is independent of [Ca2+]i [51]. Although the existence of either nPKC or aPKC remains unclear in swine, it is a possibility that calphostin C may inhibit the activity of the PKC family that is [Ca2+]i independent, affecting MPF activity and release of a low proportion of oocytes from MII stage.

In conclusion, we demonstrated for the first time that the parthenogenetic activation of porcine oocytes was mediated by the inactivation of p34cdc2 kinase via a calcium-dependent pathway and thereafter by the inactivation of MAP kinase via a PKC-dependent pathway. We argue that the new method, a combined treatment with Ca2+ ionophore and PMA, is superior for porcine oocyte activation, contributing to either intracytoplasmic sperm injection or nuclear transfer.


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


   FOOTNOTES
 
1 This work was partly supported by Grant-in-Aid for Scientific Research (M.S., No. 14760179) and Research Fellowship for Young Scientists (J.I., No. 08254) from the Japan Society for the Promotion of Science (JSPS) Back

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

Received: 8 April 2003.

First decision: 22 April 2003.

Accepted: 30 June 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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