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Regular Article |
a Departments of Obstetrics and Gynecology,
b Physiology, and
c Urology,
d Reproductive Sciences Program, University of Michigan, Ann Arbor, Michigan 48109-0617
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, kinases, meiosis, phosphatases
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The family of MAPKs (p44MAPK1 and p42MAPK2), also known as extracellular signal-regulated kinases (ERK1 and ERK2, respectively), are serine/threonine kinases that require threonine and tyrosine phosphorylation to become active [14]. Both ERK1 and ERK2 are phosphorylated and activated by MAPK kinase (MEK) [15, 16], which is believed to be activated by protooncogene c-mos phosphorylation [17–19]. Evidence suggests that MAPKs are important to meiotic progression. In mouse oocytes, ERK1 and ERK2 are activated after GBVD and remain activated throughout the MI/MII transition [20]. During oocyte meiosis, changes in microtubule/spindle organization, spindle migration, and chromatin condensation are correlated with MAPK activation [21–23]. In addition, nonphosphorylated ERK1 and ERK2 associate with MTOCs during mouse oocyte meiotic maturation [24]. However, whether this association involves active MAPK is unknown.
Serine/threonine protein phosphatases (PPs), which antagonize protein kinase phosphorylation, have been implicated in the regulation of oocyte meiosis [25–30]. Classification of serine/threonine PPs is based on substrate specificity and sensitivity to a defined set of inhibitors and activators [31]. Type 1 PP is sensitive to the heat- and acid-stable inhibitors 1 and 2 (I1 and I2), whereas type 2 PPs are insensitive to I1 and I2 [32]. Type 2 PPs can be subclassified into PP2A, PP2B, and PP2C based on their cation requirements. Both PP1 and PP2A do not require divalent cations for activity, whereas PP2B and PP2C require Ca2+/calmodulin and Mg+, respectively, for activity [33, 34]. Okadaic acid (OA) is a specific inhibitor of PP1 and PP2A [31, 35]. Activities of PP1 and PP2A are extremely sensitive to OA inhibition and can be differentiated in cell extract experiments depending on the concentrations of OA administered [33, 36, 37].
Extended treatment (2–16 h) or microinjection of immature oocytes with OA stimulates GVBD yet results in oocytes with severe cytoplasmic abnormalities, compromised spindle assembly, and inability to progress to MII [25–30]. However, transient exposure (10 min) of monkey immature oocytes to OA enhanced GVBD rates, did not result in cytoplasmic aberrations, and allowed development to MII [30]. Collectively, this indicates that inhibition of PP1 and/or PP2A stimulates GVBD, yet some PP1 and/or PP2A activity is important for normal spindle assembly, cytoplasmic function, and progression to MII. In addition, the predominant localization of PP2A in the cytoplasm and predominance of PP1 within the nucleus of germinal vesicle-intact (GVI) oocytes suggests that these two PPs may have distinct regulatory roles during meiosis [38]. In somatic and neuronal cells, PP2A is associated with microtubules and appears to be important for microtubule assembly [12, 39]. Whether oocyte cytoplasmic PP2A regulates microtubule polymerization and meiotic spindle formation remains to be elucidated.
The objectives of this study were to begin to clarify the functional importance of active MAPK and PP2A in regulating oocyte meiosis. In this regard, we focused on identification of MAPK activation, PP2A activity, intracellular kinase/phosphatase trafficking, and ultrastructural associations during oocyte meiosis. In addition, we addressed the question of requisite kinase and/or phosphatase activity and chromatin condensation, microtubule polymerization, and normal meiotic spindle formation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fully grown GVI oocytes were collected from 5-wk-old female mice (CF-1) preinjected with eCG utilizing a protocol approved by the University of Michigan Committee on Use and Care of Animals. Oocytes were collected by puncturing antral follicles in a HEPES-buffered human tubule fluid (HTF) medium (Irvine Scientific, Santa Ana, CA) and cultured in an HTF medium (Irvine Scientific) containing 0.4% BSA for 1.5 h. Oocytes that had undergone GVBD were selected and incubated for an additional 5, 8, and 10 h for progression to MI, AI, and MII, respectively. A subset of oocytes was assessed by immunocytochemical analysis using anti-ß-tubulin antibodies and propidium iodide to confirm the predominant meiotic stage for each length of incubation.
The GVBD oocytes were selected and treated in HTF medium containing 0.4% BSA with addition of one of the following chemicals: 20 µM U0126 (Sigma, St. Louis, MO), 1 µM OA (Calbiochem Novabiochem Corp., La Jolla, CA), or 50 nM calyculin-A (CL-A; Gibco BRL Life Technologies, Baltimore, MD) for an additional 5 h. As a control, GVBD oocytes were incubated in a drug-free HTF medium containing 0.4% BSA for 5 h.
Immunoblot Analysis
For phosphorylated (i.e., active) MAPK analysis, extracted proteins from different meiotic stages (GVI, GVBD, MI, AI, and MII) were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Immun-Blot; Bio-Rad Laboratories, Hercules, CA) as previously described [38]. A total of 25 oocytes were loaded in each lane. After washing with TBST (10 mM Tris, 150 mM NaCl, and 0.1% Tween 20; pH 7.4), blots were blocked overnight at 4°C in 5% nonfat milk in TBST, then incubated for 1 h at room temperature (RT) with agitation in a rabbit polyclonal anti-phospho-MAPK antibody (New England Biolabs, Inc., Beverly, MA) diluted to 0.05 µg/ml. After complete washing, blots were incubated for 1 h at RT with agitation in an anti-rabbit horseradish peroxidase-conjugated IgG diluted to 0.16 µg/ml. Following complete washing, phosphorylated MAPKs were detected with ECL Plus reagents (Amersham Life Sciences, Buckinghamshire, UK) per the manufacturer's instructions. To investigate expression of MAPK, blots were stripped for 30 min at 55°C with agitation in a stripping buffer (62.5 mM Tris-HCl [pH 6.7], 100 mM ß-mercaptoethanol, and 2% SDS). Completely stripped blots were blocked overnight at 4°C in 5% nonfat milk in TBST, then incubated with 0.4 µg/ml rabbit polyclonal anti-ERK2 antibody (Santa Cruz Biotech, Inc., Santa Cruz, CA) for 1 h at RT with agitation and processed further as described above.
To detect PP2A expression during oocyte maturation, 200 oocytes of each stage (GVI, GVBD, MI, AI, and MII) were extracted for loading. Separated proteins were transferred to a PVDF membrane and processed as previously mentioned with rabbit polyclonal anti-PP2A antibody (Upstate Biotechnology, Lake Placid, NY) at 1 µg/ml. Nonstimulated A431 cell lysates (Upstate Biotechnology) were used as a positive control.
PP Assay
The PP assay system employed (Gibco BRL) detects both PP1 and PP2A using 32P-labeled glycogen phosphorylase a as a substrate. The 32P-labeled phosphorylase a was prepared per the manufacturer's instructions. Two hundred oocytes of each stage (GVI, GVBD, MI, and MII) were extracted in 100 µl of extraction buffer (50 mM Tris-HCl [pH 7.0], 2 mM EDTA, 2 mM EGTA, 0.1% ß-mercaptoethanol, 1 mM PMSF, 2 µg/ml of leupeptin, 2 µg/ml of aprotinin, and 2 µg/ml of pepstatin A) by repeated freezing-thawing (five times). After spinning (12 000 x g, 5 min, 4°C), 5 µl of the supernatant were taken for detecting total soluble protein concentration using a bicinchoninic acid assay (BCA assay; Pierce, Rockford, IL). For the PP1 activity assay, 1 nM OA was added to half the remaining supernatant and incubated for 10 min at 30°C to inhibit PP2A activity. The other half of the remaining supernatant was also incubated for 10 min at 30°C in the absence of OA to detect PP1 plus PP2A activity. Oocyte extracts pretreated with or without OA were added to tubes containing 32P-labeled phosphorylase a and reaction buffer and then incubated for 10 min at 30°C. Reactions were stopped with addition of trichloroacetic acid. After spinning (12 000 x g, 3 min, 4°C), radioactivity (cpm) of the supernatant was counted. Meanwhile, total radioactivity of the 32P-labeled phosphorylase a used was also counted. Protein phosphatase 2A activity (nM-1 min-1 mg-1) was calculated by subtracting PP1 activity from PP1 plus PP2A activity.
Immunofluorescence and Confocal Microscopic Analysis
To identify localization of active MAPK and PP2A during oocyte maturation, oocytes at GVI, GVBD, MI, AI, and MII were collected and fixed in 2% paraformaldehyde with 0.04% Triton X-100. Oocytes were then blocked overnight with 0.3% BSA in PBS at 4°C and incubated with a rabbit polyclonal anti-phospho-MAPK antibody diluted to 0.25 µg/ml or a rabbit polyclonal anti-PP2A antibody diluted to 10 µg/ml for 1 h at 37°C. After washing with 0.3% BSA plus 0.1% Tween-20 in PBS, oocytes were incubated in the same wash buffer for 90 min at 37°C. Samples were then reacted with an anti-rabbit Alexa 568-conjugated secondary antibody (Molecular Probes, Eugene, OR) diluted to 2 µg/ml for 1 h at 37°C. Following complete washing, slides were mounted for fluorescence microscopic visualization.
For double-labeling, oocytes were incubated in a rabbit anti-phospho-MAPK antibody or rabbit anti-PP2A for 1 h at 37°C. After a complete wash, they were incubated in mouse monoclonal anti-ß-tubulin antibody (TUB2.1, 1:200 dilution; Sigma) for 1 h at 37°C. Following washing, oocytes were incubated with anti-rabbit Alexa 568-conjugated secondary antibody plus anti-mouse Alexa 488-conjugated (Molecular Probes) secondary antibody, each diluted to 2 µg/ml, for 1 h at 37°C. All antibodies were diluted in 0.3% BSA and 0.1% Tween-20 in PBS. Fluorescence microscopic visualization was performed following washing and mounting. All samples were visualized with a Bio-Rad MRC-600 confocal scanning laser microscope.
Microtubule Polymerization In Vitro
In vitro microtubule assembly was performed as described elsewhere [40]. HeLa cells (107) were washed with ice-cold PBS and homogenized in 500 µl of PEM buffer containing protease inhibitors (0.1 M PIPES, 5 mM MgCl2, 2 mM EGTA [pH 6.9], 1 mM PMSF, 10 µg/ml of leupeptin, 10 µg/ml of aprotinin, 10 µg/ml of pepstatin A, and 0.1 mg/ml of soybean trypsin inhibitor). Following cell extraction, samples were centrifuged at 1000 x g at 4°C for 10 min to remove insoluble material. Resulting cell extracts were centrifuged at 120 000 x g at 4°C for 1 h. Protein concentration within the supernatant (cytosolic fraction) was detected by BCA assay and normalized between samples with the addition of buffer. Normalized cytosolic fractions were incubated with 20 µM taxol and 1 mM GTP for 20 min at 20°C. In vitro-assembled microtubules were pelleted by centrifugation at 30 000 x g at 20°C for 30 min. The remaining supernatant was incubated with taxol and GTP again as described above to increase the yield of assembled microtubules. The twice-assembled microtubule pellets were collected together. After complete washing with PEM buffer, the final pellets were suspended in 100 µl of PEM buffer to identify microtubules by immunocytochemistry and confocal microscopy. Presence of ß-tubulin, phosphorylated MAPK, and PP2A in pellets was also determined by Western blot analysis. As a negative control, F-actin was added to the cytosolic fraction, followed by no in vitro microtubule polymerization. After centrifugation, resulting pellets and supernatant were collected for detecting ß-tubulin, phosphorylated MAPK, and PP2A.
Statistics
Mixed regression models were used to test for overall group effect on the activity of PP1 and PP2A, adjusting for experiments. Group pairwise comparisons were tested utilizing the Tukey-Kramer multiple-comparison adjustment. All tests were performed at the 5% significance level.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both PP1 and PP2A activities were measured in oocytes at all stages of meiosis (GVI, GVBD, MI, AI, and MII) (Fig. 1). Protein phosphatase 2A was activated throughout the first meiotic division. Results from the mixed regression model indicated an overall group effect (P = 0.005). A significant elevation in PP2A activity occurred in MI compared to GVI (P = 0.008) and GVBD (P = 0.007) oocytes, concomitant with first spindle assembly. Protein phosphatase 2A activity in MII oocytes was similar to that in GVI/GVBD (P > 0.7) oocytes and was significantly decreased in comparison to that in MI oocytes (P = 0.018). As opposed to PP2A activity, PP1 activity remained very low throughout the first meiotic division and did not significantly change between the developmental stages examined. The mixed regression model found only a borderline group effect (P = 0.07). This borderline effect was most likely due to the borderline higher activity in the MI group as compared to the MII group (Tukey-Kramer adjusted P = 0.054). All other pairwise comparisons had no significant differences (P > 0.2). Using Western blot analysis, the presence of PP2A was detected in oocytes at all stages of meiosis (Fig. 2, top panel). The expression and activation of MAPK were also investigated in this study. As previously reported [20], we confirmed that ERK2, an isoform of MAPK, was present at a constant amount throughout the first meiotic division (Fig. 2, middle panel). Additionally, activity of MAPK changed with meiotic progression (Fig. 2, bottom panel). Mitogen-activated protein kinase was inactive in GVI and GVBD oocytes, was activated at the time of first spindle formation during MI, and remained activated throughout the MI/MII transition.
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Active MAPK and PP2A Colocalize with Spindles During Oocyte Meiosis
Because both MAPK and PP2A are highly activated at the time of meiotic spindle assembly, we suspected involvement in meiotic spindle formation. We next asked whether active MAPK and PP2A colocalize to the meiotic spindles. As shown in Figure 3 (top panel), active MAPK was absent in the GVI and GVBD oocytes. When oocytes developed to the MI and AI stages, MAPK was highly activated, and activated MAPK appeared to localize to microtubules and poles of first meiotic spindles. Activated MAPK also appeared to localize to plates of second meiotic spindles. Protein phosphatase 2A was present in the cytoplasm of GVI oocytes (Fig. 3, bottom panel). When oocytes reached MI, PP2A appeared to localize to microtubules of the first meiotic spindle. In addition, PP2A appeared to remain associated with the spindle apparatus at both AI and MII.
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To confirm whether both active MAPK and PP2A colocalize with spindles, the localizations of these proteins and ß-tubulin in MI, AI, and MII oocytes were investigated by double-labeled immunocytochemistry and confocal microscopy. As shown in Figure 4, active MAPK specifically localized to the microtubules of the first meiotic spindle in the MI and AI oocytes. When oocytes developed to the MII stage, a majority of active MAPK specifically localized to an area in close proximity to condensed chromosomes. Protein phosphatase 2A also specifically localized to the microtubules of meiotic spindles in the MI, AI, and MII oocytes (Fig. 5).
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Active MAPK and PP2A Associate with In Vitro-Polymerized Microtubules
Colocalization of both active MAPK and PP2A with microtubules of meiotic spindles suggests that these proteins may regulate spindle formation by associating with microtubules. Cytosolic fractions of HeLa cells were rich in depolymerized microtubules (Fig. 6A, left panel). When incubated with taxol and GTP, they repolymerized to microtubules and formed a pellet on centrifugation (Fig. 6A, center panel), whereas very little unpolymerized microtubules remained in the supernatant (Fig. 6A, right panel). Cytosolic fractions contained large amounts of ß-tubulin, active MAPK, and PP2A (Fig. 6B, lane 1). When cytosolic fractions were incubated with taxol and GTP, almost all the ß-tubulin was present as microtubules within the centrifuged pellet (Fig. 6B, lanes 2 and 3). Both active MAPK and PP2A were also present within in vitro-assembled microtubules of the pellet, although they also remained in the supernatant (Fig. 6B, lanes 2 and 3). When F-actin was used instead of in vitro-polymerized microtubules, neither ß-tubulin, active MAPK, nor PP2A were detected in the F-actin pellet (Fig. 6B, lane 4) but, rather, remained in the supernatant (Fig. 6B, lane 5). These results indicate that both active MAPK and PP2A associate with in vitro-polymerized microtubules.
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MAPK and PP2A Are Required for Spindle Formation During Oocyte Meiotic Maturation
The temporal activation of MAPK and PP2A and their specific localization in oocytes suggest that both proteins may locally regulate meiotic spindle formation by associating with microtubules. Thus, the question arises whether active MAPK and/or PP2A are required for meiotic spindle formation. To address this question, GVBD oocytes were incubated in a medium containing the MEK inhibitor (U0126) or the PP1 and PP2A inhibitors (OA or CL-A) for 5 h. As a control, GVBD oocytes were incubated in a drug-free medium for 5 h. As shown in Figure 7, MAPK activation was completely inhibited by 20 µM U0126. Conversely, inhibition of PP1 and/or PP2A by their specific inhibitors, OA and CL-A, induced an increase of MAPK activity. Five-hour incubation in control media resulted in normal microtubule polymerization, spindle formation, and chromosome condensation (Fig. 8). No spindles and very short microtubules around loosely condensed chromosomes were observed in oocytes treated with U0126, which completely inhibited MAPK activation. When the GVBD oocytes were treated with OA and CL-A, neither spindles nor short microtubules were present, and condensed chromosomes were disorganized. All these observations following OA and CL-A treatment occurred even though MAPK activity increased under this treatment condition.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mitogen-activated protein kinase associates with MTOCs during mouse oocyte meiotic maturation [24], yet whether this association involves active MAPK is unknown. In the present study, we confirmed that MAPK was activated at the time of meiotic spindle formation. Importantly, we found that activated MAPK colocalized with microtubules, poles, and plates of meiotic spindles, suggesting the presence of MAPK spacial regulation of meiotic spindle organization, function, and/or chromosome modifications. In addition, active MAPK associated with in vitro-polymerized microtubules, suggesting that MAPK may locally regulate spindle formation by associating with microtubules and/or microtubule-associated proteins. Oncoprotein 18 is a microtubule-associated protein that is phosphorylated at ser25 and ser38 by MAPK [11], and this phosphorylation turns off the microtubule-destabilizing activity of oncoprotein 18 [50]. Microtubule-associated proteins, such as microtubule-associated proteins-1 and -2 and tau, are also phosphorylated by MAPK [8, 9, 11] microtubule dynamics [51, 52]. Thus, MAPK may influence meiotic spindle formation by phosphorylating these microtubule-associated proteins that, in turn, regulate microtubule polymerization and/or depolymerization.
Protein phosphatase 2A comprises a family of serine/threonine PPs containing a common catalytic subunit and diverse regulatory subunits responsible for causing PP2A holoenzymes to target specific intracellular localizations and specific substrates [34]. Protein phosphatase 2A associates with microtubules of mitotic spindles and regulates cell cycle-dependent microtubule functions [40]. Protein phosphatase 2A also appears to be involved with oocyte meiotic maturation. Continual inhibition of PP1 and/or PP2A stimulates GVBD, yet oocytes fail to progress to MII due to meiotic spindle abnormalities [25–30, 53, 54]. However, transient inhibition of oocyte PP1 and/or PP2A enhances GVBD and permits proper spindle formation and development to MII [30]. In GVI oocytes, PP1 and PP2A predominantly localize to the nucleus and cytoplasm, respectively, indicating that these PPs may have distinct functions during oocyte meiosis [38].
In the present study, PP1 activity was very low and without marked change throughout the first meiotic division. In contrast, PP2A was active in GVI oocytes, slightly decreased when GVBD occurred, increased at the time of first meiotic spindle formation, and decreased again when oocytes reached MII. Protein phosphatase 2A, but not PP1, colocalized with microtubules of meiotic spindles, indicating that PP2A may locally regulate microtubule polymerization. Protein phosphatase 2A also associated with in vitro-polymerized microtubules, suggesting that PP2A regulates spindle formation by associating with microtubules and regulating the phosphorylation of microtubule-associated proteins. Microtubule-associated protein tau is dephosphorylated by PP2A [7]. Therefore, PP2A may regulate meiotic spindle formation through dephosphorylating tau or some unidentified microtubule-associated protein(s). Identification of the microtubule-associated protein(s) mediating PP2A-regulated spindle formation remains to be elucidated.
Data strongly suggest that both MAPK and PP2A are regulators of spindle formation based on their activation at the time of spindle formation and localization to microtubules of developing spindles. In general, a proper balance between protein kinases and phosphatases is necessary for microtubule polymerization. Disruption of this balance will cause aberrant or no spindle assembly [7]. In this regard, the complete inhibition of MAPK activity by U0126 resulted in no normal spindle formation and only very little microtubule polymerization surrounding loosely condensed chromosomes, suggesting that MAPK is required for meiotic spindle formation and chromosome condensation. When oocytes were treated with OA and CL-A, neither spindles nor polymerized microtubules were observed, even though MAPK activity increased under these conditions. This increase in MAPK phosphorylation following OA/CL-A treatment is similar to previously reported effects of OA on MAPK electrophoretic mobility in oocytes gaining meiotic competence [55]. As mentioned previously, OA and CL-A inhibit both PP1 and PP2A. Although specific concentrations of OA and CL-A can be used in cell-free extracts to differentially inhibit PP1 or PP2A, such an approach is not feasible in intact cells.
We have concluded that the observed effect of OA and CL-A on oocyte spindle formation and microtubule polymerization is mediated through PP2A, not through PP1, for the following reasons. First, PP2A, but not PP1, colocalized and associated with developing microtubules. Close association of PPs and their substrates is essential in regulation of reversible phosphorylation and cell function [56]. Second, PP1 activity is low and unchanged during progression through meiosis, when microtubule polymerization and spindle formation are occurring. On the other hand, PP2A activity increased at the time of microtubule polymerization and spindle formation. Third, PP2A, not PP1, has been reported previously to associate with and to regulate mitotic microtubule polymerization [40]. Therefore, data presented in this report suggest that MAPK activation alone is not enough to support normal meiotic spindle formation, and that PP2A activity is absolutely necessary.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH grant HD35125 (to G.D.S). 
2 Correspondence: Gary D. Smith, 6428 Medical Sciences Building I, 1301 E. Catherine St., Ann Arbor, MI 48109-0617. FAX: 734 647 1006; smithgd{at}umich.edu
Accepted: August 9, 2001.
Received: February 6, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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