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BOR - Papers in Press, published online ahead of print April 28, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026310
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BIOLOGY OF REPRODUCTION 71, 691–696 (2004)
DOI: 10.1095/biolreprod.103.026310
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Activation of p38 MAPK During Porcine Oocyte Maturation1

Luis Gerardo Villa-Diaz2,3, and Takashi Miyano4

Graduate School of Science and Technology,3 Kobe University, Kobe, Japan Faculty of Agriculture,4 Kobe University, Nada-ku, Kobe 657-8501, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The p38 MAPK is a member of the mitogen-activated protein kinase (MAPK) family that participates in a signaling cascade in response to cytokines and stress in somatic cells. The present study was designed to investigate the expression and possible function of p38 MAPK in porcine oocytes during maturation. In immunoblots, p38 MAPK was detected in oocytes and cumulus cells. Its activity was determined during oocyte maturation in vitro by the phosphorylation of its substrate, activated transcription factor 2. As ERK1/2, oocyte p38 MAPK became active around germinal vesicle breakdown (GVBD) and maintained activity until metaphase II (MII). Immunofluorescent microscopy showed phosphorylated p38 MAPK accumulated in the nucleus before GVBD and localized in the cytoplasm and around chromosomes from metaphase I (MI) to MII. In cultured cumulus-oocyte complexes, a specific inhibitor of p38 MAPK, SB203580, inhibited phosphorylation of p38 MAPK in cumulus cells and blocked both FSH-induced cumulus expansion and meiotic resumption of oocytes. During spontaneous meiotic resumption of denuded oocytes, SB203580 did not affect GVBD, but it significantly decreased the number of oocytes reaching MII and conversely increased the number of oocytes arrested at MI. These results suggest that p38 MAPK in porcine oocytes becomes active around GVBD, remains active through MI to MII, and has a role in MI-MII transition, and that cumulus p38 MAPK might be involved in FSH-induced meiotic resumption of oocytes.

gamete biology, kinases, meiosis, oocyte development, signal transducers


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fully grown oocytes are arrested at the prophase of the first meiosis until hormonal stimulation makes them resume meiosis and progress to the metaphase of the second meiosis. Then oocytes become arrested again until they finally complete meiosis upon fertilization. This process is controlled by the activation of signal transduction pathways including maturation-promoting factor (MPF) [1]. This kinase consists of a 34-kDa catalytic subunit (Cdc2) and a cyclin B regulatory subunit and is a key kinase that leads to the resumption of meiosis.

In addition to MPF, the extracellular signal-regulated kinase (ERK) has been reported to be involved in oocyte maturation in different species [2–9]. Immature mammalian oocytes express two isoforms of nonphosphorylated ERK, referred to as ERK1 (44 kDa) and ERK2 (42 kDa), and around germinal vesicle breakdown (GVBD), both of them become active by their phosphorylation with a corresponding change in their electrophoretic mobilities [10]. ERK1/ 2 belong to the mitogen-activated protein kinase (MAPK) superfamily, which also integrates p38 MAPK and the c-Jun N-terminal kinases (JNK, also known as the stress-activated protein kinase). All of them are serine-threonine protein kinases that have functions as mediators of cellular responses to a variety of extracellular stimuli. Although the importance of ERK1/2 MAPK in meiosis has been documented, little attention has been paid to other members of the MAPK superfamily in oocyte maturation. Recently, JNK was found in Xenopus oocytes [11] and p38 MAPK in sea star and Xenopus oocytes [12, 13], but no report exists regarding these kinases in mammalian oocytes.

Like the other members of the MAPK family, p38 MAPK is activated by its phosphorylation at conserved threonine and tyrosine residues by upstream dual-specific p38 MAPK kinases (MKK3 and MKK6), which are activated by MAPKK kinases [14, 15]. Among the downstream kinases of p38 MAPK, mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2 [16, 17] and several transcription factors have been identified (reviewed in [18]).

The p38 MAPK has been associated with stress response and some apoptotic processes because it is activated by environmental stress, such as hyperosmolarity, ultraviolet radiation, inflammatory cytokines, and endotoxins [19]. The p38 MAPK is also activated by stimuli, such as growth factors [20], mitogens [21] and FSH [22], and it has been connected with various processes of differentiation, proliferation, and survival of somatic cells (reviewed in [23]). In Drosophila, p38 MAPK is required for asymmetric development of eggs [24]. In rat granulosa cells, p38 MAPK activity is involved in different events induced by FSH [22, 25, 26].

In this study, we investigated the possible role of p38 MAPK during meiotic maturation of porcine oocytes. This kinase became active around GVBD, and its activity remained high through metaphase I (MI) to metaphase II (MII). The p38 MAPK might be involved in meiotic progression of porcine oocytes because its inhibition affected the MI-MII transition.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture of Porcine Oocytes

Porcine ovaries were obtained from prepubertal gilts at a local slaughterhouse. After three washes in Dulbecco phosphate-buffered saline (PBS) containing 0.1% polyvinylalcohol (PVA; Sigma Chemical Co., St. Louis, MO), intact healthy antral follicles 4–6 mm in diameter were dissected in PBS-PVA from ovaries as described previously [27]. After being opened in 25 mM HEPES-buffered medium 199 (HEPES 199, Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) containing 0.08 mg/ml kanamycin sulphate (Sigma Chemical Co.), cumulus-oocyte complexes (COCs) were isolated from the follicles. After washing in HEPES 199, a maximum of 20 COCs were cultured in 500 µL of bicarbonate-buffered medium 199 (m199) supplemented with 10% fetal calf serum (FCS; BioWhittaker, Cambrex Bio Science, Rockland, ME), 0.1 mg/ml sodium pyruvate, 0.08 mg/ml kanamycin sulphate, and 100 ng/ml FSH (Biogenesis, Poole, UK) in an atmosphere of 5% CO2 in humidified air at 38.5°C during different periods of time. For spontaneous meiotic resumption of oocytes, cumulus cells were removed from oocytes by gentle pipetting with a small-bore pipette in HEPES 199, and denuded oocytes (DOs) were cultured as mentioned above without the supplementation of FSH.

After culture of COCs, cumulus cells were removed from the oocytes by treatment with 0.05% hyaluronidase (Type I-S; Sigma Chemical Co.) and repeated pipetting. Some oocytes were washed in PBS-PVA, mounted on slides, fixed with acetic acid-ethanol (1:3, v/v) solution, stained with 1% aceto-orcein, and observed under an interference microscope. Other oocytes were used for kinase assay, Western blot analysis, or immunostaining.

SB203580 Treatment

Inhibition experiments were carried out using SB203580 (Calbiochem; San Diego, CA), a specific inhibitor of p38 MAPK, at two different concentrations (10 and 20 µM) during 42 h of culture period. SB203580 was dissolved in dimethylsulphoxide (DMSO; Sigma Chemical Co.), and stock solution of 10 mM was prepared. DMSO was added at 0.2% (v/v) to culture medium in control groups.

Electrophoresis and Western Blotting

Groups of 50 oocytes or cumulus cells from 20 COCs were rinsed in PBS-PVA, dissolved in 15 µl of SDS sample buffer [28], boiled for 5 min, frozen, and kept at –20°C until use. Samples were run on 13% SDS-polyacrylamide gels, and proteins were transferred to hydrophobic polyvinylidene difluoride membrane (Immobilon; Millipore Co., Bedford, MA). Membranes were blocked with 10% FCS in PBS containing 0.1% Tween20 (PBS-Tween) for 2 h, and then for detection of p38 MAPK, membranes were incubated in 0.5% (w/v) bovine serum albumin (BSA; Serologicals Corporation, Norcross, GA) in PBS-Tween overnight with rabbit polyclonal anti-phospho-p38 MAPK (1:100; #9211; Cell Signaling Technology Inc., Beverly, MA) or rabbit polyclonal anti-human p38 MAPK antibody (1:200, Santa Cruz Biotechnology Inc., Santa Cruz, CA) at room temperature. For detection of ERK1/2 MAPK, membranes were incubated for 4 h in 5% FCS in PBS-Tween with rabbit polyclonal anti-rat ERK1 antibody (1:500, Santa Cruz Biotechnology Inc.). After washing in PBS-Tween, membranes were treated with horseradish peroxidase-labeled donkey anti-rabbit immunoglobulin antibody (1:1000, Amersham Biosciences Corp., Piscataway, NJ) in blocking buffer for 1 h at room temperature. After three washes of 10 min each with PBS-Tween, peroxidase activity was visualized using the Western blotting luminol reagent system (Santa Cruz Biotechnology Inc.). For reprobing membranes, used membranes were stripped from previous antibodies by treatment following the instructions of the Western Blot Recycling Kit (Chemicon International Inc., Temecula, CA).

The p38 MAPK and ERK1/2 MAPK Double Kinase Assay

After denudation and washing in PBS-PVA, the exact meiotic stage of each oocyte was determined by staining oocytes with 12 µg/ml Hoechst 33342 (Polysciences Inc., Warrington, PA) for 20 min followed by observation under a fluorescent microscope. In each sample, two oocytes at the same meiotic stage were transferred into an Eppendorf tube with 1 µl of PBS-PVA. Thereafter, 4 µl of ice-cold extraction buffer was added, and samples were kept at –70°C until kinase assay. The extraction buffer was composed of 80 mM ß-glycerophosphate, 25 mM HEPES (pH 7.2), 10 mM EGTA, 15 mM MgCl2, 1 mM dithiothreitol (DTT), 1 mM APMSF, 0.1 mM Na3VO4, 1 µg/ml leupeptin (Sigma Chemical Co.), and 1 µg/ml aprotinin (Sigma Chemical Co.) [29]. After thawing, samples were centrifuged at 13 000 x g for 2 min at 2°C, added to 5 µl of kinase buffer and 5 µl of substrate solution, and incubated for 20 min at 37°C. The kinase buffer was composed of 75 mM HEPES (pH 7.2), 75 mM ß-glycerophosphate, 75 mM MgCl2, 6 mM DTT, 0.1 mM EGTA, 60 µM ATP, 15 µM cAMP-dependent protein kinase inhibitor peptide (Sigma Chemical Co.) and 0.3 µCi/µl [{gamma}-32P]ATP (250 µCi/25 µl, Amersham Corp.). The substrate solution was a mixture of 4 µl of activated transcription factor 2 (2 mg/ml; ATF-2 fusion protein; #9224; Cell Signalling Technology) for p38 MAPK and 1 µl of myelin basic protein (5 mg/ml, MBP from bovine brain; Sigma Chemical Co.) for ERK1/2 MAPK. The reaction was terminated by the addition of 5 µl of 4x SDS sample buffer. Then samples were boiled for 5 min and loaded onto a 15% gel for separation of labeled ATF-2 and MBP. After running, gels were dried, autoradiographed, and scanned with Image Master ID Elite software Version 3.00 (Amersham Corp.).

Laser-Scanning Confocal Microscopy

After being washed twice in PBS-PVA, denuded oocytes were fixed in PBS-PVA containing 4% (w/v) paraformaldehyde and 0.2% (v/v) Triton X-100 for 40 min. The fixed oocytes were washed twice in PBS-PVA for 15 min each and stored overnight in 1% (w/v) BSA in PBS-PVA (BSA-PBS-PVA) at 4°C. Next, oocytes were blocked with 10% (v/v) goat serum (Dako A/S, Glostrup, Denmark) in BSA-PBS-PVA for 45 min and then incubated in BSA-PBS-PVA containing rabbit polyclonal anti-phospho-p38 MAPK antibody (1:100; #9211; Cell Signaling Technology Inc.) at 4°C overnight. After being washed three times in BSA-PBS-PVA for 15 min each, oocytes were incubated in BSA-PBS-PVA containing Alexa Fluor 488-labeled goat anti-rabbit IgG (1:300; Molecular Probes Inc., Eugene, OR) as the conjugated second antibody for 40 min at room temperature. Negative control experiments were performed by first antibody omission. After being washed three times in BSA-PBS-PVA for 15 min each, the chromosomes were stained with propidium iodide (400 µg/ml; Sigma Chemical Co.). Following complete washing, the oocytes were mounted on slides by Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA) and observed under a laser-scanning confocal microscope (MRC 1024 system; Bio-Rad, Hercules, CA). We examined at least 15 oocytes at 0, 26, and 42 h after culture.

Analysis of Data

All the experiments were repeated three times, and the number of oocytes of each meiotic stage was compared between groups with different inhibitor concentrations by Fisher exact test. Data from the densitometric analyses were expressed as the mean ± SEM, and statistical significance was determined by ANOVA. A P value of less than 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of p38 MAPK During Oocyte Maturation

Porcine oocytes underwent GVBD after 26 h of culture. A total of 93.1% of oocytes reached MI after 30 h, and 91.3% of oocytes matured to MII after 42 h (Fig. 1). The p38 MAPK was detected by anti-p38 MAPK antibody in oocytes before culture (0 h) and 18, 30, and 42 h after culture (Fig. 2A). Densitometric analysis of immunoblots showed a tendency to augment the levels of p38 MAPK after 30 h compared with the levels at 0 and 18 h (Fig. 2B). Membranes were reprobed with anti-ERK1 antibody. Both bands (44 and 42 kDa) of ERK1/2 MAPK shifted up at 30 and 42 h, indicating the activation of this kinase (Fig. 2C).



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  		FIG. 1. Maturation of porcine oocytes in the time course. Porcine oocytes in cumulus-oocyte complexes (COCs) underwent germinal vesicle breakdown (circles) after 26 h, and by 42 h, the majority of oocytes matured to metaphase II (squares). n = number of oocytes examined at each time



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  		FIG. 2. The p38 MAPK in porcine oocytes. The p38 MAPK was detected in Western blots of oocytes during maturation (A), and the densitometric analysis showed the level of p38 MAPK augmented after 30 and 42 h (B). Membranes were reprobed to detect ERK1/2 MAPK (C). Both bands of ERK1/2 MAPK shifted up (arrows) at 30 and 42 h, indicating the activation of this kinase. The intensity of the band from oocytes before culture (0 h) was arbitrarily set to 100% and the other intensity levels were expressed relative to that as a mean percentage ± SEM in Figure 2B

We evaluated the activation pattern of p38 MAPK in detail during maturation according to its ability to phosphorylate ATF-2 in an in vitro kinase assay. Autoradiographs showed that ATF-2 phosphorylation appeared after 26 h of maturation culture, coinciding with the increase of GVBD oocytes (Fig. 3). We observed a high level of ATF-2 activity from this time until the end of culture. The activation pattern of ERK1/2 MAPK was similar to this pattern.



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  		FIG. 3. Change in the phosphorylation of ATF-2 and MBP during porcine oocyte maturation reflecting p38 MAPK and ERK1/2 MAPK activities, respectively. Oocytes at the germinal vesicle (GV) stage (0–22 h) showed no phosphorylation of either ATF-2 or MBP. Phosphorylation of ATF-2 and MBP was detected in oocytes at metaphase I (MI, 26 and 30 h), anaphase I- telophase I (AI-TI, 34 h), and metaphase II (MII, 38 and 42 h)

Subcellular localization of phosphorylated p38 MAPK is shown in Figure 4. In oocytes before maturation culture (0 h), phosphorylated p38 MAPK was detected as a small number of dots in the cytoplasm (Fig. 4A). In the oocytes just before GVBD cultured for 26 h, phosphorylated p38 MAPK was concentrated in the GV (Fig. 4B). In MI and MII oocytes, phosphorylated p38 MAPK was localized in the cytoplasm and around chromosomes (Fig. 4, C and D). No staining was observed when the primary antibody was omitted (Fig. 4, insets).



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  		FIG. 4. Subcellular localization of phosphorylated p38 MAPK in porcine oocytes during maturation. Phosphorylated p38 MAPK (p-p38; green) was detected as a small number of dots in the cytoplasm in oocytes before maturation culture (0 h) (A). Just before germinal vesicle breakdown in oocytes cultured for 26 h, p-p38 was concentrated in the germinal vesicle (B). In metaphase I (C) and metaphase II (D) oocytes, p-p38 was localized in the cytoplasm and around chromosomes. Insets show the image of negative controls where anti-phospho p38 MAPK antibody was omitted and only chromatin and chromosomes (red) were stained by propidium iodide. Scale bars in A–D = 15 µm and in insets = 70 µm

Effect of SB203580 on Oocyte Maturation

When COCs were cultured in the presence of SB203580 (10 and 20 µM), the meiotic resumption of oocytes (Fig. 5A) and cumulus expansion were inhibited (Figs. 5, C–E). On the other hand, normal maturation rates were obtained in COCs cultured in SB203580-free medium: 90.0% (45/ 50) of oocytes reached MII after 42 h of culture.



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  		FIG. 5. Effect of a p38 MAPK inhibitor, SB203580, on the maturation of porcine oocytes and cumulus expansion. In cumulus-oocyte complexes (COCs), SB203580 inhibited cumulus expansion and meiotic resumption of oocytes (A, C–E). In denuded oocytes (DOs), SB203580 arrested oocytes at metaphase I significantly (B). COCs and DOs were cultured in SB203580-free medium (white bars; 50 COCs and 56 DOs) or in a medium containing SB203580 at two different concentrations: 10 µM (gray bars; 50 COCs and 62 DOs) and 20 µM (black bars; 54 COCs and 59 DOs). Photos show COCs before culture (C), COCs after culture in SB203580-free medium (D), and SB203580 (10 µM) containing medium (E). GV, Germinal vesicle stage; MI, metaphase I; AI-TI, anaphase I-telophase I; and MII, metaphase II. Values of columns with different letters within each maturational stage are significantly different (P < 0.05). Scale bars in C–E = 250 µm

In DOs, the spontaneous meiotic resumption was not affected by SB203580 because similar rates of GVBD were observed in the three groups: 85.7% (48/56) in SB203580-free medium, 83.9% (52/62) in 10 µM SB203580, and 88.1% (52/59) in 20 µM SB203580 (Fig. 5B). SB203580 caused a significant arrest at MI in DOs: 17.8% (10/56) in SB203580-free medium, 48.4% (30/62) in 10 µM SB203580, and 62.7% (37/59) in 20 µM SB203580. The percentages of DOs reaching MII were also reduced significantly from 67.8% (38/56) in SB203580-free medium to 32.2% (20/62) and 23.7% (14/59) in 10 µM and 20 µM SB203580-supplemented medium, respectively. The addition of SB203580 at either concentration did not cause degeneration of the oocytes.

The p38 MAPK in Cumulus Cells

The p38 MAPK was detected in cumulus cells surrounding oocytes before culture, and the concentration increased during culture (Fig. 6A). Treatment of COCs with SB203580 (10 µM) inhibited the phosphorylation of p38 MAPK (Fig. 6B).



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  		FIG. 6. The p38 MAPK in porcine cumulus cells. The p38 MAPK of cumulus cells was detected in Western blots, and it accumulated during culture of COCs (A). The phosphorylation of p38 MAPK in cumulus cells during culture after 30 h was inhibited by 10 µM SB203580 (B)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The p38 MAPK was detected in porcine oocytes in immunoblots, and densitometric analyses showed that p38 MAPK levels increased during maturation. Immunofluorescent microscopy showed that phosphorylated p38 MAPK accumulated in GV just before GVBD and then was distributed in the cytoplasm and around chromosomes from MI to MII. In the kinase activity assay, phosphorylation of ATF-2 increased by p38 MAPK activity after 26 h of culture, when oocytes were undergoing GVBD in our culture system. These results suggest that p38 MAPK is present in porcine oocytes at the GV stage and becomes activated around GVBD, and the activity is maintained until MII.

Under in vitro experimental conditions, meiotic resumption of mammalian oocytes is induced in two different mechanisms. One is hormone-induced maturation, where a gonadotropic hormones-cumulus cell-oocyte interaction exists, and the other is spontaneous maturation, where meiotic resumption occurs spontaneously without hormonal induction and without cumulus cell influence. In this study, we investigated the role of p38 MAPK during oocyte maturation in both types of maturation using SB203580, a cell-permeable inhibitor of p38 MAPK [30, 31]. SB203580 inhibited FSH-induced cumulus expansion and meiotic resumption of cumulus-enclosed oocytes. On the other hand, SB203580 did not affect the spontaneous meiotic resumption of denuded oocytes. Based on these results, we hypothesized that p38 MAPK participates in the FSH-induced meiotic resumption in which p38 MAPK in cumulus cells plays a role in inducing oocyte GVBD. Cumulus cells synthesize some meiosis-inhibiting substances that are transported into the oocyte via gap junctions [32]. When the cumulus expands (e.g., by FSH stimulation), gap-junctional communication between cumulus cells and the oocyte is disrupted. This correlates with the meiotic resumption of the oocyte [33, 34]. In rat granulosa cells, p38 MAPK regulates the FSH-induced production of cartilage link protein (Clrt-1), a cumulus expansion-specific protein [26]. In the present study, p38 MAPK was detected in porcine cumulus cells and its phosphorylation was inhibited by SB203580.

Spontaneous meiotic resumption of denuded porcine oocytes was not affected by SB203580. The function of oocyte p38 MAPK is more likely to take place in post-GVBD events, as indicated by significant incidences of MI arrest in denuded oocytes following SB203580 treatment.

Five isoforms of p38 MAPKs have been identified, and they are divided in two groups, the p38{alpha}/ß/ß2 and the p38{gamma}/{delta}, based on their ability to respond to different stimuli [35]. ATF-2, which was used as the substrate of p38 MAPK in this study, is phosphorylated by p38ß/ß2 [36, 37], and only p38{alpha}/ß/ß2 are inhibited by SB203580 [30, 31]. Therefore, p38{alpha}/ß/ß2 probably accounts for the results obtained in this study, although we cannot rule out the possible role of p38{gamma}/{delta} during maturation of porcine oocytes. Just recently, it was found in Xenopus oocytes that p38{gamma} MAPK promotes meiotic G2/M transition [13]. In sea star oocytes, Mipk, a p38 MAPK homologue, was proposed to participate in G2 arrest of oocytes [12]. These differences in p38 MAPK functions during oocyte maturation among species may be explained by the action of different isoforms of p38 MAPK.

In summary, we showed that p38 MAPK is present in porcine oocytes and becomes active around GVBD and high activity is maintained until MII. The p38 MAPK is also present in cumulus cells and participates in the cumulus expansion induced by FSH. The inhibition p38 MAPK activity blocks FSH-induced meiotic resumption of cumulus-enclosed oocytes and the MI-MII transition of denuded oocytes.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Hitoshi Miyazaki (University of Tsukuba) for his helpful suggestions and to the staff of the Kobe Meat Inspection Office for supplying porcine ovaries.


   FOOTNOTES
 
1 Supported in part by a Grant-in-Aid for Creative Scientific Research (13GS0008) to T.M. and by the 21st Century COE Program to T.M. and L.V. from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Back

2 Correspondence: Luis G. Villa-Diaz, c/o Dr. T. Miyano, Faculty of Agriculture, Kobe University, 1-1, Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan. FAX: 81 78 803 5807; 991d924n{at}y01.kobe-u.ac.jp Back

Received: 5 December 2003.

First decision: 26 December 2003.

Accepted: 9 April 2004.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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