Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 64, 1106-1114 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Phosphatidylinositol 3-Kinase in Cumulus Cells and Oocytes Is Responsible for Activation of Oocyte Mitogen-Activated Protein Kinase During Meiotic Progression Beyond the Meiosis I Stage in Pigs

Masayuki Shimadaa, and Takato Terada1,a

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

ABSTRACT

The roles of phosphatidylinositol 3-kinase (PI 3-kinase) during meiotic progression beyond the meiosis I (MI) stage in porcine oocytes were investigated. PI 3-kinase exists in cumulus cells and oocytes, and the PI 3-kinase inhibitor, LY294002, suppressed the activation of mitogen-activated protein (MAP) kinase in denuded oocytes during the beginning of the treatment. However, in denuded oocytes cultured with LY294002, the MAP kinase activity steadily increased, and at 48 h of cultivation MAP kinase activity, p34cdc2 kinase activity, and proportion of oocytes that had reached the meiosis II (MII) stage were at a similar level to those of oocytes cultured without LY294002. In contrast, LY294002 almost completely inhibited the activation of MAP kinase, p34cdc2 kinase activity, and meiotic progression to the MII stage in oocytes surrounded with cumulus cells throughout the treatment. Treating cumulus oocyte complexes (COCs) with LY294002 produced a significant decrease in the phosphorylation of connexin-43, a gap junctional protein, in cumulus cells compared with that in COCs cultured without LY294002. These results indicate that PI 3-kinase activity in cumulus cells contributes to the activation of MAP kinase and p34cdc2 kinase, and to meiotic progression beyond the MI stage. Moreover, gap junctional communications between cumulus cells and oocytes may be closed by phosphorylation of connexin-43 through PI 3-kinase activation in cumulus cells, leading to the activation of MAP kinase in porcine oocytes.

cumulus cells, kinases, meiosis, ovum, signal transduction

INTRODUCTION

In most animal species, follicular oocytes are arrested naturally in the first meiotic prophase (prophase I) or the late G2 phase, and resume meiosis usually in response to hormonal stimuli. Following the resumption of meiotic division, the maturation/M phase promoting factor (MPF) is activated [1]. The activated MPF then induces germinal vesicle breakdown (GVBD), chromosome condensation, and spindle formation in meiosis I (MI); MPF is then transiently inactivated, and reactivated again to induce meiosis II (MII) in fish [1], starfish [2], amphibian [3, 4], mouse [5], porcine [6], and bovine oocytes [7, 8].

An MPF complex is known to consist of two subunits: catalytic subunit p34cdc2 kinase, which is the homologue of the yeast cdc2/cdc28 protein kinase; and regulatory subunit cyclin B [9–11]. In Xenopus oocytes arrested at the germinal vesicle stage, pre-MPF, an inactive MPF, is accumulated as a heterodimer composed of 14-The and 15-Tyr phosphorylated p34cdc2 kinase and cyclin B [12, 13]. It has been established that the conversion of the pre-MPF into the active MPF is needed for mitogen-activated protein (MAP) kinase [14–18], which stimulates cdc25 phosphatase activity to dephosphorylate 14-The and 15-Tyr in p34cdc2 kinase [17, 19]. During Xenopus meiotic progression, the one pathway of MAP kinase activation is through de novo synthesized Mos protein, which is stimulated by progesterone [20–22]. The other pathway stimulated by either insulin or insulin-like growth factor I (IGF-I) is that of PI 3-kinase-dependent Ras-Raf-1-MAP kinase kinase (MEK) [23, 24].

Similar to its activation in Xenopus oocytes, MAP kinase activation has been observed during meiotic progression after GVBD in mouse, porcine, and goat oocytes [25–27]. In mouse oocytes, injection of antisense oligonucleotide against c-mos mRNA results in a failure of MAP kinase activation and progress to the MII stage does not occur [28]. Moreover, in the c-mos knockout mouse in which expression of Mos protein is completely blocked, MAP kinase activity in the oocytes does not appear, and normal meiotic progression beyond the MI stage is inhibited [29–31]. Thus, in mouse oocytes as well as in Xenopus oocytes, the Mos protein signaling pathway plays a pivotal role for MAP kinase activation. However, there is little information describing the pathway that stimulates the de novo Mos protein synthesis, or regarding the PI 3-kinase signaling pathway in mammalian oocytes.

In contrast to those in Xenopus oocytes, follicular cells such as cumulus cells and granulosa cells surrounding mammalian oocytes, regulate meiotic initiation and progression to the MII stage [32–34]. In bovine oocytes exhibiting the MI stage, the attachment, even with porcine follicular cells, suppresses MAP kinase activity [35]. Cumulus cells stimulated by FSH, epidermal growth factor (EGF), or IGF-I significantly enhance oocyte maturation; the receptors of these factors exist in these cells [36–38], and the inhibition of PI-3 kinase activity within cumulus cells, which is activated by EGF or IGF-I in a variety of cells, blocks meiotic progression to the MII stage [39, 40]. These findings raise the possibility that PI 3-kinase in cumulus cells controls meiotic progression via MAP kinase in mammalian oocytes. However, the relationship between the MAP kinase activating pathway in oocytes and PI 3-kinase in cumulus cells remains to be clarified.

We tested whether the PI 3-kinase signaling pathway was present in oocytes, cumulus cells, or both, and whether PI 3-kinase is required for the activation of oocyte MAP kinase during meiotic progression in pigs. The location of the PI 3-kinase was determined by immunofluorescence analysis using anti-mouse p85 monoclonal antibody. In order to investigate the role of the PI 3-kinase in oocytes, cumulus cells, or both on the activation of oocyte MAP kinase, MAP kinase activity was determined in oocytes separated from cumulus oocyte complexes (COCs) and in denuded oocytes cultured in the presence of LY294002, a PI 3-kinase inhibitor.

MATERIALS AND METHODS

Isolation and Culture of Porcine COCs

Porcine ovaries were collected from prepubertal gilts at a local slaughterhouse and transported within 1.5 h to the laboratory in 0.85% NaCl containing 0.1 mg/ml kanamycin (Meiji Seika, Tokyo, Japan) at about 30°C. The surfaces of follicles measuring from 3 to 8 mm in diameter were cut with a razor blade and oocytes were collected by using a surgical blade to scrape the inner surface of the follicle walls. The collected oocytes were placed in prewarmed PBS pH 7.4 supplemented with 0.1% (w/v) polyvinyl-pyrrolidone (PVP; Sigma Chemical Company, 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. Oocytes were cultured for various time periods in 100-µl drops of basic medium (about 20 oocytes/drop) covered with mineral oil (Sigma) at 39°C in a humidified atmosphere of 5% CO2 in air. The basic medium was modified NCSU37 [41] containing 10% (v/v) fetal calf serum (FCS; Gibco BRL, Grand Island, NY), 0.6 µg/ml porcine FSH (pFSH; Sigma), 1.3 µg/ml equine LH (eLH; 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).

Treatment of COCs or Denuded Oocytes with LY294002 or Calphostin C

After COCs had been cultured for 24 h in the basic medium, some COCs were mechanically denuded by pipetting with flame-draw pipette tips that had inner diameters slightly larger than the oocyte diameter. COCs and denuded oocytes were cultured further in medium supplemented with 5 x 10-5 M LY294002 (Sigma) or 1 x 10-6 M calphostin C (Sigma), respectively. Previously [40], 0.5 x 10-5 M LY294002 did not significantly affect the proportion of oocytes reaching the MII stage, but the addition of 5.0 and 7.5 x 10-5 M LY294002 significantly suppressed the meiotic progression to the MII stage. Thus, the concentration of 5 x 10-5 M LY294002 was employed in this experiment. LY294002 or calphostin C were dissolved in dimethylsulfoxide (DMSO; Sigma) at 5 x 10-2 M or 1 x 10-3 M, respectively. The final concentrations of LY294002 (5 x 10-5 M) or calphostin C (1 x 10-6 M) were obtained by dilution with the basic medium. Inhibitor-free medium supplemented only with 0.098% (v/v) DMSO to the basic medium served as a control. This concentration of DMSO did not affect porcine oocyte maturation [39].

Assessment of Nuclear Maturation

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

Extract Preparation

After COCs had been cultured for 24 h in the basic medium, COCs and denuded oocytes were cultured further in medium supplemented with 5 x 10-5 M LY294002 to inhibit PI 3-kinase or 1 x 10-6 M calphostin C to inhibit protein kinase C (PKC), respectively. Oocytes or cumulus cells, respectively, 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 except PMSF were purchased from New England Biolabs (Beverly, MA). After respective suspension of oocytes or cumulus cells in the cell lysis buffer, the samples were frozen in liquid nitrogen, then sonicated three times, 25 sec each time, at 1°C. Cell extracts were frozen and stored at -80°C just before use.

Detection of PI 3-Kinase and Connexin-43 by Immunoblotting Analysis

Either oocyte or cumulus cell extract was diluted two-fold with 2x Laemmli sample buffer [42]. 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 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 either mouse monoclonal anti-human p85 antibody (Transduction Laboratories, Lexington, KY) at 1:1000 dilution or mouse monoclonal anti-connexin 43 antibody (Chemicon International, Temecula, CA) at 1:2000 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 immunoglobulin G (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. The intensity of the bands was analyzed using a Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD). Each experiment was repeated three times.

Immunofluorescent of PI 3-Kinase

COCs or denuded oocytes were fixed with 4% (w/v) paraformaldehyde-PBS (pH 7.4) at room temperature for 30 min, rinsed three times with PBS, and then permeabilized with 0.5% (v/v) Triton X-100-PBS for 30 min. COCs or denuded oocytes were blocked by 5% (w/v) BSA (Sigma) in T-PBS and then incubated with anti-human p85 monoclonal antibody (1:30) in 5% (w/v) BSA in T-PBS overnight at 4°C. Samples were rinsed with T-PBS and incubated with a fluorescein-conjugated goat anti-mouse IgG antibody (Sigma) diluted 1:50 in 5% (w/v) BSA in T-PBS for 2 h at 38°C. After rinsing with T-PBS to eliminate excess antibody, the samples were mounted on a slide using a SlowFade Antifade kit (Molecular Probes, Eugene, OR) and observed by fluorescent microscopy (Olympus, IMT-2, Tokyo, Japan).

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. [43]. Briefly, 5 µl oocyte extract (containing 20 oocytes) was mixed with 25 µl 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 drugs except ATP were purchased from New England Biolabs. The reactions were 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 Elk 1 fusion protein was detected by immunoblotting and chemiluminescence using anti-phospho-specific Elk 1 antibody.

In Vitro p34cdc2 Kinase Assay

A p34cdc2 kinase assay was performed using a MESACUP cdc2 kinase assay kit (MBL, Nagoya, Japan), according to the method described by Shoujo et al. [44]. 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 oocyte extract (containing 10 oocytes) was mixed with 45 µl 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 by 200 µl PBS containing 50 mM EGTA (MBL). Phosphorylation of MV peptides was detected using ELISA analysis (MESACUP cdc2 kinase assay kit [MBL]). Values were expressed as the fold strength of p34cdc2 kinase in oocytes immediately after collection from their follicles.

Statistical Analysis

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

RESULTS

Location of PI 3-Kinase in Porcine COCs by Immunofluorescence

Only one band of 85 kDa in porcine cumulus cell and oocyte extracts derived from COCs immediately after collection from their follicles was recognized by the monoclonal anti-human p85 antibody (Fig. 1). The recognized protein's electrophoretic mobility and estimated molecular mass was the same as that observed in HeLa cell extracts (Fig. 1).



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  		FIG. 1. Immunoblotting analysis of the p85 regulatory subunit of PI 3-kinase. #HeLa cell extract, as positive control, was purchased from the Transduction Lab. ##Cumulus cell and oocytes immediately after collection from their follicle

In situ immunofluorescence analysis of PI 3-kinase in COCs immediately after collection and after 24-h cultivation revealed that high intensity of PI 3-kinase is present in all layers of cumulus cells and in oocytes (Fig. 2, b', b'', c', and c''). Control COCs treated with only fluorescein-conjugated goat anti-mouse IgG antibody did not stain (Fig. 2a'). At 48 h of COC cultivation, the positive signal for PI 3-kinase immunoreactivity was limited to oocytes and the inner layers of cumulus cells (Fig. 2, d' and d'').



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  		FIG. 2. The localization of PI 3-kinase in porcine COCs detected by indirect immunofluorescence using anti-p85 monoclonal mouse IgG and fluorescein isothiocyanate-conjugated anti-mouse IgG. a–d) Image obtained by differential interference microscopy. a'–d', b''–d'') Image obtained by indirect immunofluorescence of anti-p85 antibody. a, a') Negative control. b, b') COCs were immediately recovered from the follicles. c, c') COCs were cultured for 24 h. d, d') COCs were cultured for 48 h. b'') After COCs were immediately recovered from the follicles the COCs were denuded mechanically. c'', d'') After COCs were cultured for 24 h (c'') or 48 h (d''), COCs were denuded mechanically. Bar = 100 µm

Time-Dependent Changes of MAP Kinase Activity in Porcine Oocytes During Maturation Culture

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. Time-dependent changes of MAP kinase activity in oocytes surrounded with cumulus cells during in vitro maturation are shown in Figure 3. MAP kinase activity was very low during the first 24 h, but significantly increased at 28 h of cultivation of COCs. At this time point most of the oocytes (75%) had undergone GVBD. Subsequently, the MAP kinase activity further increased and reached a plateau at 36 h of cultivation.



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  		FIG. 3. Time-dependent change of MAP kinase activity in porcine oocytes surrounded by cumulus cells. a) MAP kinase activity in porcine oocytes cultured in basic medium. b) Relative amounts of MAP kinase activity, which were determined by scanning densitometry. {dagger}Activity is expressed as fold MAP kinase activity in which positive control, 5 ng active MAP kinase activity, is defined as 100. Different superscripts (a–e) were significantly different at the P < 0.01 level

Effects of LY294002 on Dramatic Activation of MAP Kinase at the MI Stage

In order to define a role of PI 3-kinase for MAP kinase activation in oocytes, COCs were precultured for 24 h in the basic medium (to allow GVBD and MI maturation) then transferred to medium supplemented with 1 x 10-5 M LY294002. In controls, MAP kinase activity was very low at 24 h of cultivation, but significantly increased at 28 h (Fig. 4a). Significant differences in MAP kinase activity were observed between oocytes cultured with LY294002 and those cultured without this drug at 28 h and up to 48 h of cultivation of COCs (Fig. 4a).



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  		FIG. 4. The effects of LY294002 on MAP kinase activity in porcine oocytes. a) After COCs had been cultured for 24 h in the basic medium, COCs were cultured further in the presence or absence of LY294002. b) After COCs had been cultured for 24 h, COCs were denuded and these denuded oocytes were cultured further in the presence or absence of LY294002. {dagger}Activity is expressed as fold MAP kinase activity in which positive control, 5 ng active MAP kinase activity, is defined as 100. No common superscripts (a–e, f, g, a–c, and d–f) were significant within the same group (P < 0.01). *Significant difference in comparison with treatment with LY294002 at the same time point (P < 0.01)

When COCs were denuded after 24 h of cultivation in the basic medium then cultured further with or without LY294002, MAP kinase activity in denuded oocytes not treated with LY294002 was significantly higher compared with those exposed to LY294002 at the 28-h or 32-h time points (Fig. 4b). MAP kinase activity in denuded oocytes cultured with LY294002 then gradually rose, and at 36 h of cultivation, significant differences in MAP kinase activity between denuded oocytes cultured with and without LY294002 disappeared (Fig. 4b).

Effects of LY294002 on p34cdc2 Kinase Activity and Meiotic Progression in Porcine Oocytes at the MI Stage

A low level of p34cdc2 kinase activity was noted in oocytes from COCs immediately after collection from their follicles, which increased after 24 h of cultivation (Fig. 5). Exposure of COCs to LY294002 from 24 to 48 h of culture significantly suppressed the p34cdc2 kinase activity (Fig. 5). LY294002 had no significant effect on the proportion of oocytes undergoing GVBD, but significantly inhibited the meiotic progression to the MII stage (26%) compared with that of COCs cultured in the drug-free medium for 48 h (74%; Fig. 6a).



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  		FIG. 5. Effects of LY294002 on p34cdc2 kinase activity in porcine oocytes. 1COCs were cultured up to 48 h in the basic medium. 2After COCs had been cultured for 24 h in the basic medium, COCs were cultured further for 24 h in the presence of LY294002 (LY). 3After COCs had been cultured for 24 h in the basic medium, COCs were denuded and these denuded oocytes were cultured further for 24 h in the basic medium. 4After COCs had been cultured for 24 h in the basic medium, COCs were denuded and cultured further for 24 h in the presence of LY. Different superscripts (a–d) were significantly different at the P < 0.01 level. {dagger}Data are expressed as fold strength of p34cdc2 kinase activity in oocytes immediately after collection from their follicles



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  		FIG. 6. The effects of LY294002 on GVBD and progressing to MII stage in porcine oocytes. a) After COCs had been cultured for 24 h in the basic medium, COCs were cultured further in the presence or absence of LY294002 (LY). b) After COCs had been cultured for 24 h, COCs were denuded and cultured further in the presence or absence of LY. *Significant difference in comparison with treatment with LY at the same time point (P < 0.01)

When COCs were denuded mechanically after 24 h of cultivation in the basic medium and cultured further for 24 h with or without drug exposure, p34cdc2 kinase activity in denuded oocytes cultured with LY294002 was comparable to that of oocytes cultured without this drug (Fig. 5). No significant difference between proportions of denuded oocytes reaching the MII stage in the presence and absence of LY294002 were observed at any time points of cultivation (Fig. 6b).

Roles of PI 3-Kinase in Cumulus Cells for the Regulation of Gap Junctional Communication via the Phosphorylation of Connexin-43

In an effort to implicate PI 3-kinase in gap junctional communication between cumulus cells and oocyte during meiotic maturation, the phosphorylation state of connexin-43, a gap junctional protein was measured in cumulus cells from COCs cultured with or without LY294002.

Figure 7 shows the immunoblotting analysis of connexin-43 in cumulus cells isolated from COCs cultured with or without LY294002. In the lysates of cumulus cells from COCs cultured for 24 h, three bands of connexin-43 situated at regions 43, 45, and 47 kDa were detected on SDS-PAGE. At 48 h, the staining intensity of the lower migration form (47 kDa), the phosphorylated form of connexin-43, was increased significantly. Concomitantly, significant reductions in the intensities of the 43-kDa and 45-kDa bands were observed at 48 h of cultivation of COCs. In sharp contrast, treating COCs with LY294002 or calphostin C (a protein kinase C inhibitor) for 24 h (a total of 48 h of cultivation) produced a significant decrease in the intensity of the 47-kDa band (phosphorylated form of connexin-43) in cumulus cells compared with that in COCs cultured without these drugs.



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  		FIG. 7. Effects of LY294002 on the phosphorylation of connexin-43 in cumulus cells from porcine COCs. a) Immunoblot probed with anti-connexin-43 monoclonal antibody. b) The intensity of connexin-43 bands as determined by scanning densitometry, (the data in each band were expressed as the fold strength of the intensity in cumulus cells from COCs cultured for 24 h). Different superscripts (a–c, d, e, f, and g) were significantly different within the same migrated bands at the P < 0.01 level; 1COCs were cultured for 24 h. 2COCs were cultured without LY294002 for 48 h. 3After COCs had been cultured for 24 h, COCs were cultured further with LY294002 for 24 h. 4After COCs had been cultured for 24 h, COCs were cultured further with calphostin C for 24 h.

DISCUSSION

There is evidence that the injection of mutant PI 3-kinase into Xenopus oocytes decreases MAP kinase activity, resulting in a failure of hormone-stimulated oocyte maturation [45, 46]. Recently, Sadler and Ruderman [47] reported that PI 3-kinase was also required for starfish oocyte maturation, and that a PI 3-kinase inhibitor blocked MAP kinase activation. In mammalian oocytes, however, basic information about the role of PI 3-kinase in meiotic progression and MAP kinase activation is as yet unknown.

The present study is the first report to demonstrate that PI 3-kinase is present in both porcine oocytes and cumulus cells during the maturation of COCs using immunofluorescent and immunoblotting analysis (Figs. 1 and 2). From the MI stage, the addition of a PI 3-kinase inhibitor, LY294002, suppressed the dramatic activation of MAP kinase in porcine denuded oocytes (Fig. 4b). These results suggest that PI 3-kinase contributes to MAP kinase activation in porcine oocytes.

In mouse oocytes, it has been reported that Raf-1 is activated at the meiotic metaphase [31, 48], and that the activated Raf-1 leads to activation of MEK, MAP kinase kinase [49]. Lopez-Ilasaca et al. [50] also showed that MAP kinase was activated by PI 3-kinase through the Ras-Raf-1-MEK signaling pathway in COS 7 cells. In Xenopus oocytes, phosphorylation of Raf-1, which is induced by PDGF, is suppressed by expression of mutant PI 3-kinase [51]. These findings, taken together, argue that PI 3-kinase in oocytes may activate MAP kinase through the Ras-Raf-1-MEK signaling pathway. However, in the denuded oocytes cultured with LY294002, MAP kinase activity was steadily increased and reached a level similar to that of oocytes cultured without this drug for 48 h of cultivation (Fig. 4b). Thus, a PI 3-kinase-independent MAP kinase activating pathway may exist.

In c-mos knockout mouse oocytes, the Mos is upstream of MAP kinase [29–31]. A protein synthesis inhibitor, cycloheximide, also causes inhibition of MAP kinase activation in mouse oocytes, probably due to the suppression of Mos protein kinase synthesis during meiotic maturation [31, 52]. In porcine oocytes, it has been demonstrated that MAP kinase activity is inhibited by cycloheximide [53], and that the expression of Mos protein was noted in the oocytes [54]. These reports lead to speculations that MAP kinase cascades in porcine oocytes may be regulated by two signaling pathways; one is dependent on PI 3-kinase (mentioned above), and the other is associated with de novo synthesized Mos.

In the present study, LY294002 completely suppressed the activation of MAP kinase in porcine oocytes surrounded with cumulus cells throughout cultivation after 24 h, although in the denuded oocytes cultured with LY294002 MAP kinase activity was steadily increased (Fig. 4). These results show that PI 3-kinase in cumulus cells is acting on the MAP kinase activating pathway independent of PI 3-kinase, which may be associated with Mos in porcine oocytes.

Matten et al. [55] reported that in Xenopus oocytes the activation of MAP kinase, which was stimulated by Mos, was down-regulated by cyclic AMP (cAMP). In bovines, the elevation of cAMP level in the oocytes suppresses the meiotic progression beyond the MI stage to the MII stage [56]. Cyclic AMP is synthesized in cumulus cells and transported into oocytes via numerous gap junctions in mammalian oocytes [57]. Furthermore, it has been reported that during meiotic progression beyond the MI stage in oocytes with activated MAP kinase, gap junctional communications between cumulus cells and oocytes are disrupted in rat and pig COCs, resulting in meiotic progression to the MII stage [58–60]. In addition, the phosphorylation of connexin-43, one component of the gap junction, has been found to control the channel [61]. In the present study, the staining intensity of the phosphorylated connexin-43 band in cumulus cells was significantly increased during meiotic maturation beyond the MI stage, although with the addition of PI 3-kinase inhibitor to the culture medium, the phosphorylation of connexin-43 was almost completely blocked (Fig. 7). This finding, along with the findings noted above regarding cAMP in oocytes, may strongly suggest that PI 3-kinase in cumulus cells induces to close the channel, inhibits transport of cAMP from cumulus cells into oocytes, and leads to the activation of MAP kinase in porcine oocytes, although closing the gap junctional channel between cumulus cells and oocyte in response to the phosphorylated connexin-43 in cumulus cells was not confirmed in this study.

EGF has been shown to stimulate the phosphorylation of serine on connexin-43 via MAP kinase then to close gap junctional communication in rat liver cells [62, 63]. Recently, Hossain et al. [64] also reported that PI 3-kinase and PKC, which are upstream of MAP kinase, are required for gap junction blockade via phosphorylation of connexin-43 in the same cells. In porcine cumulus cells surrounding oocytes, however, MAP kinase did not correlate with the phosphorylation of connexin-43 during meiotic progression beyond the MI stage [65]. Lau et al. [66] demonstrated that connexin-43 was directly phosphorylated by PKC, and that the family is potently stimulated by PIP3, which is the product of PI 3-kinase in mouse fibroblast cells [67]. Moreover, in the present study, it was found that calphostin C, a PKC inhibitor, decreased the phosphorylated form of connexin-43 in cumulus cells surrounding porcine oocytes during meiotic progression beyond the MI stage (Fig. 7). Thus, we conclude that the phosphorylation of connexin-43 in cumulus cells surrounding oocytes may be induced by the PI 3-kinase pathway, including PKC, during meiotic progression beyond the MI stage.

MAP kinase activated by Mos in Xenopus oocytes is known to stimulate MPF via cdc25 phosphatase, which dephosphorylates 14-The and 15-Tyr in p34cdc2 kinase around GVBD [17, 19], and increased activities of MPF are essential for stimulating meiotic progression beyond the MI stage [68, 69]. In mammalian oocytes, however, the mechanisms of MPF activation at the MI stage remains to be clarified. The present study provided evidence that p34cdc2 kinase activity and MAP kinase activity in porcine oocytes surrounded by cumulus cells were inhibited by LY294002, and that meiotic progression to the MII stage was suppressed (Figs. 4–6). These findings indicate that PI 3-kinase in cumulus cells controls the activity of p34cdc2 kinase in porcine oocytes. However, it is unclear whether PI 3-kinase in cumulus cells independently regulates the activation of the Mos-MAP kinase-MPF pathway, or of the Mos-MAP kinase pathway and the MPF pathway in the oocytes. The relationship between Mos-MAP kinase pathway and MPF in porcine oocytes during meiotic progression beyond the MI stage is now under investigation.

In summary, PI 3-kinase is present in both oocytes and cumulus cells. The activation of MAP kinase in porcine oocytes may be regulated by two signaling pathways. One of these is dependent on the PI 3-kinase signaling pathway in oocytes. In cumulus cells, the phosphorylation of connexin-43 is mediated by the activation of the PI 3-kinase pathway, including PKC, and may block the gap junctional communication between cumulus cells and oocytes. The closing of the gap junctional communication induces the activation of MAP kinase and p34cdc2 kinase, resulting in meiotic progression beyond the MI stage to the MII stage in porcine oocytes.

ACKNOWLEDGMENTS

We are grateful to the staff of the Meat Inspection Office in Hiroshima City for supplying porcine ovaries.

FOOTNOTES

First decision: 2 October 2000.

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

Accepted: November 13, 2000.

Received: August 21, 2000.

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