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Regular Article |
a Center for Research on Reproduction and Women's Health and Department of Obstetrics & Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cyclic adenosine monophosphate, kinases, meiosis, oocyte development, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The signal(s) that initiates oocyte maturation in vivo is thought to come from cumulus cells that are intimately connected to the oocyte plasma membrane via gap junctions [4]. Oocyte cAMP is generated in part by an oocyte adenylyl cyclase and in part by transit of cAMP from the cumulus cells across these gap junctions [5]. In addition to alterations in cAMP levels within the oocyte, there are alterations in the phosphorylation status of a number of oocyte proteins during the time of commitment to the resumption of meiosis [6, 7]. PKA is responsible for at least some of the critical phosphorylation events, as experimental inhibition of PKA activity within the oocyte prevents meiotic maturation [7].
While it is clear that the PKA signaling pathway is important, it is not known how PKA activity within the oocyte is regulated such that only the appropriate downstream substrates are phosphorylated. Temporal regulation of PKA in cells is provided by cAMP that is generated by adenylyl cyclases and degraded by cAMP phosphodiesterases. The kinase also is regulated spatially by compartmentalization via A-kinase anchor proteins (AKAPs) [8], ensuring that PKA can act locally and does not have to rapidly diffuse to its physiological substrates. AKAPs tether PKA through the regulatory subunits to various cellular structures, placing the kinase in close proximity with both its upstream effector molecules and downstream targets. By compartmentalizing PKA, pockets of enzymatic activity are created in specific regions of the cell. This type of regulation is particularly important in large cells with copious amounts of cytoplasm, such as oocytes and eggs.
While a number of AKAPs have been described in ovarian follicles [9, 10], little is known about the presence and role of AKAPs in oocytes. One AKAP, ezrin, is expressed in oocytes and eggs [11–13]. Ezrin is a member of a group of scaffolding proteins known as ERM proteins (for ezrin, radixin, moesin) that may regulate interactions between the cortical cytoskeleton and plasma membrane [12, 14].
In order to gain insight into the mechanisms by which cAMP and PKA control the meiotic status of oocytes, we have immunolocalized the PKA subunits within meiotically arrested and maturing oocytes. Regulatory subunits (RI, RII) and catalytic (C) subunits all were observed and showed a polarized distribution in both oocytes and eggs, suggesting the presence of scaffolding proteins such as AKAPs. In addition, the PKA subunits were redistributed during oocyte maturation. We identified a prominent AKAP, AKAP140, which was developmentally regulated in oocytes and eggs. AKAP140 bound RI and RII, indicating that it may function as a dual-specific AKAP in these cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animals used in this study were maintained in accordance with the National Academy of Science Guide for the Care and Use of Laboratory Animals. Female CF-1 mice (6–8 wk old) were obtained from Harlan Sprague-Dawley (Indianapolis, IN). Germinal vesicle (GV)-intact oocytes and metaphase II-arrested eggs were collected from superovulated females as previously described [15]. The collection medium was modified Whitten [16] containing 15 mM Hepes, 7 mM NaHCO3, 10 µg/ml gentamicin, and 0.01% polyvinyl alcohol. For oocyte collection, the medium was supplemented with 0.25 mM dibutyryl cAMP to inhibit resumption of meiosis [6]. For maturation in vitro, oocytes were washed free of dibutyryl cAMP and cultured in CZB medium [17] in 5% CO2 for 2 h (GVBD stage), 6 h (metaphase I), or 16 h (metaphase II). In some cases, oocytes or eggs were cultured in CZB containing cytochalasin D (5 µg/ml; Sigma, St. Louis, MO), colchicine (10 µM; Sigma), or both reagents for 4 h prior to immunofluorescence processing. For maturation in vivo, oocytes were collected from superovulated mice at various times after hCG administration. In these experiments, the cumulus cells were mechanically stripped from the oocytes, the zona pellucida was removed, and the oocytes were fixed within 15 min of ovarian dissection.
Immunofluorescence Procedures
Immunofluorescence was performed as previously described [18]. Briefly, the zona pellucida was removed using acid Tyrode medium, then the cells were fixed in 3.7% paraformaldehyde and permeabilized with 0.1% Triton X-100. Primary antibodies used at the indicated final concentrations were mouse anti-PKA RI (25 µg/ml; Transduction Laboratories, Lexington, KY), rabbit anti-PKA RII (50 µg/ml; courtesy of C. Rubin, Albert Einstein Medical College, New York, NY) [19], rabbit anti-PKA RII (20 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit anti-PKA C
(2 µg/ml; Santa Cruz Biotechnology) that also immunoreacts with the Cß and C
isoforms. Nonimmune IgGs obtained from Jackson Immunoresearch (West Grove, PA) were used at the same final concentrations as the experimental primary antibody. As a control for PKA C staining, the antibody was incubated for 2 h with a 5-fold molar excess of specific blocking peptide prior to use. Actin was stained with phalloidin-FITC (0.8 µM; Sigma), and DNA was stained with 4',6-diamidino-2-phenylindole (DAPI, 1.5 µg/ml; Sigma). Secondary antibodies were anti-rabbit Alexa Fluor 488 (4 µg/ml; Molecular Probes, Eugene, OR), anti-mouse Alexa Fluor 633 (4 µg/ml; Molecular Probes), or Cy5-conjugated anti-rabbit IgG (3 µg/ml; Jackson Immunoresearch). Images were recorded using a Leica TCS confocal microscope. Unless otherwise indicated, all images within a single figure were taken using identical excitation and detection voltages.
Protein Preparation and RII Blot Overlay Assays
Isolated oocytes and eggs were solubilized directly in reducing SDS sample buffer. Protein from ELN cells (immortalized mouse granulosa cells) grown in culture was extracted and quantified according to standard protocols. Blot overlay assays were then performed as previously described [20]. Briefly, bovine heart RII subunit (Promega, Madison, WI) was labeled by incubation with PKA C subunit (Promega) and [32P]ATP. Residual [32P]ATP was separated from the labeled RII by passing the mixture through a Centri-sep column (Princeton Separations, Inc., Adelphia, NJ). Proteins were separated by SDS-PAGE on gels containing 8% (w/v) polyacrylamide and transferred to Immobilon-P. The membranes were blocked in Blotto/BSA (5% nonfat dry milk, 0.1% BSA, 150 mM NaCl, 0.02% NaN3, 10 mM potassium phosphate buffer, pH 7.4) before the addition of [32P]-RII (65 000 cpm/ml of Blotto/BSA). After incubation, blots were washed, dried, and then quantified using a Molecular Dynamics STORM system (Molecular Dynamics, Sunnyvale, CA). Results were analyzed for differences in RII binding by the Kruskal-Wallis test and for differences between specific groups by the Mann-Whitney U-test using StatView 4.0 software (Abacus Concepts, Berkeley, CA).
Immunoprecipitation with Anti-PKA RI Antibody
Immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF, 0.5% NP-40, 0.02% NaN3) was added to 600 isolated oocytes. The resulting oocyte extract was precleared using Protein A Sepharose (Sigma) and divided into two equal volumes. Two micrograms of either monoclonal anti-PKA RI antibody (Transduction Laboratories) or nonimmune mouse IgG (Jackson Immunoresearch) was added, and the samples were tumbled at 4°C for 2 h. Following the addition of 5 µg rabbit anti-mouse IgG (Jackson ImmunoResearch), the 4°C incubation continued overnight. After centrifugation, the precipitated proteins were washed using immunoprecipitation buffer, resuspended in reducing SDS sample buffer, and subjected to an RII blot overlay.
Phosphatase Treatment
Equal numbers of oocytes or eggs (200 cells per group) were lysed in 50 mM Tris-HCl, pH 8.5, 1.1 mM EDTA, 0.125 mM MgCl2, 0.025 mM ZnCl2, and Complete Protease Inhibitor Cocktail (Boehringer Mannheim, Indianapolis, IN). The lysates were incubated at 37°C for 30 min after the addition of 1) 45.3 U calf intestinal alkaline phosphatase (CIAP; Gibco BRL, Grand Island, NY), 2) no CIAP, or 3) CIAP and 1 mM sodium vanadate (to inhibit CIAP activity). The reaction was stopped by boiling in reducing SDS sample buffer, and the lysates were subjected to an RII blot overlay.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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While PKA can be soluble in somatic cells, it also can be sequestered by AKAPs, forming pockets of PKA activity in specific regions of the cell. The oocyte is a large cell with an abundant amount of cytoplasm, suggesting that PKA is localized to specific sites within this cell where it can be in close proximity with its physiological substrates. Using immunofluorescence techniques, RI showed a polarized distribution in both oocytes and eggs, as it was present at the cortical region and plasma membrane (Fig. 1). At metaphase I and metaphase II, RI also was seen at the meiotic spindle (Fig. 1, B and C), suggesting that the shifts in RI localization are functionally important. In the cortical region overlying the chromatin (that coincides with the amicrovillar region of the plasma membrane), there was less intense staining for RI than in the remainder of the cortex (data not shown).
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The cortical position of RI suggested that its localization might be mediated by the actin cytoskeleton that is particularly extensive in this region. Disruption of the actin cytoskeleton with cytochalasin D did not affect the cortical RI localization in oocytes or eggs (Fig. 2, B and F). Likewise, disruption of the microtubular cytoskeleton using colchicine had no effect on RI localization (Fig. 2, C and G). When oocytes and eggs were treated with cytochalasin D and colchicine together to disrupt both the actin and microtubular cytoskeletons, RI still was found only in the cortex (Fig. 2, D and H). Under these treatment conditions, actin was found in both cortical and noncortical areas and the meiotic spindle was disrupted, indicating that these drugs were effective. Obviously, as colchicine disrupts the meiotic spindle, RI was no longer seen in association with this structure in colchicine-treated eggs (Fig. 2, G and H).
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Previously, photoaffinity labeling of the PKA regulatory subunits with 8-N3-[32P]cAMP demonstrated that RI is the only regulatory subunit detectable in oocytes [21]. Because no other mammalian cell type has been reported to lack RII, we wanted to confirm this finding by an independent method. RII mRNA was identified in oocytes by reverse transcription-polymerase chain reaction techniques (data not shown). In addition, RII protein was observed by immunofluorescence staining in both oocytes and eggs; this staining was confirmed using a second anti-RII antibody. In the oocyte, it showed a punctate distribution within the cytoplasm but did not have a plasma membrane or cortical localization like RI (Fig. 3A). At meiosis I and meiosis II, RII was redistributed to the region surrounding, but not corresponding to, the meiotic spindle (Fig. 3, B and C). Nonimmune IgG controls had minimal background staining (Fig. 3D).
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In GV-intact oocytes, C was present in the cortical region and at the plasma membrane (Fig. 4A), in a pattern similar to RI but not RII. In metaphase I oocytes, C localization in the cortex was diminished compared with GV-intact oocytes, and staining of C in the cytoplasm had increased (Fig. 4B). In eggs, the distribution of RI and C differed while RII and C had similar patterns. C was redistributed to two locations (Fig. 4C). First, it was found diffusely in the cytoplasm. Second, it appeared to be present around, but not at, the meiotic spindle. In order to determine how these changes in C distribution occurred during oocyte maturation in vivo, we collected oocytes at various times after hCG administration to eCG-primed mice. At 1.5 h after hCG, the localization of C did not differ from that in oocytes not undergoing maturation (compare Figs. 4A and 5A). However, by 2.5 h after hCG, an increase in C immunoreactivity was seen in the center of the oocyte in the region surrounding the condensing chromatin (Fig. 5C). In these oocytes, the GV could no longer be seen by light microscopy. At 3 and 3.5 h after hCG, the amount of C in the oocyte cortex was decreased and the enrichment of C in the center of the oocyte was no longer seen (Fig. 5, D and E).
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To determine if this shift in localization of C to the GV was mediated by either RI or RII, we repeated the same in vivo maturation experiment but divided the maturing oocytes into three groups for immunofluorescence staining for C, RI, or RII. The RI and RII immunofluorescence staining patterns during maturation in vivo did not differ from those seen during in vitro maturation (data not shown). At the same time points that C was present in the GV, there was no indication that either RI or RII was present in this location, suggesting that neither of these proteins was responsible for relocalizing C to the GV.
AKAPs Present in Mouse Oocytes and Eggs
The presence of RI, RII, and C in restricted regions of oocytes and the movement of these proteins during oocyte maturation suggest that the PKA subunits are preferentially localized. Because association with actin or tubulin did not appear to mediate this localization, we examined whether AKAPs were present in oocytes and eggs and could potentially serve to anchor PKA in the different regions of these cells. One prominent Mr 
140 000 protein (to be called AKAP140) was identified based on its ability to bind RII (Fig. 6A). While an AKAP of a similar size has been identified in rat granulosa cells [22], AKAP140 had a different apparent molecular size from all AKAPs observed in mouse granulosa cells (Fig. 6A). A less intense RII-binding band of Mr 37 000 also was present, as well as several other faint bands.
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AKAP140 was developmentally regulated in the oocyte and egg. RII binding to AKAP140 appeared to be high in oocytes and slightly lower in eggs (Fig. 6A). The relative changes in RII binding between oocytes and eggs could reflect changes in protein expression of AKAP140 and/or changes in ability to bind RII. In addition, the apparent molecular weight of AKAP140 appeared to increase slightly between oocytes and eggs (Fig. 6A), suggesting a posttranslational modification of the protein (see below).
AKAP140 Interactions with the RI Subunit In Vivo
AKAP140 was identified as an AKAP by its ability to bind RII in vitro. However, because oocytes and eggs contain both RI and RII, we determined whether AKAP140 could bind RI. When an oocyte protein extract was immunoprecipitated with anti-RI antibody and the precipitated protein probed with radiolabeled RII on a blot overlay assay, AKAP140 was the only band detected (Fig. 6B, lane 1). This was a specific interaction, as control nonimmune IgG did not precipitate AKAP140 (Fig. 6B, lane 2). This result indicates that AKAP140 may serve as a dual-specific AKAP, localizing PKA via either RI or RII subunits in these cells.
AKAP140 RII-Binding and Phosphorylation Status During Oocyte Maturation
The increase in the apparent molecular weight of AKAP140 during oocyte maturation suggested that the protein was posttranslationally modified (Fig. 6A). In order to examine the timing of this modification, we collected oocytes at various times during maturation and performed RII blot overlays. A shift in size of AKAP140 occurred in oocytes that had undergone GVBD, and a further increase occurred by metaphase II (Fig. 7A). In addition to the size shifts, we noted significant differences in RII binding to AKAP140 at the different stages of maturation (Fig 7B). RII binding had increased by GVBD, was maximal at metaphase I, and decreased again at metaphase II (Fig. 7B).
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After treating protein from oocytes and eggs with calf intestinal alkaline phosphatase, AKAP140 migrated slightly faster, suggesting that it was phosphorylated (Fig. 8). The effect of phosphatase treatment was abrogated in the presence of vanadate (data not shown), indicating that the difference was due to phosphatase activity. There was no obvious correlation between RII binding and AKAP140 phosphorylation, i.e., the unphosphorylated oocyte protein bound less RII while the unphosphorylated egg protein bound more RII than their phosphorylated counterparts. Of note, the dephosphorylated egg AKAP140 protein still migrated more slowly than the untreated oocyte AKAP140 (Fig. 8). One explanation for this is that the phosphatase treatment may not have completely dephosphorylated the egg proteins. Alternatively, a posttranslational modification of AKAP140 in addition to phosphorylation could occur during maturation and be responsible for maintaining a small shift in apparent size after phosphatase treatment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our work showing cortical localization of PKA RI and C subunits prior to meiotic maturation demonstrates that the kinase is positioned appropriately for it to respond to cAMP coming from the cumulus cells to the oocyte. Local elevation of cAMP in the cortex would cause the release of active C from RI tethered in this region. Such spatial regulation could sequester PKA near downstream substrates in the cortex during the period of meiotic arrest and might also prevent degradation of cortical cAMP by cytoplasmic phosphodiesterases prior to the activation of the kinase. Redistribution of C to the region of the GV at the time of GVBD would allow for phosphorylation of proteins normally present in nuclei, e.g., the cAMP response element binding protein. The fact that neither RI nor RII was found in the GV at the same time as C suggests that the C relocalization was a result of cAMP-mediated release of C from the R subunits. This is consistent with the finding that cAMP is a positive mediator of gonadotropin-induced reinitiation of meiotic maturation in preovulatory follicles [23] and supports the idea that, while sustained elevated levels of cAMP maintain oocytes in meiotic arrest, a transient increase may trigger GVBD [24]. Subsequent movement of RI, RII, and C to the spindle and surrounding regions indicates a possible role for PKA in meiosis. Similar to RI, other signaling molecules, e.g., MAP kinase, polo-like kinase 1, and protein kinase C [25–28], move to the spindle region during meiosis. The localization of RI at the meiotic spindle is consistent with a similar localization observed during mitosis and suggests that PKA could have a role in a number of mitotic functions, e.g., centrosome duplication and/or segregation, sister chromatid separation, or cytokinesis [29]. Furthermore, the phosphorylation of the centrosomal protein, centrin, by PKA during mitosis has been proposed to signal the separation of centrosomes at prophase [30]. Whether PKA has similar roles during meiosis awaits further examination.
The restricted distribution of the PKA subunits suggests that they are sequestered in subcellular locations by specific proteins. In this regard, a prominent AKAP, AKAP140, was present in oocytes and eggs. The difference in apparent size between AKAP140 and granulosa cell AKAPs [9] suggests that these are distinct proteins. It remains possible, however, that alternative splicing or posttranslational modification in the two cell types could be responsible for these differences. In fact, several AKAPs are known to have various splice forms [8] and/or to be posttranslationally modified [31–34]. The identification of AKAP140 is being pursued, but we have not yet determined its primary sequence. Immunoblot analyses using antibodies to known AKAPs, e.g., AKAP-KL [32] and MAP-2 [35], were negative (data not shown). In addition to AKAP140, several other relatively weak bands were seen on the RII overlay that likely represent additional AKAPs present in oocytes and eggs. The mobility of one of these bands was consistent with the known size of ezrin, a Mr 78 000 AKAP previously identified as being present in oocytes [11]. The presence of several AKAPs in these cells may be important for the different spatial localizations of RI and RII at different developmental stages.
AKAP140 bound both RI and RII, indicating that AKAP140 is a dual-specific AKAP similar to AKAP1, AKAP4, and AKAP10 [36–38]. Although the relative affinity of AKAP140 for RI and RII is not known, mammalian AKAPs typically bind RII with a higher affinity [39, 40]. However, there may be mammalian AKAPs that bind RI with high affinity, similar to a Caenorhabditis elegans AKAP [41]. Within a particular dual-specific AKAP, RI-binding sites may overlap with known RII-binding sites or they may be distinct [36, 38]. It is not known if AKAP140 in oocytes and eggs binds both RI and RII or preferentially localizes only one of these subunits in vivo. Other AKAPs present in these cells also may modulate differential localization of the regulatory subunits.
A number of AKAPs are phosphorylated [33, 42], suggesting that this posttranslational event may modulate PKA regulatory subunit binding. AKAP140 increases in apparent molecular weight during oocyte maturation, and this increase is at least in part due to phosphorylation. Of interest, two-dimensional gel electrophoresis experiments have shown that a protein similar in size to AKAP140 is phosphorylated during commitment to GVBD [6, 7]. If AKAP140 phosphorylation affects RI and/or RII binding, the localization of PKA might be altered by this posttranslational modification in oocytes. Obviously, the distribution of PKA will influence its ability to phosphorylate target substrates.
Differences in RII binding were observed depending on the stage of oocyte maturation and the phosphorylation status of AKAP140. The correlation between AKAP140 phosphorylation and RII binding was dependent on the source of protein. When oocyte proteins were treated with phosphatase, AKAP140 bound less RII. However, when egg proteins were treated similarly, more RII was bound by AKAP140. These findings could be explained if AKAP140 phosphorylation at discrete sites in oocytes as compared with eggs results in differences in RII-binding ability. Further studies on the effects of phosphorylation await a more complete characterization of AKAP140, including mapping of the phosphorylation sites and examination of the phosphorylation of these sites at the different stages of oocyte maturation.
In addition to anchoring PKA, AKAPs can bind other protein kinases, phosphatases, and phosphodiesterases [8, 43], thereby serving as a scaffold for the localization of multiple proteins. These protein complexes can coordinately regulate the phosphorylation state of substrate proteins in specific subcellular compartments. For example, local degradation of cAMP by a phosphodiesterase tethered to an AKAP could result in the inactivation of C in that specific region. The recent identification of a nontransmembrane adenylyl cyclase [44] suggests that cAMP also could be produced in cellular regions away from the plasma membrane and cortex, providing a mechanism for maintaining C activity in the cytoplasm and the area surrounding the meiotic spindle. Thus, the localization and redistribution of AKAPs including AKAP140 in oocytes and eggs would spatially regulate PKA activity and possibly other signaling molecules in these cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a Howard Hughes Medical Institute Research Training Fellowship to R.L.B., a Burroughs Wellcome Fund Career Award in the Biomedical Sciences to C.J.W., and NIH PO1 HD06274 to S.B.M. Part of this work was done in the Intermediate Voltage Electron Microscopy and Biomedical Image Analysis Facility at the University of Pennsylvania, supported by NIH grant RR-2483. 
2 Correspondence: Carmen J. Williams, Center for Research on Reproduction & Women's Health, 1313 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6142. FAX: 215-573-7627; cjwill{at}mail.med.upenn.edu
3 Current address: Department of Medicine, University of Chicago Hospitals, 5841 S. Maryland Ave., Chicago, IL 60637-1419
4 These authors contributed equally to the work described in this manuscript
Accepted: April 18, 2002.
Received: December 28, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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