Regulation of spontaneous meiosis resumption in mouse oocytes by various conventional PKC isozymes depends on cellular compartmentalization

Nathalie Avazeri, Anne-Marie Courtot and Brigitte Lefevre*

Institut National de la Santé et de la Recherche Médicale Unité 566 Commissariat à l'Energie Atomique, 92260 Fontenay-aux-Roses CEDEX, France

* Author for correspondence (e-mail: brigitte.lefevre{at}cea.fr)

Accepted 22 June 2004


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 Materials and Methods
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In this study, we investigated the spatio-temporal distribution of conventional protein kinases C (cPKC) isoforms PKC-{alpha}, PKC-ßI, PKC-ßII and PKC-{gamma} in mouse oocytes. The cPKCs were present in the cytoplasm at the start of the process and migrated to the nucleus (or germinal vesicle) before germinal vesicle breakdown, except for PKC-{gamma} which remained cytoplasmic. In both compartments, the fully phosphorylated form corresponding to the `mature' enzyme was revealed for PKC-{alpha}, PKC-ßI and PKC-ßII. Microinjection of specific antibodies against each isozyme in one or the other cell compartment at different times of the meiotic process, permitted us to observe the following: (1) When located in the cytoplasm at the beginning of the process, PKC-{alpha} is not implicated in germinal vesicle breakdown, PKC-ßI and PKC-{gamma} are involved in maintaining the meiotic arrest, and PKC-ßII plays a role in meiosis reinitiation. Furthermore, just before germinal vesicle breakdown, these cytoplasmic cPKCs were no longer implicated. (2) When located in the germinal vesicle, PKC-{alpha}, PKC-ßI and PKC-ßII are involved in meiosis reinitiation. Our data highlight not only the importance of the nuclear pathways in the cell cycle progression, but also their independence of the cytoplasmic ones. Further investigations are however necessary to discover the molecular targets of these cPKCs to better understand the links with the cell cycle progression.

Key words: Oocyte, Meiosis, Nucleus, cPKCs


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mammalian oocytes are arrested at the G2 stage of the cell cycle and in vivo resumption of meiosis is triggered by the luteinizing hormone (LH), while these cells are enclosed in a follicle with a large antrum. The first step in resumption of meiosis is germinal vesicle breakdown (GVBD), corresponding to the breakdown of the nuclear envelope. A cascade of protein phosphorylation-dephosphorylation events, regulated by protein kinases and protein phosphatases, controls meiotic arrest and resumption. The resumption of meiosis is associated with a decrease in the intracellular cAMP concentration, resulting in the inactivation of cAMP-dependent protein kinase A (PKA). Although the way in which LH induces this process is still unclear, several authors have demonstrated the involvement of the phosphoinositide-PLCCa2+ pathway (Avazeri et al., 2000Go; Carroll and Swann, 1992Go; Pesty et al., 1998Go; Su et al., 2002Go).

Involvement of the PKC pathway in oocyte meiosis has been demonstrated in several species since 1985. However, the results obtained seem to depend on how meiosis resumption occurs: either spontaneously (mammals oocytes resume spontaneously meiosis when released from their ovarian follicles) or following gonadotropin induction (oocytes are maintained in meiotic arrest, and FSH is added to induce meiosis). Considering the so-called spontaneous meiotic maturation, it is stimulated by PKC activators in rat (Aberdam and Dekel, 1985Go), monkey (Lefevre et al., 1988Go) and bovine oocytes (Rose-Hellekant and Bavister, 1996Go), and is inhibited by such activators in mouse oocytes (Alexandre and Mulnard, 1988Go; Bornslaeger et al., 1986Go; Lefevre et al., 1992Go). By contrast, PKC inhibitors stimulate meiosis in mouse oocytes (Lefevre et al., 1992Go) and inhibit this process in bovine oocytes (Rose-Hellekant and Bavister, 1996Go).

PKCs exists as a family of serine/threonine kinases and the activators and inhibitors that are generally used are not selective for a particular type of enzyme. PKCs can be broadly categorized into three groups depending on their cofactor requirements. Conventional PKCs (cPKC-{alpha}, -ß and -{gamma}) require Ca2+ and diacylglycerol (DAG) for maximal activity, novel PKCs (nPKC-{delta}, -{epsilon}, -{eta} and -{theta}) are Ca2+-independent but require DAG like the related protein kinases D, atypical PKCs (aPKC-{zeta}, -{iota} and -{lambda}) require neither Ca2+ nor DAG for activity.

In this paper, we focus on cPKCs only, because these enzymes require Ca2+ for activity, nuclear and cytoplasmic [Ca2+] oscillates before GVBD (Pesty et al., 1998Go) and Ca2+ chelation inhibits meiosis resumption (Avazeri et al., 2003Go). It is unclear whether cPKCs are present in the mouse oocytes: according to Downs et al. and Luria et al. they are present (Downs et al., 2001Go; Luria et al., 2000Go), whereas Gangeswaran and Jones claim they are absent (Gangeswaran and Jones, 1997Go). We analyzed changes in the subcellular distribution of the four cPKC isozymes (PKC-{alpha}, -ßI, -ßII and -{gamma}) by immunofluorescence, immunocytochemistry and western blotting, throughout the process of spontaneous meiosis resumption. We then checked whether these isozymes were involved in the regulation of spontaneous meiosis resumption and whether the role they played was related to the subcellular distribution of the enzymes.


    Materials and Methods
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 Materials and Methods
 Results
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Oocyte recovery and culture conditions
Ovaries were stimulated by injecting 5 IU of pregnant-mare serum-gonadotrophin (Chronogest; Intervet International, Boxmeer, Holland) into 6-week-old female CD1 mice (Charles River, Saint-Aubin-Lès-Elbeuf, France). Forty hours after injection, cumulus-enclosed oocytes were retrieved from the ovarian follicles and placed in M2 medium (Sigma, Saint-Quentin Fallavier, France). As soon as oocytes are released from their ovarian follicles, they spontaneously enter the meiosis process: molecular events necessary for meiosis resumption began immediately. Oocytes were denuded by repeated aspiration through a glass capillary to remove cumulus cells. Groups of 15 oocytes were immediately added to 30 µl of M2 medium covered by mineral oil (Sigma) in chambered coverslip dishes (Lab-Tek II, Nalge Nunc International, Naperville, IL) and incubated at 37°C on the heating stage of the stereomicroscope (MZ12.5; Leica Microsystèmes, Paris, France) for 0, 30, 60 or 90 minutes before experiments.

Localization of `conventional' isozymes of PKC in the oocyte Immunofluorescence studies by confocal microscopy
The subcellular distribution of each cPKC isozyme (cPKC-{alpha}, -ßI, -ßII and -{gamma}) was evaluated in oocytes cultured for 0, 30, 60 or 90 minutes after their release from the follicles. At the end of the culture period, the oocytes were prepared as previously described (Avazeri et al., 2000Go). Briefly, oocytes were freed from the zona pellucida and fixed in 2% paraformaldehyde (Sigma) in PBS for 1 hour at 37°C. They were then incubated overnight at 4°C with the primary antibody. Isozyme-selective rabbit IgG polyclonal primary antibodies against cPKC-{alpha}, -ßI, -ßII, and -{gamma} were obtained from Santa Cruz Biotechnology (sc-208, sc-209, sc-210 and sc-211, respectively) (Santa Cruz, CA) and used diluted 1:200. For each isozyme, the antibodies were raised against a peptide that mapped the C-terminus. The antibodies directed against each isoform of cPKC had already been demonstrated to be specific, in competition experiments involving the corresponding blocking peptide (Das et al., 1997Go; Fan et al., 2002Go; Luria et al., 2000Go).

Oocytes were immunostained for 45 minutes at 37°C with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit secondary antibody (1:300 in the secondary blocking solution) (Amersham, Saclay, France). All incubations were carried out in the presence of saponin (0.5%) to ensure membrane permeability. The oocytes were then examined by using confocal microscopy (LSM5 Pascal, Carl Zeiss S.A., Göttingen, Germany) through a 40x objective (Planapo 40/0.95). After scanning, the sections through the germinal vesicle (GV) were selected. We checked the specificity of immunolabeling by incubating control oocytes with the secondary antibody alone. Control oocytes were maintained in meiotic arrest with hypoxanthine (4 mM) during 1 or 3 hours of culture before immunostaining.

In addition, isolated nuclei have also been studied. Oocytes cultured for 30 or 90 minutes were mechanically disrupted with a fine micropipette to recover the nucleus. Isolated nuclei were treated as described above, and examined with a 40x objective. We used double-staining to check that the isolated nuclei retained their nuclear envelopes. Following the immunostaining described above, the nuclei were incubated for 1 hour at room temperature with mouse monoclonal anti-lamin A/C antibody (Sc-7292) (Santa Cruz, CA) and then with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG secondary antibody (1:400) (Jackson ImmunoResearch Laboratories, West Grove, PA) for 45 minutes at 37°C.

Immunocytochemistry by electron microscopy
After 30 minutes or 90 minutes of culture, the oocytes were prepared as previously described (Avazeri et al., 2000Go). The cPKC-isozyme-specific primary antibodies were diluted to 1:200 in 5% BSA in PBS, the 10 nm in diameter gold-labeled goat anti-rabbit IgG (British Biocell International) were diluted 1:50 in 5% BSA in PBS. The sections were counterstained with saturated aqueous uranyl acetate for 30 minutes and examined under a Jeol-1010 electron microscope. We checked that the labeling was specific by incubating the control oocytes with the gold-conjugated goat anti-rabbit IgG secondary antibody alone. No staining was observed. Several sections of the same oocyte were photographed, and, for each section, the gold particles were counted in different areas of similar size. We then calculated the ratio between nucleus and cytoplasm contents for each oocyte.

Western blotting
Cell fractionation: We used 250 cumulus-free oocytes, cultured for 30 or 90 minutes, after their release from follicles for each western blot. They were homogenized by repeatedly frozen and thawed in 10 µl of homogenization buffer (50 mM Tris-HCl, 75 mM KCl, 50 mM NaF, 10 mM Na2HPO4, 1 mM EDTA, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride) and then suspended in 10 µl of 2x Laemmli sample buffer (Sigma). Whole cell lysate was centrifuged at 1000 g for 10 minutes. The resulting supernatant (a mixture of cytoplasm and plasma membrane) was separated into membrane and cytosolic fractions by centrifugation at 100,000 g for 60 minutes at 4°C. The nuclei in the initial pellet obtained in this way had intact membranes and were free from significant cytoplasmic contamination (Wooten et al., 1997Go). This nuclear pellet was resuspended in lysis buffer containing 0.8% Triton X-100, left on ice for 45 minutes and centrifuged at 100,000 g for 60 minutes at 4°C, to separate the membrane-stripped nuclei and nuclear membrane fractions.

Western blot analysis: Nuclear, cytoplasmic and particulate fractions were assessed for cPKC isoenzyme content by western blotting, using the same isozyme-specific antibodies (Santa Cruz) and horseradish peroxidase-conjugated anti-rabbit IgG (1:2000) (Amersham, Saclay, France). Blots were developed by enhanced chemiluminescence (ECL) (Amersham). The molecular mass of each isozyme detected was determined by comparison with known molecular mass markers (Sigma).

Cross-contamination between cytoplasm, nucleus and nuclear membranes was monitored by testing for ß-tubulin (mouse monoclonal antibody Sc-5274) (Santa Cruz, CA) and lamin A/C (mouse monoclonal antibody Sc-7292) (Santa Cruz).

Involvement of cPKCs in the regulation of spontaneous meiosis resumption
We investigated the effect of each cPKC isozyme on GVBD kinetics. The antibody for each cPKC isozyme [diluted 1:50 in microinjection medium (140 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.2)] was microinjected with sterile, ready-made needles (Femtotips; Eppendorf, Hamburg, Germany) either into the GV or the cytoplasm of oocytes, that had been cultured for 30 or 90 minutes after release from the follicle. The injection volume was 0.5 nl in the nucleus and 2.0 nl in the cytoplasm. The oocyte mortality rate was about 15%, 4 hours after the microinjections. The oocytes were then maintained in M2 medium with or without 3.5 mM hypoxanthine (HX) at 37°C and were examined by light microscopy every hour until 4 hours after release from the follicle to check for GVBD. For each microinjection experiment, control groups were always processed in parallel.

Statistical analysis
Each of the above protocols was carried out at least in triplicate, and control oocytes, collected from the same mice, were always analyzed simultaneously. Oocytes showing signs of degeneration were excluded from the statistical analysis. The data given, are means for at least three experiments and are expressed as a total percentage (pooled data) or as means±s.e.m. For data expressed as means±s.e.m., values were considered to differ significantly if a P-value less than 0.05 was obtained in a one-way analysis of variance (ANOVA) with Fisher's protected least significant different (PLSD) test.


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Immunolocalization of the cPKCs in the oocyte Confocal immunofluorescence microscopy
Confocal microscopy of whole oocytes immunostained with anti-cPKC isozyme-specific antibodies at various times between release from the follicle and GVBD, revealed differences in the changes in subcellular localization of these isoforms during the course of meiosis. Similar patterns of staining were observed for PKC-{alpha}, PKC-ßI and PKC-ßII (Fig. 1A): (1) intense labeling around the nuclear envelope (type I); (2) labeling around the nuclear membrane associated with the appearance of nucleoplasm-staining (type II); (3) intense immunoreactivity in the form of large dots within the nucleus (type III). In all observed oocytes, immunolabeling was diffuse in the cytoplasm. The frequencies of these patterns were estimated for each cPKC isozyme in oocytes maintained in culture for 0, 30, 60 or 90 minutes (Fig. 1B). If the oocytes were fixed immediately after release from the follicle, type-I-staining was observed almost exclusively, regardless of the cPKC isozyme tested. After 30 minutes in culture, when staining was still mostly of type I for PKC-ßII (97.1±2.8%; n=40), type-II-staining was most frequently observed for the PKC-{alpha} and PKC-ßI isozymes, (68.3±9.4% and 52.7±1.6%, respectively). When oocytes were fixed after 60 minutes in culture, then type-II-staining predominated for PKC-{alpha} (67.1±12.3%), PKC-ßI (52.7±1.6%), and PKC-ßII (43.9±15.8%). Finally, after 90 minutes in culture, type III staining was detected in most of the oocytes immunostained with the antibody against PKC-{alpha} (64.4±18.2, n=37), PKC-ßI (93.5±4.4%, n=60), and PKC-ßII (71.9±10.4%, n=52). These observations showed that these three PKC isozymes were becoming progressively more concentrated in the nucleus during the progression of the oocytes to the GVBD stage of meiosis. It has to be noticed that, compared to the others, PKC-ßII-staining was stronger near the plasma membrane and fainter in the nucleus. By contrast, for PKC-{gamma} the pattern of staining remained constant (100%; n=25) throughout meiosis: the staining was intense around the plasma membrane, in the cytoplasm (with large dots), faint around the nuclear envelope and absent from the nucleoplasm (Fig. 1A,B).



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Fig. 1. Intracellular redistribution of the various cPKCs isoforms in mouse oocytes during the period preceding germinal vesicle breakdown (GVBD). (A) The photographs show the three different types of immunolabeling observed with the four antibodies, against PKC-{alpha}, PKC-ßI, PKC-ßII and PKC-{gamma}, by confocal microscopy of whole mouse oocytes during spontaneous resumption of meiosis. These observations were recorded for a single optical section through the GV. Type I: spots of dense staining in the cytoplasm, intense labeling around the nuclear envelope, no staining in the nucleoplasm; type II: increased labeling in the nucleoplasm; type III: considerable immunoreactivity almost exclusively within the nucleus and around the cortex; isolated spots of fluorescence scattered throughout the cytoplasm. Scale bar, 40 µm. (B) The histograms indicate the distribution of the three different types of immunostaining with respect to time after meiosis reinitiation for each cPKC isoform. White columns: type I; gray columns: type II; black columns: type III. (C) The photographs show the immunolabeling of whole mouse oocytes, observed with the antibodies against PKC-{alpha}, PKC-ßI and PKC-ßII when the meiotic arrest was maintained by hypoxanthine during 1 to 3 hours.

 

When the meiotic arrest was maintained by hypoxanthine, in almost all the oocytes type-I-staining was observed for the three cPKC isozymes, [PKC-{alpha} (17/17), PKC-ßI (13/18) and PKC-ßII (13/14)] after 1 or 3 hours of culture (Fig. 1C). We then investigated the intranuclear distribution of cPKCs by determining the level of immunoreactivity in nuclei isolated from oocytes previously cultured for 30 or 90 minutes. The pattern of immunostaining observed, although slightly less intense, was similar for the various cPKCs (Fig. 2) than that in the nuclei of intact cells, indicating no significant loss of any of the isozymes during the isolation procedure. Furthermore, the nuclear membrane labeling with anti-lamin A/C antibody indicated clearly that the different PKC isozymes were localized at this cellular level before to be concentrated in the nucleus (Fig. 2).



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Fig. 2. Temporal patterns of staining with the various anti-cPKC antibodies in isolated nuclei. For each cPKC studied, the selected photographs are representative of the redistribution of immunostaining observed by confocal microscopy in isolated nuclei. The nuclei were isolated from oocytes cultured for 30 or 90 minutes and then immediately immunostained with an antibody against one of the cPKCs studied. The bottom panel shows control nuclei immunostained with an antibody against lamin A/C. (Left) The nuclear envelope is retained during the nuclear isolation procedure. (Right) Corresponding transmission image of the nucleus. Bar scale, 10 µm.

 

Electron microscope immunocytochemistry
We carried out a more detailed analysis of the distribution of the four cPKCs by electron microscope immunogold labeling on oocytes cultured for 0-30 or 60-90 minutes. This made it possible to determine the mean amount of enzyme in the two cell compartments considered - the cytoplasm and nucleus (Figs 3, 4).



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Fig. 3. Visualization of the subcellular distribution of PKC-{alpha} and PKC-ßI by electron microscope immunocytochemistry in oocytes 30 and 90 minutes after meiosis reinitiation. Oocytes treated with anti-PKC-{alpha} antibody 0-30 minutes (A) or 60-90 minutes (B, C) after release from the follicle; Oocytes treated with anti-PKC-ßI antibody 0-30 minutes (D) or 60-90 minutes (E, F) after release from the follicle. For each cPKC isoform, changes in the distribution of gold particles in the cytoplasm (gray columns) and nucleus (black columns) over time are shown in the two histograms: for both principal isoforms, the number of gold particles in the nucleus increased over time; the particles were distributed on the nuclear envelope (NE, white arrow), in the nucleoplasm (Np) and in the interchromatin granules (IG). The number of gold particles remained constant in the cytoplasm (Cy) for PKC-{alpha}, but decreased with increasing time in culture for PKC-ßI. Black arrows indicate the gold particles on the NE. Magnification, 20,000x; *, statistically significant difference between cytoplasm and nucleus at P<0.05.

 


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Fig. 4. Visualization of the subcellular distribution of PKC-ßII and PKC-{gamma} by electron microscope immunocytochemistry in oocytes 30 and 90 minutes after meiosis reinitiation. Oocytes treated with anti-PKC-ßII antibody 0-30 minutes (A) or 60-90 minutes (B,C) after release from the follicle. Oocytes treated with anti-PKC-{gamma} antibody 0-30 minutes (D) or 60-90 minutes (E) after release from the follicle. For each cPKC isoform, changes in the distribution of gold particles in the cytoplasm (gray columns) and in the nucleus (black columns) over time are shown in the two histograms: for PKC-ßII, the number of gold particles increased in the nucleus [in the nucleoplasm (Np) as well as in the interchromatin granules (IG)] and in the cytoplasm (Cy); for PKC-{gamma}, the nuclear envelope (NE, white arrow) and the nucleus were free of gold particles but the number of gold particles, although smaller than for other isoforms, increased over time. Black arrows indicate the gold particles on the NE. Magnification 20,000x. *, statistically significant difference between cytoplasm and nucleus, P<0.05.

 

Oocytes treated with the anti-PKC-{alpha} antibody after 0-30 minutes in culture (n=3) had more gold particles in the cytoplasm than in the nucleus (4.95±0.38 and 1.90±0.24 particles/µm2, respectively; P<0.05); conversely, those treated after 60-90 minutes in culture (n=2) had a similar number of gold particles in the cytoplasm and in the nucleus (5.33±0.59 and 7.30±0.88 particles/µm2, respectively; P=0.05).

Oocytes treated with the anti-PKC-ßI antibody after 0-30 minutes in culture (n=2) also had more gold particles in the cytoplasm than in the nucleus (6.80±0.76 and 2.03±0.32 particles/µm2, respectively; P<0.05); by contrast, those treated after 60-90 minutes in culture (n=2) displayed the opposite distribution (3.67±0.56 versus 6.80±0.69 particles/µm2, respectively; P<0.05).

An oocyte treated with the anti-PKC-ßII antibody after 0-30 minutes in culture (n=1) had significantly fewer gold particles in the cytoplasm and the nucleus (1.45±0.65 and 0.55±0.24 particles/µm2, respectively) than did those treated after 60-90 minutes in culture (n=3; 6.31±0.56 and 7.08±0.84 particles/µm2, respectively). The mean number of gold particles was also significantly higher in the cytoplasm after 60-90 minutes than after 0-30 minutes, indicating a large increase of the synthesis of this isozyme.

Concerning these three cPKCs, when oocytes were maintained for 60-90 minutes in culture, gold particles were observed not only in the nucleoplasm but also in interchromatin granules (IG). These experiments confirmed the nuclear accumulation of these three cPKCs before GVBD and specified their colocalization with some nuclear regions.

By contrast, fewer gold particles (1.28±0.38; n=1) were detected in the cytoplasm of an oocyte treated with the anti-PKC-{gamma} antibody after 0-30 minutes in culture than in those treated after 60-90 minutes in culture (2.90±0.54, P<0.05). A negligible number of gold particles was detected in the nucleus, regardless of the duration of culture (0.40±0.30 and 0.26±0.20, respectively). Thus, it seems that, compared to the other isozymes, PKC-{gamma} is present at lower levels in mouse oocytes.

Western blots
Each of the antibodies used was directed against the COOH-terminal region of the corresponding isozyme. With these antibodies, it was possible to differentiate between the three forms of each isozyme - the inactive dephosphorylated precursor with an apparent molecular weight of 74 kDa, the transient 77-kDa phosphorylated form and the final 80-kDa mature form (Keranen et al., 1995Go). We used western blots to determine, for each of the four isozymes, which forms were present in the cytoplasmic and nuclear compartments (Fig. 5A).



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Fig. 5. Western blot analysis of the four cPKCs isoforms. (A) Cellular extracts of GV oocytes cultured for 30 or 90 minutes were subjected to biochemical fractionation (cytoplasmic, nucleoplasmic and nuclear envelope fractions) and analyzed by immunoblotting. For each cPKC isoform, western blots were probed with an antibody against the catalytic domain that reacted equally strongly with dephosphorylated and fully phosphorylated protein. The double asterisk indicates the 80-kDa mature PKC. The single asterisk denotes the presence of the 77-kDa form, which is phosphorylated at the two C-terminal sites. The 74-kDa form, indicated by a hyphen, has either no phosphate groups or a single phosphate on the carboxyl terminus. (B) Control blots demonstrating the purity of the subcellular fractions: ß-tubulin, used as a specific marker of the cytoplasm is absent from the nuclear fraction; lamin A/C, used as a specific marker of the nuclear envelope, is present in the nuclear envelope fraction and absent from the nucleoplasm.

 

The cytoplasmic fraction of oocytes maintained for 30 minutes in culture gave a strong signal for PKC-{alpha}, PKC-ßI and PKC-ßII, whereas it gave only a faint signal for PKC-{gamma}. These isozymes migrated as two bands: the faster migrating band corresponds to the 77-kDa precursor form (single asterisk) and the slower migrating band to the protein phosphorylated at all three `maturation' positions (double asterisk). After 30 minutes in culture, the phosphorylated forms of PKC-{alpha} and PKC-ßI were detected in both the cytoplasmic and nuclear envelope fractions, whereas the transient 77-kDa phosphorylated form was detected exclusively in the cytoplasmic fraction. However, the 80-kDa immunoreactive band gave a faint signal in the nuclear fraction. By contrast, for PKC-ßII the 77-kDa phosphorylated form was the major band detected in the cytoplasm. No signal was detected in the nuclear envelope and the nucleoplasmic fractions. PKC-{gamma} was detected neither in the cytoplasm nor in the nuclear fractions.

In oocytes previously cultured for 90 minutes for PKC-{alpha}, -ßI and -ßII, the 77/80-kDa doublet was systematically detected in the cytoplasm, with an intensity similar to that observed after only 30 minutes in culture. However, the 80-kDa form was detected in the nuclear membrane and in the nucleoplasmic fraction. For PKC-{gamma}, three small bands were detected only in the cytoplasmic fraction. These results confirm that all the isozymes except PKC-{gamma} were relocated to the nucleus during oocyte maturation.

We assessed the purity of the cellular subfractions used by reprobing the blots for several marker proteins specific for the cytoplasm (ß-tubulin) and for the nuclear envelope (lamin A/C). As expected, only the nuclear envelope fraction displayed staining for lamin A/C, the nucleoplasm remaining unstained. The nuclear fractions showed no staining for ß-tubulin whereas the cytoplasm fractions gave a positive signal for this protein (Fig. 5B).

Characterization of the cPKCs involved in spontaneous meiosis resumption
Among the different cPKC isoforms, we tried to identify which one(s) were involved in regulating the meiotic process, which role they played, and whether their actions differed according to their cellular compartmentalization. For these reasons, we inhibited isozymes PKC-{alpha}, PKC-ßI, PKC-ßII and PKC-{gamma} and analyzed the kinetics of meiotic maturation. We microinjected the various antibodies into the cytoplasm or nucleoplasm of oocytes that had been previously maintained in culture for 30 minutes. Then, we continued to maintain the oocytes in culture, in the presence or absence of HX, and observed them under a light microscope every hour for 4 hours.

Control oocytes
Because we had previously demonstrated that PKC-{gamma} was absent from the nucleus, oocytes in which anti-PKC-{gamma} antibody was injected into the nucleus were considered as controls. After microinjection, the oocytes were cultured in M2 medium with or without HX. When antibodies were microinjected into the GV, the rates of GVBD observed for oocytes cultured with or without HX were similar to those of their non-microinjected counterparts (Fig. 6A). For example, at the end of the culture period, meiotic resumption occurred in 75.1±5.5% (n=39) of microinjected-oocytes in absence of HX versus 77.2±4.2% (n=49) for controls and in 6.2±3.8% (n=32) of microinjected-oocytes in presence of HX versus 0.0±0.0% (n=53) for controls. These control experiments showed also that the medium in which the antibodies were diluted did not affect the meiotic process when microinjected into the nucleus.



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Fig. 6. Effects on spontaneous meiosis resumption or on HX-maintained meiotic arrest of the microinjection into the cytoplasm or into the GV of antibodies specific for the various cPKCs. (A) Control oocytes. Because PKC-{gamma} is absent from the nucleus, anti-PKC-{gamma} was used as a control for the microinjection procedure. The microinjection affected neither spontaneous meiosis resumption (-HX) nor maintained meiotic arrest (+HX). (B) Inhibition of the nuclear isoform of each cPKC except PKC-{gamma} had a negative effect on spontaneous meiotic maturation. By contrast, the inhibition of the cytoplasmic isoforms of these enzymes had no effect on meiosis. (C) Top: oocytes were first cultured for 30 minutes, then microinjected into the cytoplasm and finally maintained in meiotic arrest by adding HX. Inhibition of the cytoplasmic isoforms affected meiotic arrest in different ways: the {alpha} and ßII isoforms were not involved whereas the ßI and {gamma} isoforms acted with HX to maintain meiotic arrest, the inhibition of these enzymes resulting in meiosis resumption. Bottom: oocytes were first cultured for 90 min, then microinjected and maintained in meiotic arrest by adding HX. Inhibition of the various cytoplasmic cPKC isoforms had no effect on meiotic arrest. The frequencies±s.e.m. of GVBD were calculated for at least three experiments. *, statistically significant difference versus control, P<0.05. White columns: control oocytes; gray columns: intracytoplasmic microinjection of the antibody; black columns: intranuclear microinjection of the antibody.

 

Effect of blocking cPKC-{alpha}, cPKC-ßI and cPKC-ßII by means of the corresponding specific antibodies
With the exception of the antibody directed against PKC-{gamma}, microinjection of isozyme-specific antibodies into the GV of oocytes during the first 30 minutes after release from the follicle significantly delayed GVBD (P<0.05) (Fig. 6B). This inhibition was maintained during the 4 hours in culture: 43.18±8.9% (for PKC-{alpha}, n=43), 56.2±5.1% (for PKC-ßI, n=55), and 42.3±8.6% (for PKC-ßII, n=43) of the oocytes resumed meiosis versus more than 80% in the control groups.

However, the microinjection of isozyme-specific antibodies into the cytoplasm of oocytes during the first 30 minutes after release from the follicle had no significant effect on GVBD kinetics for PKC-{alpha} (66.6±6.6% n=48), PKC-ßI (74.9±5.4%, n=48) and PKC-{gamma} (75.2±9.7%, n=46). By contrast, microinjection of the anti-PKC-ßII antibody into the cytoplasm slightly delayed GVBD (P<0.05). Indeed, after 2 hours in culture, the rate of GVBD was 47.8±4.6% for treated oocytes versus 69.5±5.5% for the control group, whereas at the end of the culture time, GVBD rates were similar for both treated and control groups (60.6±6.6% versus 79.8±4.0%).

Because the cytoplasmic microinjection of antibodies against the different cPKCs isoforms did not affect oocyte maturation except for PKC-ßII, we went on to investigate whether this inhibition overcame the inhibitory effect of HX on meiosis resumption. We microinjected the isozyme-specific antibodies into the cytoplasm of oocytes maintained in culture for 30 or 90 minutes. The oocytes were then cultured in HX-supplemented medium (Fig. 6C). (1) When microinjected into the cytoplasm 30 minutes after the start of culture, the inhibition of PKC-ßI (n=62) partly overcame the HX-maintained meiotic arrest (20.9±5.9% versus 3.2±2.4% for control oocytes; n=77). As for the anti-PKC-ßI antibody, injection of the anti-PKC-{gamma} antibody into the cytoplasm (n=35) overrode HX-maintained meiotic arrest (P<0.05), and this effect was maintained until the end of the culture (25.71±8.1% versus 0.0±0.0%). By contrast, the intracytoplasmic injection of antibodies directed against the other two isoforms (PKC-{alpha} and PKC-ßII) had no effect on GVBD rates in HX-treated oocytes. (2) When microinjected into the cytoplasm 90 minutes after the start of culture, the antibodies against each isoform did not induce release of HX-maintained meiotic arrest.


    Discussion
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We have previously demonstrated the involvement of the nuclear PI-PLC pathway in the control of mouse oocyte maturation (Pesty et al., 1998Go; Avazeri et al., 2000Go; Lefevre, 2003Go). We wanted to verify whether generation of the second messenger DAG induces the activation of the cPKCs in the nucleus. Until now, the involvement of the PKC pathway in the control of mouse oocyte meiosis had been studied by using activators or inhibitors of the enzyme (Downs et al., 2001Go; Lefevre et al., 1992Go; Lu et al., 2001Go; Luria et al., 2000Go; Viveiros et al., 2001Go). But these reagents are unable to discriminate between the different types of PKC and their subcellular localization. In this paper, the spatio-temporal distribution of the four isozymes {alpha}, ßI, ßII and {gamma} of the cPKCs as well as their functions are studied for the first time during the period preceding the GVBD.

Subcellular localization of conventional PKCs
The combination of immunofluorescence, immunocytochemistry and western blot experiments permit us to demonstrate the changes in the subcellular distribution of cPKCs before GVBD. Immediately after release from the follicle, i.e. at the beginning of meiosis reinitiation, all cPKC isozymes are mainly distributed throughout the cytoplasm, with stronger signals obtained around the nuclear envelope. Later on, the staining corresponding to each of the isozymes other than PKC-{gamma} gradually increased in the nucleus, PKC-{gamma} remaining cytoplasmic. The examination of isolated nuclei reveals the colocalization of the four cPKCs with the nuclear envelope at least at the beginning of the process. By electron microscope immunocytochemistry, all cPKCs except PKC-{gamma} are well detected in the nucleoplasm just before GVBD, and more particularly in the interchromatin (IG) and perichromatin (PG) granules that correspond to nuclear speckles (Spector et al., 1991Go). These granules are ribonucleoprotein (RNP) structures forming part of the nuclear matrix and corresponding to sites at which transcription products are processed (Maraldi et al., 1993Go; Puvion and Puvion-Dutilleul, 1996Go; Santella and Kyozuka, 1997Go; Takeuchi et al., 1990Go). It has also been shown in several cell types that nuclear speckle domains are not only associated with PKC (Capitani et al., 1987Go; Payrastre et al., 1992Go; Zhu et al., 2003Go; Zini et al., 1993Go), but also with some elements of the phosphoinositide cycle (Boronenkov et al., 1998Go; Didichenko and Thelen, 2001Go; Osborne et al., 2001Go). For instance, in the cell nucleus, PKC colocalizes with PtdIns(4,5)P2 and PLC-ß1, at the RNP clusters of the interchromatin domains (for a review see Maraldi et al., 1999Go), as we also observed in the mouse oocyte (Avazeri et al., 2000Go).

It was recently reported that cPKCs are present only in the cytoplasm and never in the nucleus of GV-stage mouse oocytes (Luria et al., 2000Go), a finding that appears to contradict our data. However, the experiments in this previous study were performed at only one time point, immediately after oocyte recovery from the ovaries. The immunostaining patterns obtained are entirely consistent with our own for the same time point, with no cPKCs present in the GV. More surprisingly, Gangeswaran and Jones did not detect cPKCs in mouse GV oocytes by RT-PCR and western blotting (Gangeswaran and Jones, 1997Go). This discrepancy may be owing to differences in the sensitivity and purity of the antibodies used and technical difficulties with polymerase chain reaction techniques.

Consistent with our findings, a number of reports have highlighted the presence of PKC-{alpha}, PKC-ßI and PKC-ßII in the nuclei of cells of various types (Buchner, 1995Go). However, it has been shown that each type of cells produces a defined set of PKC isozymes, relocated to the nucleus in response to signaling events (Asaoka et al., 1992Go). Indeed, in many instances a stimulus-dependent relocation to the nuclear compartment takes place, but it is not clear how isozyme-specific migration happens. PKC-transport into the nucleus seems to differ from the transport system used by classic NLS-bearing proteins (Schmalz et al., 1996Go; Schmalz et al., 1998Go). It might occur via PKC-binding proteins (such as PICK1). However, as for mouse oocytes, contradictory findings have been reported concerning the nuclear localization of PKC isozymes in a same cell model. For example, some authors have reported PKC-ß to be present in the nucleus of rat liver cells (Rogue et al., 1990Go), whereas others have detected this isoenzyme exclusively in the cytoplasm of these cells (de Moel et al., 1998Go). These differences may be accounted for by the use of antibodies raised against different peptide sequences, recognizing the unphosphorylated and/or phosphorylated cPKC forms or by experiments being performed at different stages of the cell cycle.

However, these different studies of the intracellular distribution of the different cPKCs do not permit to discriminate between their inactive and active forms. It was possible only by western blots to detect the three forms of each isoenzyme: the 74-kDa inactive dephosphorylated precursor, the transient 77-kDa phosphorylated form and the final 80-kDa mature form. Comparison of the cytoplasmic and nucleoplasmic fractions at two time points after meiosis reinitiation reveals the 80-kDa form in the nucleoplasm only 90 minutes after release from the follicles for all three of these cPKCs. One other report demonstrates the presence of phosphorylated PKC isoforms in the nucleus of other cell types (Neri et al., 2002Go). Furthermore, these results and those obtained by electron microscope immunocytochemistry showed that all cPKCs were not only relocated into the nucleus, but also synthesized in the cytoplasm during oocyte maturation. It is also noticeable that the cPKCs migration into the nucleus did not occur when the meiotic arrest was maintained suggesting the involvement of nuclear cPKC isozymes in the control of meiosis resumption.

It is noticeable that, as in rat liver cells (Kuriki et al., 1992Go), the migration of PKC-{alpha}, PKC-ßI and PKC-ßII into the nucleus closely coincides with that of PLC-ß1 in the mouse oocyte (Avazeri et al., 2000Go). Furthermore, our observations of both inositol trisphosphate receptors (Pesty et al., 1998Go) and PLC-ß1 (Avazeri et al., 2000Go) in the GV of the mouse oocyte provides evidence for a nuclear phophoinositide-cycle in the female gamete (for a review see Lefevre, 2003Go), as has been demonstrated in other cell types (Alessenko and Burlakova, 2002Go; Cocco et al., 1996Go; Mazzotti et al., 1995Go; Vann et al., 1997Go; Zini et al., 1994Go). Thus, in the GV of the mouse oocyte, as in other cell types (Divecha et al., 1991Go; Mallia et al., 1997Go; Neri et al., 1998Go; Neri et al., 2002Go), PKC-{alpha}, PKC-ßI and PKC-ßII could be attracted and activated by DAG generated by PLC-ß1. Originally, Divecha et al. proposed that cPKC isoforms (such as PKC-{alpha}) might be continuously cycled to and from the nucleus and might become `fixed' in the nuclear compartment by an increase in DAG levels (Divecha et al., 1993Go). Indeed, in Swiss 3T3 cells, it has been shown that the DAG produced in the nucleus by phosphoinositide-specific PLC drives the nuclear translocation of PKC-{alpha} (Neri et al., 1998Go) and also that PKC-{alpha} is responsible for the negative feedback-regulation of PLC-ßI (Xu et al., 2001Go).

Cytoplasmic and nuclear cPKC and meiosis resumption
The location of PKC-{alpha}, PKC-ßI and PKC-ßII in the nucleus of the mouse oocyte before GVBD suggests that these enzymes might play a specific role in this cell compartment. The microinjection of antibodies against each cPKC isozyme into the cytoplasm or the nucleus provided, for the first time, insight into the respective roles of these enzymes, according to their subcellular localization, on the GVBD kinetics. Indeed, we found a relationship between the cellular distribution of PKC-{alpha}, PKC-ßI, PKC-ßII and PKC-{gamma}, and their roles in meiosis resumption. On one hand, at the beginning of the process, when the cPKCs were present in the cytoplasm, their roles in the control of GVBD differed according to the isoform: for instance, PKC-{alpha} was not involved in this process whereas PKC-ßI and PKC-{gamma} prevented GVBD and PKC-ßII promoted it. Our data are consistent with several reports describing the antagonistic effects of various cPKC isoforms on the regulation of biological functions in other cell types (Chen and Mochly-Rosen, 2001Go; Efendiev et al., 1999Go; Gusovsky and Gutkind, 1991Go; Rosales et al., 1998Go; Song et al., 2002Go). For example, for the cPKC family, PKC-ßI and PKC-ßII have been shown to have opposite effects in vascular smooth muscle cell proliferation (Yamamoto et al., 1998Go). On the other hand, concerning the oocyte, in the period preceding GVBD, although the cytoplasmic isoforms appeared to play no further role, the {alpha}, ßI and ßII isoforms detected in the nucleus played an inducing role on GVBD. This is also in agreement with the fact that the cPKC isozymes did not translocate into the nucleus when oocytes were maintained in meiotic arrest.

In conclusion, this study provides the first detailed spatio-temporal investigation of cPKCs and their respective roles on mouse-oocyte maturation. Whereas the PKC-{gamma} isozyme appears to be minimally expressed and to remain in the cytoplasm, the three other cPKC isozymes, PKC-{alpha}, PKC-ßI and PKC-ßII, are strongly expressed and all concentrate into the nucleus before GVBD. According to their subcellular distribution, they appear to behave differently, and this observation explains the difficulty to study the cell complexity with tools that are not specific enough. When activators or inhibitors were used, the PKC pathway was mainly demonstrated to be responsible for maintaining the meiotic arrest (Downs et al., 2001Go; Lefevre et al., 1992Go; Lu et al., 2001Go; Luria et al., 2000Go; Viveiros et al., 2001Go). However, these reagents are always added to the culture medium at the very beginning of the process. This could mean that only the cytoplasmic isoforms are impaired because, at the start of the process, they are exclusively found in the cytoplasm. Later, when they migrate to the nucleus, except for PKC-{gamma}, the cytoplasmic enzymes are no more involved in the meiotic maturation, whereas the nuclear ones are all able to induce meiosis resumption. At this particular period of meiosis, the nuclear signaling appears to play a major role. This idea is supported by the fact that PKC plays a role in the phosphorylation of lamins that accompanies the mitotic nuclear breakdown at the G2-M transition (Fields and Thompson, 1995Go). In the case of the oocyte, the cPKCs could also play such a role, thereby participating to the germinal vesicle breakdown. Indeed, in preliminary results, we observed the colocalization of PKC-ßI and lamin A/C on the nuclear envelope (N.A., A.-M.C. and B.L., unpublished data). We hypothesize that, at the beginning, the cytoplasmic cPKCs (at least the ßI and {gamma} isoforms) are involved in meiotic arrest, whereas later on, before GVBD, the nuclear cPKCs are responsible for meiosis resumption. However, among them, PKC-ßII is particular because either in the cytoplasm or in the nucleus it is responsible for meiosis resumption.

Our data highlight not only the importance of the nuclear pathways in the cell cycle progression, but also their independence of the cytoplasmic ones. We are still in the beginning of understanding the role of the nuclear lipid signaling in this process and further investigations are necessary to discover the molecular targets of these cPKCs to better understand the links with the cell cycle progression.


    Acknowledgments
 
We thank `Alex Edelman & Associates' for editing the English text. Nathalie Avazeri was supported by a postdoctoral fellowship from the Commissariat à l'Energie Atomique. The work was supported by a Grant from the Nuclear Toxicology program of the Commissariat à l'Energie Atomique. The ultrastructural observations were carried out at the `Service de Neurologie' with the help of Josette Bacci (Pr Saïd, Hôpital Kremlin-Bicêtre, Paris XI University).


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 References
 

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