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a Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
b Institute of Zoology, Chinese Academy of Sciences, Beijing, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, PKC-ßI, and PKC-ßII, as well as RACK1, are expressed in mouse oocytes (germinal vesicle [GV]) and mature eggs (metaphase II [MII]). In GV oocytes, PKC-, PKC-ßII, and RACK1 were uniformly distributed in the cytoplasm, while PKC-ßI was localized in the cytoplasm and in the plasma membrane as well. Treatment of GV oocytes with the biologically active phorbol ester, 12-o-tetradecanoyl phorbol-13-acetate (TPA), resulted in a rapid translocation of the cytosolic PKC-, but not PKC-ßI, PKC-ßII, or RACK1, to the plasma membrane. This was associated with inhibition of GV breakdown. In MII eggs (17 h post-hCG), PKC- was uniformly distributed in the cytoplasm while PKC-ßI and -ßII were distributed in the cytoplasm and in the plasma membrane as well. Treatment with TPA resulted in a rapid translocation of PKC- from the cytoplasm to the plasma membrane and a significant decrease of PKC-ßI throughout the cytoplasm, while it also remained in the cell periphery. No change in the distribution of PKC-ßII or RACK1 was observed. TPA also induced pronucleus formation. Physiological activation of MII eggs by sperm induced cortical granule exocytosis associated with significant translocation of PKC- and -ßI, but not -ßII, to the plasma membrane. Overall, these results suggest a possible involvement of cPKC isoforms in the mechanism of mouse oocyte maturation and egg activation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ß, and 
, which require Ca2+ and diacylglycerol (DAG) for maximal activity [3]; 2) novel (nPKC) 
, 
, 
, µ, and 
isoforms, which are Ca2+ independent but require DAG [4–7]; and 3) atypical (aPKC) 
, 
, and 
, which are Ca2+ and DAG insensitive [8, 9]. Expression of individual PKC isoforms depends on the cell type and on its developmental stage [10]. Stimulation by specific hormones or by phorbol ester leads to translocation of some of the PKCs to new subcellular sites where they phosphorylate their specific target proteins [11]. Recent studies have shown that activated PKC isoforms bind to anchoring proteins termed RACKs [12–14], which are thought to be positioned in close proximity to the PKC substrate. Moreover, the functional specificity of the PKC isoforms are determined, in part, by the differing localization of the isoform-specific RACK anchors [15].
The involvement of PKC in mammalian egg activation has been previously suggested. Pharmacological agents such as phorbol esters or synthetic diacylglycerols have been used in some studies to successfully mimic events of egg activation, such as cortical granule exocytosis [16–19] and second polar body initiation [20, 21]. Conversely, inhibition of PKC by antagonists prior to activation results in inhibition of these events [16, 17]. However, pharmacological studies using PKC activators or inhibitors on mammalian eggs have yielded inconsistent results. It has been suggested that phorbol esters induce initiation of second polar body-like structures in hamster eggs, while in mouse eggs such structures could not be detected [22]. In mouse oocytes, meiotic resumption was inhibited in the presence of phorbol esters [23]. Recently, Gangeswaran and Jones [24] showed the presence of PKC- and PKC- in mouse oocytes, but they failed to detect cPKC isoforms although these isoforms have been detected in rat eggs [25]. Since early fertilization events are accompanied by a rise in intracellular calcium [26, 27], which in turn stimulates the exocytosis of egg cortical granules (CGs) [28], we assumed that cPKCs are likely to participate in these processes. Two questions were addressed in this study. First, are cPKC isoforms expressed in mouse oocytes and eggs? Second, which of these isoforms are activated during egg stimulation by phorbol ester and sperm penetration? In this study we show that cPKCs are expressed and selectively activated during oocyte maturation and egg activation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovarian oocytes at GV stage were released from antral follicles of 4- to 6-wk-old BALB/c mice into defined M16 medium supplemented with BSA (1 mg/ml). This medium contains all factors and nutrients known to be required for in vitro maturation, including Ca2+ (2 mM). The cumulus cells were removed by gentle pipetting through a narrow-bore glass pipette.
Cumulus cell-enclosed MII-arrested eggs were obtained from 8-wk-old BALB/c mice. Females were superovulated by injection of 10 IU of eCG; 48 h later they were injected with 10 IU of hCG. Mice were killed by cervical dislocation at 12–14 h post-hCG for fertilization or 17 h post-hCG for 12-o-tetradecanoyl phorbol-13-acetate (TPA) treatment. The cumulus cell masses were released from the oviducts. The cumulus cells surrounding the eggs were removed by brief exposure to 300 IU/ml hyaluronidase (H4272; Sigma Chemical Co., St. Louis, MO), and the eggs were immediately washed 3 times with M16 medium (Sigma M7292) supplemented with 1 mg/ml BSA (Sigma A2153). Sperm cells were obtained from the caudal epididymis and vas deferens of 14- to 16-wk-old BALB/c mice and capacitated (1 x 106 cells/ml) for 1–2 h in M16 medium with BSA (3 mg/ml) at 37°C in a humidified atmosphere of CO2 (5%) and air (95%).
Electrophoresis and Immunoblotting
Oocytes or eggs (as indicated) were lysed in SDS sample buffer [29] and boiled for 5 min. After cooling on ice and centrifuging at 13 000 x g for 5 min at 4°C, samples were frozen at -70°C until use.
The proteins were separated by SDS-PAGE with a 4% stacking gel and a 10% separating gel (50 min at 188 volts) and then electrophoretically transferred onto a Protran nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) for 1 h (200 mA, 4°C). After blocking for 1 h with low-fat milk (1%), the membrane was incubated overnight at 4°C with polyclonal isotype-specific anti-PKC antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1000 in TBS (137 mM NaCl, 20 mM Tris, pH 7.6) or with monoclonal anti-RACK1 antibodies (Transduction Laboratories, Lexington, KY). After 3 washes of 15 min each in TBS-T (TBS supplemented with 0.1% Tween 20), the membrane was incubated for 1 h at room temperature with donkey anti-rabbit IgG-horseradish peroxidase-labeled antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:10 000 in TBS-T, or with anti-IgM-ADS (AFF)-Perox-labeled antibodies (The Binding Site, Birmingham, UK) diluted 1:1000 in TBS-T, respectively. The membrane was washed 3 times in TBS-T and then processed using the enhanced chemiluminescence super signal detection system (Amersham, Little Chalfont, UK). Specificity of the anti-PKC antibodies was confirmed by preabsorbing the antibody with its peptide antigens for 1 h before incubating the antibody with the nitrocellulose membrane.
Experimental Treatments of Oocytes and Eggs
Stock solution of TPA was prepared in dimethyl sulfoxide. TPA-treated oocytes or eggs were incubated for 4 h for GV breakdown (GVBD) evaluation or for 8 h for pronuclear (PN) formation. GVBD was evaluated under a stereomicroscope, and PN formation was evaluated under an inverted phase-contrast microscope.
For the fertilization experiments, eggs collected at 12–14 h post-hCG administration were removed from the oviducts as described above and cultured with 106 capacitated sperm/ml. In order to identify CGs, eggs at differing times of fertilization were fixed and stained with Lens Culinaris agglutinin (LCA) coupled to biotin followed by incubation with fluorescein isothiocyanate (FITC)-streptavidin-conjugated antibody (Vector Labs., Burlingame, CA) according to Cherr et al. [30] and Ducibella et al. [31]. Briefly, eggs were fixed with paraformaldehyde (4%) followed by blocking and permeabilization (1% BSA, 0.2% Triton 15 min, respectively). The zona pellucida was removed mechanically after fixation, and the eggs were double-stained for PKC isoforms and CGs evaluation (see below).
Immunohistochemistry
Oocytes and eggs were fixed for 30 min (37°C) with paraformaldehyde (4% in PBS). The fixed cells were permeabilized by treatment with 0.2% Triton (37°C) for 15 min, followed by two washes in PBS and blocking in 1% BSA for 30 min. After fixation the zona pellucida was removed mechanically. For double staining, oocytes and eggs were incubated overnight at 4°C with both polyclonal anti-PKCs (1:100) and mouse monoclonal anti-RACK1 (1:100; Transduction Laboratories). The antibodies were detected using rhodamine-labeled anti-rabbit IgG and FITC-labeled anti-mouse IgM, respectively, both diluted 1:500. For double staining of PKC isoforms and CGs, eggs were incubated with both polyclonal anti-PKCs and LCA-biotin (1:100) overnight. After extensive washes, eggs were incubated with rhodamine-labeled anti-rabbit IgG and FITC-streptavidin (1:500). Cells were mounted on slides using GVA-mount (Zymed, San Francisco, CA). Nonspecific staining was determined by incubation without primary antibody or in the presence of an excess of the appropriate peptide. The meiosis stage of each oocyte or egg was determined from its chromatin configuration by staining with 10 µg/ml Hoechst 33258 (Molecular Probes, Eugene, OR).
Oocytes were examined under a confocal laser scanning microscope (Bio-Rad MRC-1024; Richmond, CA) equipped with argon-krypton laser. In each experiment at least 10 oocytes were examined, and each was repeated 3 times.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To establish the presence of PKC isoforms and RACK1 in mouse oocytes, SDS-extracted proteins were separated by SDS-PAGE, subjected to Western immunoblot analysis, and detected by isozyme-specific antipeptide antibodies. We detected PKC- (Fig. 1A) and PKC-ßI (Fig. 1B) in GV oocytes and MII eggs. The antibody preparation directed against PKC- or -ßI specifically recognized a single protein of approximately 80 kDa. The specificity of the antibody was demonstrated by its preabsorption with the specific peptide antigen, which completely abolished immunodetection of the 80-kDa protein band (data not shown). The amount of PKC- derived from the same number of cells was higher in GV oocytes compared to MII eggs, while the amount of PKC-ßI derived from the same number of cells was consistently higher in MII eggs as compared to GV oocytes. To determine whether the receptor for activated PKC, RACK1, is also present in oocytes, proteins extracted from GV oocytes were subjected to Western immunoblot with a RACK1-specific antibody (Fig. 2). We detected a single band of the expected apparent mass (36 kDa) in GV oocyte preparations. In order to avoid contamination by other cell types such as ovarian, follicular, or cumulus cells of our oocyte preparations, extensive washing and careful monitoring were applied to each oocyte preparation.
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Thus, mouse GV oocytes and MII eggs expressed cPKC isoforms as well as the PKC-binding protein RACK1.
Effects of TPA on Distribution of PKCs and Oocyte Maturation
Further confirmation of the presence of PKC-, PKC-ßI, PKC-ßII, and RACK1 in GV oocytes was established by immunocytochemical staining followed by the subcellular localization of these proteins in mouse GV oocytes stimulated by phorbol ester. Fixed GV oocytes were incubated with primary PKC-, -ßI, -ßII, or RACK1 antibodies followed by fluorescently labeled second antibodies, and the cells were examined by confocal microscopy. Nonstimulated GV oocytes exhibited cytosolic staining for PKC-, -ßI, -ßII, and RACK1. No staining was seen in the GV (Fig. 3, B and C). Antibodies preabsorbed with the specific peptide antigen did not stain the cells (Fig. 3A). TPA is frequently used to activate Ca2+-dependent PKC isoforms. Therefore, we treated GV oocytes with 100 ng/ml TPA and followed its effects on cPKC distribution. After TPA treatment, PKC- staining was eliminated from the cytoplasm and localized to the cell periphery, suggesting its translocation to the plasma membrane and its activation (Fig. 3C). After TPA treatment, PKC-ßI, -ßII, or RACK1 did not show any significant change in their distribution (Fig. 3C). Furthermore, treatment with TPA inhibited the spontaneous resumption of meiotic maturation (GVBD). We found that 88% of control oocytes underwent spontaneous GVBD, whereas after treatment with TPA, only 13% of the oocytes underwent GVBD (Fig. 4). Detection of GVBD was evaluated by the disappearance of the GV. Since PKC- was the only kinase that responded to TPA treatment, it is possible that GVBD inhibition is modulated by PKC- activation.
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Effects of TPA on Distribution of PKC Isoforms and Egg Activation
Nonstimulated MII eggs stained with the various anti-PKC antibodies exhibited cytoplasmic staining for the three PKC isoforms and RACK1, while for PKC-ßI and -ßII, staining was also seen in the cell periphery (Fig. 5A). Activation of these eggs with TPA for 5 min revealed a significant depletion of staining for PKC- and -ßI from the cytoplasm and a prominent pattern of staining throughout the plasma membrane. No significant change in the distribution of PKC-ßII and RACK1 was observed (Fig. 5B). Antibodies preabsorbed with the specific peptide antigen did not stain the cells (data not shown). Under the same conditions, TPA also induced interphase transition in eggs. As compared to the value in unstimulated eggs, TPA (100 ng/ml) increased PN formation from 4.1% to 58.3% (Fig. 6). The activation of mouse eggs by TPA was dependent on the egg's age [32]; optimal results were obtained when eggs were isolated 17 h post-hCG administration (Fig. 6). Together, these results suggest the possible involvement of PKC- and -ßI, but not PKC-ßII, in egg activation.
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PKC Distribution After Egg Activation by Sperm
Although TPA is an attractive agent as a Ca2+-dependent effector of cPKC and egg activation, treatment of eggs with phorbol esters does not serve as a physiological agent of egg activation. Therefore, we followed localization of PKCs in response to sperm-induced egg activation. Early and late fertilization events were defined by CG exocytosis and PN formation, respectively [33, 34]. Low density and large CG-free domain constitute a marker for the early-event stages of the fertilization process [35]. High density of CGs together with MII chromatin configuration was observed in unfertilized eggs (Fig. 7). As early as 10 min after egg-sperm interaction, fertilized eggs exhibited a low density of CGs in the entire cortex associated with translocation of both PKC- and PKC-ßI toward the plasma membrane (Fig. 8). After 1 h of incubation, PKC- was still localized to the plasma membrane, while PKC-ßI redistributed to the egg cytosol (Fig. 8). Localization of PKC-ßII did not change after sperm-egg activation (Fig. 8). These results suggest a putative role for PKC- and -ßI but not -ßII in the events leading to fertilization.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, PKC-ßI, or PKC-ßII, during oocyte maturation and egg activation using both Western blot analysis and immunocytochemistry. Furthermore, we show for the first time the specific expression of RACK1, the receptor for activated PKC in oocytes. Gangeswaran and Jones could not detect expression of cPKC in mouse oocytes and eggs [24]. This discrepancy could relate to differences in the sensitivity and purity of the antibodies as well as technical difficulties utilizing polymerase chain reaction techniques. Nevertheless, another study using the same antibodies as those employed by Gangeswaran and Jones demonstrated the presence of these PKCs in rat eggs [25]. We show that PKC- expression was consistently higher in GV oocytes, while PKC-ßI expression was somewhat increased in MII eggs (Fig. 1, A and B). The differences in expression were also associated with selective activation of PKC isoforms as demonstrated by different distribution of specific isoforms following PKC activation. These differences might suggest a selective role for each kinase in regulating different stages of oocyte development.
Treatment of GV oocytes with the PKC activator TPA induced depletion of PKC- from the cytoplasm followed by the accumulation of this isoform at the plasma membrane. The distribution of PKC-ßI, PKC-ßII, or RACK1 did not change (Fig. 3). It is known that TPA promotes a remarkably high affinity interaction between PKCs and their membrane receptors leading to enzyme activation [36]; thus the localization of PKC- in the cell periphery (Fig. 3) can now be explained in terms of its translocation to the plasma membrane followed by activation. Moreover, the spontaneous GVBD inhibition induced by TPA treatment (Fig. 4 and [37]) suggests the possible involvement of PKC- in GVBD inhibition.
Treatment of MII eggs with TPA revealed a different distribution of the PKC isoforms as compared to that in TPA-treated GV oocytes. In these eggs, PKC-, -ßI, -ßII, and RACK1 were all present in the cytoplasm, and PKC-ß isoforms were also present in the egg plasma membrane (Fig. 5A). Upon TPA activation, PKC- was eliminated from the cytoplasm and accumulated in the cell periphery (Fig. 5B), suggesting its translocation from the cytoplasm to the plasma membrane. No changes were observed in PKC-ßII or RACK1 distribution. As for PKC-ßI, treatment with TPA caused a complete drop in staining throughout the cytoplasm, while the cell area surrounding the cytoplasm remained stained (Fig. 5B). Since PKC-ßI is already localized in the cell periphery in untreated eggs, it is difficult to conclude whether this isoform was translocated after TPA treatment. The staining for PKC-ß revealed some differences between eggs at 17 h and 12 h post-hCG administration (Figs. 5 and 8, respectively). In older eggs (17 h post-hCG), PKC-ßI was localized in the cytoplasm and in the plasma membrane, while in younger eggs (12 h post-hCG), most of this isoform was located in the cytoplasm. Therefore the conclusion regarding translocation of PKC-ßI from the cytoplasm to the plasma membrane can be supported from the fertilization study (Fig. 8). In this experiment, eggs at 12 h post-hCG revealed significant localization of PKC-ßI throughout the cell periphery during 10-min incubation with sperm. For our TPA experiment we used older eggs (17 h post-hCG), since younger eggs (12 h post-hCG) do not respond well to TPA treatment [32].
Since TPA induced PN formation in MII eggs (Fig. 6), we suggest that PKC- and PKC-ßI might be involved in this stage of egg activation.
The most convincing results for the importance of cPKC involvement in egg activation were revealed by the fertilization assay. Incubation of eggs with sperm induced translocation of PKC- and PKC-ßI, but not PKC-ßII, from the cytosol to the plasma membrane. This effect was accompanied by CG exocytosis (Fig. 8). Nevertheless, while PKC- remained constantly in the plasma membrane after sperm-egg interaction for at least 60 min, PKC-ßI was redistributed to the cytoplasm after 60 min of incubation, suggesting another role for PKC- in modulating later events of fertilization. A recent publication has shown that although PKC antagonists block TPA-induced CG loss, they do not inhibit CG release in fertilized eggs [38]. In order to explain this discrepancy, a possible hypothesis was advanced by Ducibella and LeFevre [38], suggesting that after fertilization, a very small amount of PKC activity is necessary for CG release or that other activation pathways can compensate for PKC inhibition. Together, our results may suggest a role for PKC-ßI and PKC- in early events of fertilization like CG exocytosis or later events of fertilization like PN formation. This suggestion is supported by previous studies showing that short-term activation of PKC seems to play an important role in the activation of signal transduction pathways leading to short-term events like secretion or ion fluxes, while prolonged activation may play a critical role in the regulation of long-term cellular events such as proliferation, differentiation, or tumorigenesis [39, 40]. In GV oocytes, the translocation of PKC- to the plasma membrane without any change in RACK1 localization suggests that RACK1 is not the receptor for PKC-. Furthermore, in MII eggs, the translocation of PKC- and -ßI to the plasma membrane without any change in RACK1 localization indicates that RACK1 is not the receptor for PKC-ßI (Fig. 5). In light of these conclusions we suggest that RACK1 might be the protein receptor for PKC-ßII in mouse oocytes and eggs. This conclusion is supported by a recent study in which PKC-ßII and RACK1 were shown to be colocalized in the cytosol prior to their translocation to the plasma membrane of CHO cells [41]. Moreover, previous reports showed the interaction of PKC-ßII-RACK1 as a complex that mediates binding F-actin and rearrangement of the cytoskeleton [42], suggesting a possible role for PKC-ßII in cytoskeletal organization in the advanced stages of egg development.
In conclusion, in this study we identified members of the cPKC family in mouse oocytes and eggs and demonstrated their activation by phorbol ester and during egg activation by sperm. Taken together, the physiological changes in oocyte maturation can now be explained in terms of PKC translocation and activation by distinct isoforms. While the specific contribution of each PKC isoform to the development of the oocyte and the mature egg is still unknown, our results may suggest a role for PKC- in GV maintenance and a role for PKC- and -ßI in egg activation and CG exocytosis. An aim in future studies will be to identify the specific roles of each of these isoforms in egg development and their effects on egg maturation and fertilization.
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FOOTNOTES |
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1 This work was supported by a grant from the Ihel Foundation to H.B. 
2 Correspondence. FAX: 972 3 5344766; breith{at}mail.biu.ac.il
Accepted: January 11, 2000.
Received: September 9, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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