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BOR - Papers in Press, published online ahead of print November 13, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.008839
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BIOLOGY OF REPRODUCTION 68, 968–977 (2003)
DOI: 10.1095/biolreprod.102.008839
© 2003 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Characterization of Ribosomal S6 Protein Kinase p90rsk During Meiotic Maturation and Fertilization in Pig Oocytes: Mitogen-Activated Protein Kinase-Associated Activation and Localization1

Heng-Yu Fana, Chao Tonga, Li Liana, Shi-Wen Lia, Wen-Xue Gaoa, Yong Chenga, Da-Yuan Chena, Heide Schattenb, and Qing-Yuan Sun2,a

a State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China b Department of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, Missouri 65211


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitogen-activated protein kinase (MAPK) becomes activated during the meiotic maturation of pig oocytes, but its physiological substrate is unknown. The 90-kDa ribosome S6 protein kinase (p90rsk) is the best known MAPK substrate in Xenopus and mouse oocytes. The present study was designed to investigate the expression, phosphorylation, subcellular localization, and possible roles of p90rsk in porcine oocytes during meiotic maturation, fertilization, and parthenogenetic activation. This kinase was partially phosphorylated in oocytes at germinal vesicle (GV) stage through a MAPK-independent mechanism, but its full phosphorylation is dependent on MAPK activity. After fertilization or electrical activation, p90rsk was dephosphorylated shortly before pronucleus formation, which coincided with the inactivation of MAPK. A protein phosphatase inhibitor, okadaic acid, accelerated the phosphorylation of p90rsk during meiotic maturation and induced its rephosphorylation in activated eggs. MAPK kinase (MAPKK or MEK) inhibitor U0126 inhibited the activation of MAPK and p90rsk in both cumulus-enclosed and denuded pig oocytes, but prevented GV breakdown (GVBD) only in cumulus-enclosed oocytes. Active MAPK and p90rsk were detected in pig cumulus cells, and U0126 induced their dephosphorylation. In meiosis II arrested eggs, U0126 led to the inactivation of MAPK and p90rsk, as well as the interphase transition of the eggs. P90rsk was distributed evenly in GV oocytes, but it accumulated in the nucleus before GVBD. It was localized to the meiotic spindle after GVBD and concentrated in the spindle mid zone during emission of the polar bodies. All these results suggest that p90rsk is downstream of MAPK and plays functional roles in the regulation of nuclear status and microtubule organization. Although MAPK and p90rsk activity are not essential for the spontaneous meiotic resumption in denuded oocytes, activation of this cascade in cumulus cells is indispensable for the gonadotropin-induced meiotic resumption of pig oocytes.

fertilization, kinases, meiosis, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fully grown mammalian oocytes are arrested at prophase of the first meiotic division, a stage known as the germinal vesicle (GV) stage. When the oocyte resumes meiosis, it undergoes GV breakdown (GVBD), extrudes the first polar body, and develops to the metaphase of second meiosis (MII), where it maintains a second meiotic arrest until fertilization or parthenogenetic activation. The physiological trigger for meiotic resumption is the estrus surge of gonadotropins, and the removal of the oocytes from their follicular environment also induces GVBD. The molecular control of oocyte maturation and activation has been thoroughly investigated, and protein phosphorylation/dephosphorylation, which is regulated by both protein kinases and protein phosphatases, is considered to be the most important mechanism regulating meiotic cell cycle progression.

Mature oocytes have elevated levels of maturation promoting factor (MPF) activity and mitogen-activated protein kinase (MAPK) activity. MPF has been identified as a heterodimeric protein kinase composed of a catalytic subunit p34cdc2 kinase and a regulatory subunit cyclin B [1]. The activation of MPF induces entry into metaphase in eukaryotes [2]. MAPK, which is also termed extracellular-regulated kinase (ERK), is another principal regulator of oocyte maturation, and its action in regulating cell cycle events may be uncoupled from MPF in mammalian oocytes. MAPK appears to facilitate meiotic resumption, maintain the normal morphology of the meiotic spindle, inhibit interphase transition between the two meiosis events, and prevent the release from MII arrest [3, 4]. Despite the multifunctional roles of MAPK in meiotic maturation of oocytes, we still know very little about the substrates of the MAPK cascade.

The best known physiological substrates of MAPK are the p90 ribosomal S6 kinase (p90rsk), a family of serine/threonine kinases that were cloned originally on the basis of their ability to phosphorylate the S6 protein of the 40S ribosomal subunit in maturing Xenopus oocytes [5–7]. These enzymes differ from most other protein kinases in that they contain two distinct kinase domains [8]. Phosphorylation of p90rsk substrate is mediated by the N-terminal domain, although the C-terminal domain is necessary for full activity [9]. P90rsk is activated by ERK1 and 2 in vitro and in vivo via phosphorylation on Ser369 and Thr577 [10].

Functional roles for p90rsk in the regulation of meiotic cell cycle progression were revealed in the past decade. During maturation of Xenopus oocytes, MAPK and p90rsk activity is required at the onset of meiosis I to suppress entry into S phase, to regulate the anaphase promoting complex, to facilitate cyclin B accumulation, and to support spindle formation [11]. In vertebrates, the cytostatic factor (CSF) is postulated to maintain elevated levels of MPF activity and hence may be responsible for meiotic arrest at the MII stage [12, 13]. In Xenopus, p90rsk was shown to be the sole mediator of CSF arrest: constitutively active p90rsk caused CSF arrest in the absence of an active MAPK pathway [14] and depletion of p90rsk from egg extracts removed CSF activity, which could be restored by readding p90rsk [15].

The timing of MAPK activation during oocyte maturation, unlike that of MPF, varies in different species. In some species, MAPK is activated before GVBD and might stimulate GVBD. For example, in Xenopus oocytes, progesterone-dependent entry into meiosis I is accompanied by the synthesis of Mos [16–18]. Accumulation of Mos above a threshold level activates the MAPK pathway [19], which leads to MPF activation, most likely through activation of p90rsk [20]. Activated p90rsk inactivates Myt1, a negative regulating kinase of MPF [21], thereby promoting activation of MPF and entry into meiosis I. Experimental treatments that block activation of MAPK significantly inhibit GVBD [22, 23]. In other species by contrast, MAPK activation is not required for the initial activation of MPF. In the oocytes of species such as mice [24] and rats [25], MAPK activation occurs only after GVBD. However, accumulating information suggests that rodents may be atypical with regard to regulating mechanisms of mammalian oocyte maturation and fertilization. In oocytes from large domestic species, MAPK appears to be activated simultaneously with [26] or after GVBD [27, 28]. Whether the function of MAPK is critical for initiation, progression, or MII arrest has not been determined. We reported recently that MAPK failed to be phosphorylated after maturation culture in incompetent pig oocytes [29]. Microinjection of active MAPK into germinal vesicle markedly accelerates GVBD of oocytes [30], but there is no direct evidence that MAPK activity is indispensable for meiotic resumption of porcine oocytes.

Others [31, 32] and we [33] reported MAPK-associated activation of p90rsk in rodent oocytes. This kinase is partially activated shortly before GVBD by a mechanism independent of MAPK, followed by an MAPK-dependent event required for full activation of p90rsk after GVBD. Recently, Sugiura et al. [34] investigated the activation of p90rsk during pig oocyte maturation. However, the characterization of p90rsk after fertilization in mammalian oocytes other than those of rodents has never been described. Besides, although the changes in subcellular distribution of MAPK have been investigated in rodent [35, 36] and porcine [27] oocytes, no report is available concerning the subcellular localization of p90rsk in oocytes throughout the animal kingdom.

In this study we investigated for the first time the expression, phosphorylation, and subcellular localization of p90rsk in porcine oocytes during meiotic maturation, fertilization, and parthenogenetic activation. We also provide evidence that its activity and subcellular migration is regulated by MAPK and protein phosphatase. These studies allowed us to systematically evaluate the roles of the MEK/MAPK/p90rsk cascade in meiotic progression of pig oocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Vitro Maturation and Fertilization of Oocytes

Ovaries were collected from gilts at a local slaughterhouse and transported to the laboratory within 1 h. Oocytes were aspirated from antral follicles (2–6 mm in diameter) with an 18-gauge needle fixed to a 20-ml disposable syringe. After washing three times with maturation medium (see below), oocytes with a compact cumulus and evenly granulated ooplasm were selected for maturation culture. The medium used for maturation culture was improved TCM-199 (Gibco, Grand Island, NY) supplemented with 75 µg/ml potassium penicillin G, 50 µg/ml streptomycin sulphate, 0.57 mM cysteine, 0.5 µg/ml FSH, 0.5 µg/ml LH, and 10 ng/ml epidermal growth factor. A group of 20 oocytes was cultured in a 100-µl drop of maturation medium for 46 h at 38.8°C in an atmosphere of 5% CO2 and saturated humidity.

After maturation culture, oocytes were freed of cumulus cells by treatment with 300 IU/ml hyaluronidase (Sigma Chemical Company, St. Louis, MO) and repeated pipetting. The denuded eggs were then washed twice in TCM-199 and used for either in vitro fertilization (IVF) or drug treatment. IVF was carried out according to a method reported previously [37]. Oocytes were inseminated in a 100-µl drop of modified Tris-buffered medium (mTBM) containing 0.4% BSA (A7888, Sigma) and 2.5 mM caffeine with freshly ejaculated spermatozoa (1 x 106 cells/ml) that were incubated for 2 h in the same medium. Six hours after insemination, eggs were removed from the fertilization drop and cultured in 500 µl of North Carolina State University (NCSU)-23 medium containing 4 mg/ml BSA (A8022, Sigma) for 14 h.

Electrical Activation of Eggs

The method used for egg activation by an electrical pulse was essentially the same as that reported by Onishi et al. [38]. Briefly, after washing three times in the electroporation medium (0.28 M mannitol, 0.05 mM CaCl2, 0.1 mM MgSO4, and 0.01% w/v BSA), cumulus-free eggs were placed in a fusion chamber. An 80-µsec pulse at 120 V/mm DC was exerted to the eggs. The eggs were then washed three times and cultured in NCSU-23 medium containing 0.4% BSA.

Evaluation of Nuclear Status

Orcein staining was conducted according to the procedures described by Sun et al. [29] with a minor modification. Denuded oocytes were mounted on slides, fixed in acetic acid:ethanol (1:3 v/v) for at least 48 h, stained with 1% orcein, and examined with a phase-contrast microscope.

SDS-PAGE and Western Blot Analysis

Proteins from 50 oocytes or cumulus cells of 50 cumulus-oocyte complexes (COCs) were collected in SDS sample buffer and heated to 100°C for 4 min. Oocytes used for measurement after GVBD were unselected earlier because pig oocytes are opaque and their nuclear status is unable to be evaluated in living conditions. After cooling on ice and centrifuging at 12 000 x g for 4 min, samples were frozen at -20°C until use. The total proteins were separated by SDS-PAGE with a 4% stacking gel and a 10% separating gel for 20 min at 56 V and 4.5 h at 110 V, respectively, and then electrophoretically transferred onto nitrocellulose membranes for 2 h at 200 mA at 4°C. The membrane was then blocked overnight at 4°C in TBST buffer (20 mM Tris, 137 mM NaCl, 0.1% Tween-20 pH 7.4) containing 5% low-fat milk. To detect both p90rsk and ERK1/2, blots were cut in two parts containing the proteins above and below the 68-kDa molecular weight marker and were incubated separately for 1 h in TBST with 1:300 polyclonal rabbit anti-mouse p90rsk antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for the upper part of the membrane and with 1:500 mouse anti-p-ERK1/2 antibody (Santa Cruz Biotechnology) for the lower part of the membrane. After three washes for 10 min each in TBST, the upper and lower parts of the membrane were incubated for 1 h at 37°C with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) and HRP-conjugated rabbit anti-mouse IgG diluted 1:1000 in TBST, respectively. The membranes were washed three times in TBST and then processed using the enhanced chemiluminescence (ECL) detection system (Amersham, Buckinghamshire, U.K.).

For reprobing total ERK2, the lower part of the membrane was washed in stripping buffer (100 mM ß-mercaptoethanol, 20% SDS, and 62.5 mM Tris pH 6.7) to strip off bound antibody after ECL detection at 50°C for 30 min. The membrane was reprobed with polyclonal rabbit anti-ERK2 antibody (Santa Cruz Biotechnology) diluted 1:300, incubated with HRP-labeled goat anti-rabbit IgG diluted 1:1000, and finally processed as described above. All experiments were repeated at least three times.

Confocal Microscopy

After removing the zona pellucida in acidified Tyrode medium (pH 2.5), oocytes were fixed with 4% paraformaldehyde in PBS (pH 7.4) for at least 30 min at room temperature. Cells were permeabilized with 1% Triton X-100 overnight at 37°C, followed by blocking in 1% BSA for 1 h and incubation overnight at 4°C with rabbit anti-mouse p90rsk antibody or rabbit anti-mouse ERK antibody diluted 1:100 in blocking solution. After three washes in PBS containing 0.1% Tween-20 and 0.01% Triton X-100 (washing solution) for 5 min each, the eggs were labeled with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG diluted 1:100. The nuclear status of oocytes was evaluated by staining them with 10 µg/ml propidium iodide (PI) for 10 min. Following extensive washings, samples were mounted between a coverslip and a glass slide supported by four columns of a mixture of vaseline and paraffin (9:1). The slides were sealed with nail polish. Cells were observed with a Leica confocal laser scanning microscope (TCS-4D) on the same day.

For meiotic spindle staining, fixed oocytes were incubated with FITC-conjugated anti-{alpha}-tubulin anitibody (Sigma) for 1 h. The cells were then stained with PI and sealed as mentioned above. For colocalization of p90rsk and {alpha}-tubulin, fixed oocytes were sequentially incubated with rabbit anti-mouse p90rsk antibody, tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA), and FITC-conjugated anti-{alpha}-tubulin antibody. Nonspecific staining was determined by substituting primary antibodies with normal rabbit IgG. Each experiment was repeated 3 times and at least 20 oocytes were examined each time.

Experimental Designs

In experiment 1, the phosphorylation/dephosphorylation of MAPK and p90rsk during meiotic maturation (both in cumulus-enclosed oocytes and in denuded oocytes), IVF, and electrical activation was studied by Western blot. The subcellular localization of ERK2 and p90rsk in these processes was detected with confocal microscopy.

In experiment 2, we studied the possible regulation of protein phosphatases on MAPK and p90rsk phosphorylation in pig oocytes and activated eggs by using okadaic acid (OA), a specific inhibitor of phosphatases 1 and 2A. Oocytes that had been freshly isolated from ovaries were denuded of cumulus cells and cultured in the medium containing 2 µM OA for different times and then subjected to nuclear status examination and Western blot analysis. Twenty hours after electrical activation, some activated eggs were treated with 2 µM OA for 30 min or 1 h and collected for analysis.

Experiment 3 was conducted to determine the requirement of the MEK/MAPK/p90rsk cascade for meiotic resumption in pig oocytes. Cumulus-enclosed or denuded pig oocytes at GV stage were cultured in maturation medium containing 10 µM U0126, a specific inhibitor of MEK [39]. GVBD and MAPK/p90rsk phosphorylation were examined at different times of culture.

In experiment 4, expression and phosphorylation of MAPK and p90rsk in pig cumulus cells were studied. Cumulus cells from 50 COCs were collected at various times of maturation culture and then subjected to Western blot analysis. To reveal the regulatory roles of MEK on MAPK and p90rsk activation in pig cumulus cells, some COCs were cultured in medium containing 10 µM U0126 before cumulus cell isolation.

Experiment 5 was conducted to elucidate the necessity of MEK/MAPK on the maintenance of p90rsk phosphorylation as well as MII arrest. Denuded MII eggs were incubated in the medium containing 10 µM U0126 for 6, 14, or 20 h before orcein staining or Western blotting.

Statistical Analysis

All percentages from three repeated experiments are expressed as means ± SEM and the number of oocytes observed is labeled in parentheses as (n =). The rates of GVBD were subjected to arcsin transformation. The transformed data were analyzed by ANOVA followed by the Student-Newman-Keuls test. Differences at P < 0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phosphorylation of p90rsk During Meiotic Maturation

In our culture system, 21.18% (n = 85) and 43.37% (n = 83) of the cumulus-enclosed oocytes underwent GVBD at 20 h and 24 h after maturation culture, respectively, and 86.42% (n = 141) of them developed to MII stage at 44 h. The kinetics of MAPK and p90rsk phosphorylation during pig cumulus-enclosed oocyte maturation is shown in Figure 1A. Phosphorylation of p90rsk was assessed by examining its electrophoretic mobility shift on SDS-PAGE and phosphorylation of ERK1/2 was evaluated by both mobility shift and a specific antibody against phospho-MAPK. MAPK was inactive in fully grown GV oocytes, it was first detected 18 h after culture, and it was fully phosphorylated at 30 h. In oocytes just obtained from ovaries, p90rsk was observed as double bands. A new staining above the two existing band was detected 18 h after culture, with lesser staining of the lowest band. The band with the slowest electrophoretic mobility increased in intensity after the maturation culture and culminated at 44 h. However, staining of the lowest and middle bands disappeared at 24 h and 44 h, respectively.



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  		FIG. 1. Phosphorylation of MAPK and p90rsk during meiotic maturation of cumulus-enclosed and denuded pig oocytes. A) MAPK was inactive in fully grown GV oocytes, began to be detected 18 h after culture, and was fully phosphorylated at 30 h. In oocytes just obtained from ovaries, p90rsk was observed as double bands. A new staining above the two existing bands was detected 18 h after culture, with decreased staining of the lowest band. The band with slowest electrophoretic mobility increased in intensity following the maturation culture and culminated at 44 h. However, the staining of the lowest and middle bands disappeared at 24 h and 44 h, respectively. B) Although phosphorylated ERK1/2 could be detected from 18 to 44 h after culture in denuded oocytes, the staining of the total ERK2 demonstrated that most molecules of this kinase existed as the dephosphorylated form. Only weak staining of the fully phosphorylated p90rsk could be detected in these oocytes at 44 h, with the partially phosphorylated form being the major type of p90rsk

Denuded pig oocytes did not complete maturation in vitro, but most of them underwent GVBD (84.75%, n = 59 in our system) after maturation culture. As shown in Figure 1B, although phosphorylated ERK1/2 could be detected from 18 to 44 h after culture in these oocytes, the staining of the total ERK2 demonstrated that most molecules of this kinase existed in the dephosphorylated form. Only weak staining of the fully phosphorylated p90rsk could be detected in these oocytes at 44 h, with the partially phosphorylated form being the major type of p90rsk.

Dephosphorylation of P90rsk after In Vitro Fertilization and Electrical Activation

In vitro fertilized pig eggs typically form pronuclei 14–20 h after insemination, but electrically activated eggs form pronuclei faster than fertilized eggs, with 43.33% (n = 60) of them forming one or two pronuclei at 14 h after activation. MAPK and p90rsk were kept phosphorylated until 14 h after fertilization, but were partly dephosphorylated at 17 h and were completely dephosphorylated at 20 h after fertilization (Fig. 2A). In oocytes that underwent parthenogenetic activation, MAPK and p90rsk remained phosphorylated at 6 h after electrical stimulation. Dephosphorylation of these two kinases was detected at 14 h (Fig. 2B, left panel) after electrical activation, just at the same time that pronuclear formation occurred.



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  		FIG. 2. Dephosphorylation of MAPK and p90rsk after fertilization and electrical activation were detected by Western blotting. In oocytes subjected to in vitro fertilization (A), MAPK and p90rsk remained active until 14 h of insemination (A, lanes 1–3). The partial dephosphorylation of both kinases was detected 17 h after fertilization (A, lane 4), and the complete dephosphorylation of p90rsk and MAPK was observed at 20 h after fertilization (A, lane 5). Among oocytes activated by electrical pulse (B), p90rsk and MAPK were kept active at 6 h of activation (B, lane 2), but were dephosphorylated as early as 14 h after electrical stimulation (B, lane 3). The inactive status of p90rsk and MAPK was sustained at 20 and 24 h (B, lanes 4 and 5) after activation. If the activated eggs were transferred into medium containing 2 µM OA 24 h after electrical stimulation, however, OA led to the rephosphorylation of both the MAPK and the p90rsk after 30 min or 2 h of treatment (B, lanes 6 and 7)

Regulation of p90rsk Phosphorylation by Protein Phosphatase

In GV oocytes, OA greatly accelerated MAPK and p90rsk phosphorylation (Fig. 3). MAPK and p90rsk were fully phosphorylated as early as 10 min after OA treatment. However, the amount of MAPK and p90rsk in oocytes is significantly decreased by 2 h of continuous exposure to OA. ERK2 and p90rsk maintained the phosphorylation state at 10 h and 24 h of OA treatment, but they could not be detected at 44 h. Meiotic resumption was greatly accelerated by OA treatment. More than half the oocytes (55%, n = 80) underwent GVBD 2 h after OA treatment. In parthenogenetically activated pig eggs with dephosphorylation of MAPK and p90rsk, OA led to the rephosphorylation of both MAPK and the p90rsk after 30 min or 2 h of treatment (Fig. 2B, right panel). Nuclear envelope breakdown (83.33%, n = 90) and chromatin recondensation were also induced 2 h after OA treatment (data not shown).



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  		FIG. 3. Effect of OA treatment on MAPK and p90rsk phosphorylation during meiotic maturation. Denuded oocytes at GV stage were cultured in maturation medium containing 2 µM OA and were collected for immunoblotting at 5 min, 10 min, 2 h, 10 h, 24 h, and 44 h after OA treatment. ERK2 and p90rsk were promptly phosphorylated as early as 10 min after OA treatment (lane 3), and their phosphorylation status was maintained at 2 h (lane 4), 10 h (lane 5), and 24 h (lane 6) after culture with 2 µM OA. However, the amount of ERK2 and p90rsk in oocytes was significantly decreased by 2 h or longer of continuous exposure to OA (lanes 4–6). ERK2 and p90rsk could not be detected at 44 h (lane 7)

Effects of U0126, the MEK Inhibitor, on Meiotic Maturation, MII Arrest, and MAPK and P90rsk Activity

When the cumulus-enclosed GV oocytes were cultured in medium containing 10 µM U0126, GVBD was significantly inhibited (Fig. 4). Only 23.66% (n = 301) of them underwent GVBD at 44 h. Correspondingly, the phosphorylation of MAPK and p90rsk was inhibited in these oocytes (Fig. 5A). Most denuded pig oocytes underwent GVBD (83.18%, n = 94) when they were observed at 44 h of maturation culture, although only a few of them developed to the MII stage (15.15%, n = 94). Treatment with 10 µM U0126 could not inhibit GVBD in denuded oocytes with the GVBD rate of 85.32% (n = 236) at 44 h, although the phosphorylation of MAPK and p90rsk had been effectively inhibited (Fig. 5B). The difference in GVBD rate of denuded oocytes cultured with 10 µM U0126 or in a drug-free medium was not statistically significant. Expression and phosphorylation of MAPK and p90rsk were detected in pig cumulus cells (Fig. 5C). Active MAPK existed in cumulus cells surrounding GV oocytes that were freshly isolated from ovaries, but the phosphorylation levels of MAPK and p90rsk increased following maturation culture. A significant increase in MAPK and p90rsk activation was detected at 12 h of culture and it culminated at 24 h. Both kinases were dephosphorylated in cumulus cells after treatment of cumulus-oocyte complexes with U0126 for 12, 20, or 24 h.



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  		FIG. 4. Effect of U0126 on meiotic maturation of cumulus-enclosed and denuded pig oocytes. Each bar represents the average GVBD rate of three parallel experiments. The rates of GVBD were subjected to arcsin transformation and were analyzed by ANOVA followed by the Student-Newman-Keuls test. Differences at P < 0.05 were considered to be statistically significant. Cumulus-enclosed or denuded pig oocytes were cultured in maturation medium for 44 h with the presence of 10 µM U0126, and the nuclear status was examined by orcein staining. GVBD was significantly inhibited in cumulus-enclosed oocytes in comparison with the control group. However, there was no statistically significant difference in GVBD rate between the denuded oocytes cultured with or without U0126. CEO, Cumulus-enclosed oocytes; DO, denuded oocytes



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  		FIG. 5. Effect of the MEK inhibitor U0126 on MAPK and p90rsk phosphorylation was studied in pig oocytes, eggs, and cumulus cells. In experiments shown in A and B, cumulus-enclosed or denuded GV oocytes were cultured in maturation medium containing 10 µM U0126, and collected for immunoblotting after 18 h, 30 h, and 44 h of culture. MII eggs were loaded as the positive control (lane 4 in A and B). The phosphorylation of MAPK and p90rsk was inhibited at 18 h, 30 h, and 44 h of culture in both cumulus-enclosed oocytes (A, lanes 1–3) and denuded oocytes (B, lanes 1–3). Expression and phosphorylation of MAPK and p90rsk was detected in pig cumulus cells (C). Cumulus cells isolated from 50 COCs were loaded into each lane. Active MAPK existed in cumulus cells surrounding GV oocytes freshly isolated from ovaries (C, lane 1). Significant increases in MAPK and p90rsk activation were detected at 12 h of culture (C, lane 3) and culminated at 24 h (C, lane 5). Both kinases were dephosphorylated in cumulus cells after COCs were treated with 10 µM U0126 for 12, 20, or 24 h (C, lanes 6–8). In MII-arrested pig eggs in which p90rsk and MAPK existed as the phosphorylated form (D, lane 1), treatment with 10 µM U0126 led to the dephosphorylation of both kinases as early as 6 h of culture (D, lane 2) and culminated at 14 and 20 h after incubation (D, lanes 3 and 4)

When MII-arrested pig eggs were cultured in the medium containing 10 µM U0126 for 20 h, 41.44% (n = 111) of the eggs were released from MII arrest and entered interphase with pronucleus formation. The results of Western blots showed that the dephosphorylation of MAPK and p90rsk occurred as early as 6 h and culminated at 14 h (Fig. 5D).

Subcellular Localization of p90rsk in Pig Oocytes During Meiotic Maturation and Activation

Subcellular distribution of p90rsk and ERK2 in porcine oocytes before and around GVBD are shown in Figure 6. In GV oocytes immediately aspirated from follicles, ERK2 staining was observed primarily in the cytoplasm (Fig. 6A'), and p90rsk was almost evenly distributed in the oocytes (Fig. 6A). In oocytes cultured for 20 h, just before GVBD, the positive signals for both ERK2 and p90rsk were clearly detected in the GV (Fig. 6, B' and B). At 24 h after culture in oocytes undergoing GVBD, the area around condensed chromatin was preferentially stained with ERK2 and p90rsk (Fig. 6, C' and C). However, in cumulus-enclosed oocytes cultured in medium containing 10 µM U0126 for 24 h, GVBD was not observed, and no accumulation of ERK2 and p90rsk in the GV was detected (Fig. 6, D' and D). No specific signal was observed with normal rabbit IgG (data not shown).



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  		FIG. 6. Localization of p90rsk (upper panels) and ERK2 (lower panels) before and around GVBD were compared in pig oocytes. Green shows FITC-conjugated antibodies; red shows PI-stained chromatin; orange shows overlapping of green and red (also in Figs. 7 and 8). Black dots in the cytoplasm of some oocytes are unstained cytoplasmic areas resulting from the existence of fat drops. In GV oocytes immediately aspirated from follicles, anti-ERK2 staining was observed primarily in the cytoplasm with a weak staining in the nucleus (A'), and p90rsk was almost evenly distributed in the oocytes (A). In oocytes cultured for 20 h, just before GVBD, the positive signal for both ERK2 and p90rsk was clearly detected in the GV (B' and B). In oocytes undergoing GVBD at 24 h after maturation culture, the area around condensed chromatin was preferentially stained with ERK2 or p90rsk (C' and C). However, in cumulus-enclosed oocytes cultured in medium containing 10 µM U0126 for 24 h, GVBD was significantly inhibited, and no accumulation of ERK2 or p90rsk in the GV was detected (D' and D)

Most pig oocytes developed to pre-metaphase I or MI stage at 30 h of culture. In these oocytes, prominent staining for p90rsk was observed around the dispersed or aligned chromosomes, putatively the position of pre-MI or MI spindle, as shown in Figure 7. Localization of p90rsk and ERK2 during the MI/MII transition, as well as their spatial relationship with microtubules at this time are compared in Figure 8. During the process of PB1 emission, with the progression of anaphase I and telophase I, p90rsk (Fig. 8, A and B) and ERK2 (Fig. 8, C and D) were detected at the mid zone of the elongated spindle, as confirmed by the staining of {alpha}-tubulin (Fig. 8, E and F). To further prove the colocalization of p90rsk and meiotic spindle, p90rsk and {alpha}-tubulin were double stained in eggs arrested at MII stage or activated with an electrical pulse. In MII eggs, p90rsk was present at the entire spindle except the equatorial region (Fig. 9, B and C), whereas in activated eggs at anaphase II, p90rsk translocated to the center of the elongating spindle (Fig. 9, E and F). Staining for {alpha}-tubulin showed that in both stages p90rsk always concentrated to the region with the most abundant distribution of microtubules (Fig. 9, A and D). In activated eggs with pronuclear formation, p90rsk distributed evenly in the cells, with no differences in staining between cytoplasm and nucleus (data not shown).



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  		FIG. 7. Localization of p90rsk in pig oocytes at pre-MI (upper panels) and MI (lower panels) stages was detected with confocal microscopy. DNA and p90rsk were stained with PI (red) and FITC (green), respectively. The double-stained samples were then scanned with red and green channels separately and the images obtained in each channels were merged. Prominent staining for p90rsk (A and C) was observed around the dispersed chromosomes (B) at pre-MI stage. In oocytes at MI stage, p90rsk (D and F) was localized where chromosomes were aligned (E), putatively the position of the MI spindle



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  		FIG. 8. Localization of p90rsk and ERK2 at anaphase (left panels) and telophase (right panels) of meiosis I, as well as microtubule organization at the same time. Oocytes were collected at 32 h after maturation culture, and the cell cycle stages were determined by observing the chromosome status and microtubule configurations after PI and FITC-anti-{alpha}-tubulin staining. During the process of PB1 emission, with the progression of anaphase I and telophase I, p90rsk (A and B, stained in green) and ERK2 (C and D, stained in green) were detected at the mid zone of the elongated spindle, as confirmed by the staining of {alpha}-tubulin (E and F, stained in green)



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  		FIG. 9. P90rsk and {alpha}-tubulin were double-stained in pig eggs arrested at MII stage (upper panels) or activated with an electrical pulse (lower panels). Green indicates staining of {alpha}-tubulin with FITC-labeled anti-{alpha}-tubulin, red indicates staining of p90rsk with TRITC-conjugated antibody, orange indicates the overlap of green and red. Cells were scanned with green and red channels separately and then merged together. In MII eggs, p90rsk was present at the entire spindle except the equatorial region (B and C), whereas in activated eggs at anaphase II (2 h after electrical stimulation), p90rsk disappeared from spindle poles but it translocated to the center of the elongating spindle (E and F). Staining for {alpha}-tubulin showed that in both stages p90rsk always concentrated to the region with the most abundant distribution of microtubules (A and D)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we determined that p90rsk, the best-known substrate of MAPK, was expressed in porcine oocytes during meiotic maturation and fertilization. Three p90rsk forms with different electrophoretic mobilities exist in pig oocytes. These forms may be the result of two phosphorylation events and may be correlated with changes in the catalytic activity of p90rsk as reported previously [31, 33, 34]. The absence of MAPK activity until 18 h after maturation culture is in agreement with our previous studies [40] and provides evidence that MAPK does not play a role in the first step of p90rsk activation. It was reported that the partially phosphorylated p90rsk is not an active kinase, and may be the result of autophosphorylation [41]. However, active MAPK is required for the complete phosphorylation of p90rsk. In both cumulus-enclosed and denuded oocytes, MEK inhibitor U0126 prevents the full phosphorylation of p90rsk even at 44 h of culture. It was reported in mouse [32] and pig [34] oocytes that p90rsk activity was low at the GV stage, but it was significantly elevated after maturation. High levels of p90rsk activity in maturing pig oocytes may facilitate spindle formation, support cyclin B accumulation, and maintain MII arrest, as shown in Xenopus oocytes [11].

In activated eggs stimulated by fertilization or electrical pulse, p90rsk is slowly dephosphorylated by a phosphatase that is activated shortly before pronuclear formation. The rapid decrease of MPF activity is involved in the initiation of egg activation, whereas little is known about the importance of the delayed inactivation of the MAPK/p90rsk cascade. A recent report suggests that MAPK plays an essential role in inducing PB2 emission and the transition from meiosis to mitosis [42]. We propose that this effect may be mediated by the active p90rsk at this time, and it is supported by our results with confocal microscopy, which revealed that p90rsk concentrates in the spindle mid zone at anaphase of meiosis II.

It is evident that p90rsk is the sole mediator of CSF activity in the maintenance of MII arrest in Xenopus eggs [43], but its importance in mammalian oocytes is uncertain. We showed here that in pig MII-arrested eggs, MEK inhibitor U0126 induced the inactivation of MAPK and p90rsk, and subsequently, the interphase transition of cell cycle. This result suggests that p90rsk activity might be necessary for the maintenance of MII arrest in porcine eggs. Furthermore, MEK/MAPK activity is essential not only for the phosphorylation of p90rsk, but also for the maintenance of its active state, because U0126 led to the dephosphorylation of p90rsk, which has already been fully phosphorylated in MII eggs.

Serine/threonine protein phosphatases, which antagonize protein kinase phosphorylation, have been implicated in the regulation of oocyte meiosis. In our experiment, OA induced the prompt phosphorylation of MAPK and p90rsk in GV oocytes. Because OA blocks protein synthesis as a by-effect, a long OA treatment time may lead to a decrease in ERK quantity. Arrighi et al. [44] and Lu et al. [4] reported in mouse oocytes that a 1-h pulse of OA treatment induced the irreversible activation of MAPK, and thus may avoid the apparent toxic effects. Although this approach was not tried in pig oocytes, the results from our previous report [45] and this report suggest that the changes in MAPK phosphorylation and cell cycle progression after long-term OA treatment were almost similar to those reported in mouse oocytes after short-term OA treatment. This fact infers that fully grown GV oocytes have acquired the ability to phosphorylate MAPK/p90rsk, but that an OA-sensitive phosphatase-dependent inhibitory mechanism prevents the MAPK/p90rsk activation. Our hypothesis is supported by the reports of Inoue et al. [30], who found that active MAPK injected into the cytoplasm of pig GV oocytes was quickly dephosphorylated and failed to induce GVBD. In parthenogenetically activated eggs, OA induced the rephosphorylation of MAPK and p90rsk, which was also reported in mice [46] and rats [33]. The regulation of MAPK and p90rsk inactivation after egg activation has been poorly understood until now, but this evidence suggests that even in activated eggs with pronuclear formation, the mechanism responsible for MAPK activation still exists. The inactivation of MAPK cascade may be the result of enhanced activity of OA-sensitive phosphatase before pronucleus formation.

Reports on the role of the MEK/MAPK/p90rsk cascade during mammalian oocyte meiotic resumption are not definitive. During spontaneous maturation of mouse and rat oocytes, MAPK and p90rsk are activated after GVBD and MPF activation. We (unpublished results ) and others [3] found that in denuded mouse oocytes treated with U0126, GVBD could occur normally without MAPK and p90rsk activity. These results suggest that the MEK/MAPK/p90rsk cascade is not a prerequisite for spontaneous meiotic resumption, but rather it is involved in post-GVBD events. In contrast, MAPK is activated before GVBD and may be required for meiotic resumption of equine [47] and bovine [26] oocytes. But some authors have argued for the necessity of the MAPK cascade in oocyte meiotic resumption of farm animals. Injection of MKP-1 mRNA, which encodes a dual specific MAPK phosphatase, into GV stage bovine oocytes prevents MII arrest, but not the resumption and progression through meiosis [48]. MAPK activity is down-regulated by U0126 in pig oocytes, but GVBD is not prevented [42]. These results taken together have convinced many researchers that although activation of MAPK cascade is necessary for resumption of meiosis in lower vertebrates such as Xenopus, it is dispensable for meiotic resumption in mammals.

Some recent experiments have provided deeper insights into the roles of the MAPK cascade in meiotic resumption of mammalian oocytes. MEK inhibitors U0126 and PD98059 inhibited FSH-induced meiotic resumption in cumulus-enclosed mouse oocytes, but not the spontaneous GVBD in denuded oocytes [3]. MAPK was activated in mouse cumulus cells after FSH stimulation. We also observed the phosphorylation of MAPK and p90rsk during pig oocyte maturation. Although MAPK and p90rsk phosphorylation were inhibited by U0126 in both cumulus-enclosed and denuded pig oocytes, only the gonadotropin-induced meiotic resumption of cumulus-enclosed oocytes was prevented. Considering the function and regulation of the MAPK cascade in meiotic maturation of Xenopus, mouse, and pig oocytes/COCs, a unified understanding has emerged. The meiotic resumption of Xenopus oocytes is induced by progesterone, which exerts its effect on the oocytes directly and stimulates the MAPK cascade [49–51]. However, there are two pathways leading to meiotic maturation in mammalian oocytes: spontaneous meiotic resumption when released from inhibitory follicular environment, which is MAPK-independent; and gonadotropin-induced meiotic resumption by overcoming the inhibitory effects of the follicular environment, which is MAPK-dependent. In the latter, cumulus cells, instead of the oocytes, are the primary targets of meiosis-inducing signal (gonadotropin). Thus, we hypothesized that the MAPK cascade is indispensable for the mediation of meiotic resumption-inducing signal among all vertebrates, either in oocytes or in cumulus cells, depending on the primary targets of the stimulations.

The subcellular localization of ERK2 and p90rsk was investigated in our experiments. These two kinases appeared to share the same pattern of distribution (i.e., migrating from the cytoplasm to the GV before GVBD and associating with the meiotic spindle at metaphase and anaphase). The similarity of ERK and p90rsk localization implies that p90rsk is the putative target of MAPK in pig oocytes, and the concentration of p90rsk in these areas may lead to a local increased activity that could participate in meiotic cell cycle regulation. In porcine oocytes, the inhibition of MAPK activity with U0126 during the meiosis I/meiosis II transition suppressed chromosome separation, first polar body emission, and formation of the metaphase II spindle [52]. We propose that the spindle-associated p90rsk also plays an important role in the events that occur during the meiosis I/meiosis II transition as the mediator of MAPK effects, such as chromosome separation, spindle elongation, and cleavage furrow formation in pig oocytes. Both kinases failed to translocate from the cytoplasm to the GV when their activation was inhibited by U0126 treatment, suggesting that the MAPK-dependent activation of p90rsk is the prerequisite for its nuclear accumulation.

In conclusion, we showed that p90rsk was partially phosphorylated before the meiotic resumption of porcine oocytes, was fully phosphorylated after GVBD following MAPK phosphorylation, and was dephosphorylated before pronuclear formation. The distinct subcellular distribution of p90rsk during meiotic cell cycle progression suggests its functional roles in the regulation of nuclear status and microtubule organization. Although MAPK and p90rsk activities are not essential for the spontaneous meiotic resumption in denuded oocytes, activation of this cascade in cumulus cells is indispensable for the gonadotropin-induced meiotic resumption of pig oocytes.


   FOOTNOTES
 
1 This study was supported by grants from the Special Funds for Major State Basic Research (973) Project (G1999055902), Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-SW-303), and Grant for Outstanding Young Scientists from the National Natural Science Foundation of China (30225010). Back

2 Correspondence. FAX: 8610 6256 5689; e-mail: sunqy{at}panda.ioz.ac.cn Back

Received: 26 June 2002.

First decision: 1 August 2002.

Accepted: 1 October 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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