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Gamete Biology |
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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calcium, fertilization, kinases, meiosis, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ß, 
, and 
, ranging in molecular weight from 52 kDa () to 58–61 kDa (ß, , and ) [3, 4]. CaMKII is normally maintained in an inactive state by the interaction of the catalytic domain with an autoinhibitory domain located on the same polypeptide. In the nonphosphorylated form, CaMKII requires calcium and calmodulin for its activity. In the presence of calcium and calmodulin, the enzyme is autophosphorylated on the threonine 286 and becomes calcium and calmodulin independent [5]. Thus, the generation of this autonomous kinase may underlie some long-term potentiation of transient calcium signals. Calcium is thought to be involved in regulating mitotic transitions by mechanisms mediated by calmodulin and CaMKII, but studies on calcium as a possible candidate for an overriding signal that induces germinal vesicle breakdown (GVBD) in oocyte meiotic maturation have not produced conclusive evidence, probably as a consequence of the use of different systems and culture conditions [6, 7]. Some researchers have suggested that meiotic cell division is not associated with an increase in intracellular calcium and that artificial elevation of calcium level in the oocytes is not capable of inducing meiotic resumption in rodent oocytes [8]. In addition, chelation of the intracellular calcium does not block GVBD in mouse, rat, and cow oocytes [8–10]. However, there are also several lines of evidence from amphibian or mammalian models suggest that calcium-mediated and CaMKII-dependent signaling pathways are essential for the normal progression of meiotic cell cycles in oocytes [11]. Meiotic maturation was induced in Xenopus oocytes when the intracellular Ca2+ concentration was raised in the presence of ionophore [12]. Injection of calcium chelator BAPTA or CaMKII inhibitor autocamtide-2 related inhibitory peptide (AIP) inhibited GVBD in Xenopus oocytes [13].
Fully grown mammalian oocytes are arrested at the diplotene stage of first meiotic prophase, which is also termed germinal vesicle (GV) stage. The GV stage–arrested oocytes can resume meiosis spontaneously when they are released from the inhibitory environment of follicles. Oocytes also mature in vitro under the stimulation of gonadotropin when spontaneous maturation is prevented by meiotic inhibitors [14]. In these two experimental models, different mechanisms are employed in regulating the progression of meiotic cell cycle [15, 16]. Calcium-dependent pathways are essential for gonadotropin-induced oocyte meiotic resumption of mouse oocytes [7]. Spontaneous calcium oscillations occur in the GV-stage mouse oocytes [17, 18], and chelation of intracellular calcium blocks FSH-induced meiotic resumption in mouse and pig cumulus-enclosed oocytes (CEOs) [19, 20]. Calcium probably interacts with calmodulin to regulate oocyte maturation, since calmodulin antagonists inhibit GVBD in mouse and rabbit oocytes, and prevent meiotic progression to the second meiotic metaphase (MII) stage in cow and rabbit oocytes [21, 22]. Recent reports indicated that CaMKII was a possible molecular linkage between calcium signal and cell cycle regulatory molecules in the oocytes. The FSH-induced GVBD of mouse CEO, rather than spontaneous meiotic resumption, is inhibited by CaMKII inhibitors KN-93 and myristoylated AIP [23].
Calcium-dependent pathway is also involved in the progression of meiosis beyond metaphase I. CaMKII inhibitors and calmodulin antagonist W7 prevent the emission of first polar body and arrest the oocytes at the MI stage [23]. Culture of pig and bovine oocytes in a calcium-deficient medium suppresses polar body formation [24–26]. These results suggest that CaMKII is involved in not only the initiation of meiotic resumption but also the MI to MII transition of oocytes. Furthermore, there is evidence that the influx of extracellular calcium, instead of the release of intracellular calcium, is essential for activation of CaMKII and polar body extrusion because nifedipine, a membrane calcium ion channel blocker, inhibits polar body emission and blocks the meiosis I at metaphase [23]. However, evidence for the role of CaM and CaMKII in GVBD and polar body emission of pig oocytes remains absent.
In most vertebrates, unfertilized oocytes are arrested at MII by a cytostatic factor (CSF) [27]. CSF prevents ubiquitin-dependent degradation of metaphase cyclins and thus inactivation of the M phase-promoting factor (MPF). The evolutionarily conserved stimulus that releases the egg from cell cycle arrest in all animal species studied so far is a rise in intracellular free Ca2+, which is triggered by fertilization or parthenogenetic activation [28]. An undefined mechanism activated by Ca2+ inactivates both CSF and MPF and releases oocytes from meiotic metaphase arrest [29]. An early and transient activation of CaMKII was detected in Xenopus eggs after parthenogenetic activation. Microinjection of the constitutively active CaMKII into MII-arrested Xenopus oocytes inactivated MPF and triggered Mos degradation. Conversely, unfertilized Xenopus oocytes microinjected with the CaMKII inhibitory peptide failed to undergo cyclin degradation and inactivation of either Cdc2 kinase or CSF upon parthenogenetic stimulation [30]. These results indicate that CaMKII mediates the Ca2+-dependent inactivation of MPF and CSF accompanying fertilization or egg activation.
CaMKII is also activated in mouse oocytes following a rise in intracellular calcium concentration, induced by calcium ionophore A23187 [31], ethanol [32], or sperm penetration [33]. Calmodulin forms a tight association with CaMKII on the meiotic spindle immediately after activation in mouse oocytes. Further, CaMKII becomes localized in the midzone microtubules between anaphase II and telophase II [31]. However, the participation of CaMKII in egg activation and its association with meiotic apparatus have never been proven in other mammals.
Mitogen-activated protein kinase (MAPK), also termed extracellular regulating kinase (ERK), is another kind of protein kinase that plays key roles in the regulation of oocyte meiosis [34, 35]. MAPK is activated at the same time (pig and horse) [16] as or shortly after (rodents) [36–38] GVBD, and its activity remains high after egg activation until pronuclear formation [16, 39]. A study on mouse oocytes revealed that MAPK and CaMKII were colocalized on the meiotic spindle, suggesting their potential mutual regulation at vicinity. Suppression of CaMKII activity during egg activation resulted in reduction in the amount and kinase activity of MAPK [40]. CaMKII could serve to potentiate MAPK activity and the colocalization of these two kinases may facilitate such an interaction. Whether the same mechanism is also employed by other mammalian species still needs to be elucidated.
Although it has been confirmed that calmodulin and CaMKII are among the most important mediators of calcium signal during meiotic maturation and fertilization of mouse oocytes, the importance of CaMKII in oocytes of mammalian species other than mouse is not known. The oocyte development is under the control of multiple signaling pathways that related to numerous important protein kinases, such as MPF, protein kinase A, protein kinase C, MAPK, and 90 kDa ribosome S6 protein kinase (p90rsk). How CaMKII regulates the activity of these kinases is still an unsolved problem in revealing the mechanism of meiotic maturation and fertilization.
In this study, we evaluated in pig oocytes 1) the subcellular distribution of CaM and CaMKII during meiotic maturation and activation; 2) the roles of CaM and CaMKII in meiotic maturation and activation; and 3) the possible regulation of CaM/CaMKII pathway on the activity of MPF and MAPK, two protein kinases that play key roles in the progression of meiotic cell cycles. Our results provide evidence suggesting that CaM and CaMKII are involved in multiple steps of meiotic resumption and activation of pig oocytes as well as the regulation of other protein kinase activities.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals used in this study were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise noted. Stock solutions of BAPTA-AM (50 mM), KN-93 (20 mM), KN-92 (20 mM), cell-permeable autocamtide-2-related inhibitory peptide II (Ant-AIP-II, 10 mM), thimerosal (Thi, 200 mM) were prepared in dimethyl sulfoxide and stored frozen at -20 °C in a dark box until use. Stock solutions of W7 (20 mM) and dithiothreitol (DTT, 8 M) were prepared in sterile water and stored at -20°C. The final working solutions of all the above chemicals were diluted in culture media just before use.
In Vitro Maturation of Oocytes
Ovaries were collected from gilts at a local slaughterhouse and transported to the laboratory within 1 h. Oocytes were aspirated from antral follicles (2–6 mm in diameter) with an 18-gauge needle fixed to a 20-ml disposable syringe. After three washes with maturation medium (see below), oocytes with compact cumulus and evenly granulated ooplasm were selected for maturation culture. The medium used for maturation culture was TCM-199 (Gibco, Grand Island, NY) supplemented with 75 µg/ml potassium penicillin G, 50 µg/ml streptomycin sulphate, 0.57 mM cysteine, 0.5 µg/ml FSH, 0.5 µg/ml LH, and 10 ng/ml epidermal growth factor. A group of 25 oocytes was cultured in a 100-µl drop of maturation medium for 44 h at 39°C in an atmosphere of 5% CO2 and saturated humidity.
After maturation culture, oocytes were freed of cumulus cells by treatment with 300 IU/ml hyaluronidase (Sigma) and repeated pipetting. The denuded oocytes were then washed twice in TCM-199 and used for either parthenogenetic activation or drug treatment.
Parthenogenetic Activation of Pig Oocytes
In vitro matured pig oocytes were induced to undergo parthenogenetic activation by either physical (electrical pulse) or chemical (thimerosal followed by DTT) stimulation. The method of electrical activation was essentially the same as that reported by Onishi et al. [41]. Briefly, after washing three times in the electroporation medium (0.28 M mannitol, 0.05 mM CaCl2, 0.1 mM MgSO4, and 0.01% (w/v) BSA), cumulus-free oocytes were put in a fusion chamber. An 80-µsec pulse at 120 V/mm DC was exerted to oocytes. The oocytes were then washed three times and cultured in NCSU-23 medium containing 0.4% BSA.
Chemical activation was conducted as described by Máchaty et al. [42]. Mature pig oocytes were exposed to 200 µM thimerosal for 10 min, with subsequent incubation with 8 mM DTT for 30 min to reverse the oxidation of sulfhydryl groups induced by thimerosal and then were cultured in NCSU-23 medium.
Nuclear Status Examination
To determine the nuclear status of pig oocytes, oocytes were fixed in 4% paraformaldehyde for 30 min and permeabilized in PBS containing 1% Triton X-100 overnight at 37°C. The samples were stained with 1:50 diluted fluorescein isothiocyanate (FITC)-conjugated anti--tubulin (Sigma) for 1 h, and then with 10 µg/ml propidium iodide (PI). After extensive washing, the cells were mounted on the glass slide and examined by confocal microscopy (for more details, see below). The nuclear status was determined by checking the microtubule organization (stained in green) and the chromosome configuration (stained in red) at the same time.
CaMKII Activity Assay
CaMKII activity before and after electrical activation was assayed by using Signa TECT calcium/calmodulin-dependent protein kinase assay system (Promega, Madison, WI) according to the manufacturer's protocol with minor modifications. Briefly, 20 MII-arrested or electrically activated eggs were collected in 5 µl extraction buffer (20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 2 mM EGTA, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 2 mM DTT, 25 mM benzamidin, and 1 mM PMSF) and frozen at -80°C until use. Just before the assay of CaMKII activity, the frozen samples were thawed and added with 2.5 µl CaMKII biotinylated peptide substrate, 5 µl CaMKII reaction 5 x buffer, 5 µl CaMKII control 5 x buffer (both buffers were provided by the CaMKII assay kit), 5 µl [-32P] ATP mix, and 2.5 µl deionized H2O. After thorough mixture, the samples were incubated at 30°C for 5 min and then were added with 12.5 µl of termination buffer to stop the reactions. A 10-µl sample was spotted onto a square of the SAM biotin capture membrane (Promega) and the washing and rinsing steps followed as described by the manufacturer. Then the radiation intensity of each membrane was quantitated by a Bechman scintillation counter (Fullerton, CA) as the relative endogenous CaMKII activity of a sample.
Western Blot Analysis
For detection of p90rsk and active ERK1/2, proteins from 50 oocytes were collected in SDS sample buffer and heated to 100°C for 4 min. After cooling on ice and centrifuging at 12 000 x g for 3 min, samples were frozen at -20°C until use. The total proteins were separated by SDS-PAGE with a 4% stacking gel and a 10% separating gel for 20 min at 90 V and 4.5 h at 110 V, respectively, and then electrophoretically transferred onto nitrocellulose membrane for 2.5 h, 200 mA, at 4°C. Then the membrane was blocked overnight at 4°C in TBST buffer (20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.4) containing 5% low-fat milk [43]. To detect both p90rsk and active ERK1/2, blots were cut into two parts containing the proteins above and below the 68 x 10-3 kDa molecular weight marker and incubated separately for 2 h in TBST with 1:300 polyclonal rabbit anti-mouse p90rsk antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for the upper part of the membrane and with 1:500 mouse anti-p-ERK1/2 antibody (Santa Cruz Biotechnology) for the lower part of the membrane. After three washes of 10 min each in TBST, the upper and lower parts of the membrane were incubated for 1 h at 37°C with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated rabbit anti-mouse IgG diluted 1:1000 in TBST, respectively. The membranes were washed three times in TBST and then processed using the enhanced chemiluminescence (ECL) detection system (Amersham, Little Chalfont, UK).
For reprobing of total ERK2, the lower part of the membrane was washed in stripping buffer (100 mM ß mercatoethanol, 20% SDS, 62.5 mM Tris, pH 6.7) to strip off bound antibody after ECL detection at 50°C for 30 min. The membrane was reprobed with polyclonal rabbit anti-ERK2 antibody (Santa Cruz Biotechnology) diluted 1:300, incubated with HRP-labeled goat anti-rabbit IgG, and finally processed as described above.
For detection of cyclin B1, proteins from 200 oocytes were extracted, separated, and transferred onto the nitrocellulose membrane as mentioned above. The membrane were blocked with 5% low-fat milk in TBST for 1 h at 37°C, and then incubated overnight with polyclonal rabbit anti-cyclin B1 antibody (Santa Cruz Biotechnology) diluted 1:300 at 4°C. The steps of the second antibody binding, washing, and ECL processing were the same as those of ERK2 detection.
Confocal Microscopy
After removing the zona pellucida in acidic Tyrodes medium (pH 2.5), oocytes were fixed with 4% paraformaldehyde in PBS (pH 7.4) for at least 30 min at room temperature. Cells were permeabilized with 1% Triton X-100 overnight at 37°C, followed by blocking in 1% BSA for 1 h and incubation overnight at 4°C with rabbit anti-mouse CaM (Calbiochem, La Jolla, CA) antibody or mouse anti-rabbit CaMKII (Zymed, San Francisco, CA) antibody diluted 1:50 in blocking solution. After three washes in PBS containing 0.1% Tween 20 and 0.01% Triton X-100 (washing solution) for 5 min each, the oocytes were labeled with FITC-conjugated goat anti-rabbit IgG or rabbit anti-mouse IgG diluted 1:100. Nuclear status of oocytes was evaluated by staining with 10 µg/ml PI for 10 min. Following extensive washing, samples were mounted between a coverslip and glass slide supported by four columns of a mixture of petroleum jelly and paraffin (9:1). The slides were sealed with nail polish. Cells were observed under a Leica confocal laser-scanning microscope (TCS-NT) (Leica Lasertechnik GmBH, Heidelberg, Germany) on the same day. Nonspecific staining was determined by substituting primary antibodies with normal rabbit IgG. Each experiment was repeated three times, and at least 20 oocytes were examined each time.
Statistical Analysis
All percentages from three repeated experiments were expressed as mean ± SEM and the number of oocytes observed was labeled in brackets as (n =). All frequencies were subjected to arcsin transformation. The transformed data were statistically compared by ANOVA using SPSS software (SPSS Inc., Chicago, IL) followed by Student-Newman-Keuls test. Differences at P < 0.05 were considered statistically significant.
Experimental Design
Experiments 1–4 were designed to study the roles of CaMKII in the meiotic maturation of pig oocyte.
Experiment 1 To study the possible involvement of calmodulin and CaMKII in the initiation of meiotic maturation in pig oocytes, cumulus-enclosed or denuded oocytes isolated from follicles were cultured in maturation medium containing membrane-permeable calcium chelator BAPTA-AM, CaM antagonist W7, CaMKII inhibitor KN-93, or cell permeable CaMKII inhibitory peptide Ant-AIP-II for 36 h. Then the nuclear status of the oocytes was determined. As the control, KN-93 was substituted by its biologically inactive analog KN-92 at the same concentration in some experiments (the same control was also used in the following experiments).
Experiment 2 The necessity of CaMKII activity to the activation of MAPK/p90rsk cascade and the accumulation of cyclin B, the regulatory subunit of MPF, during pig oocyte maturation was studied by Western blotting. Oocytes matured in drug-free or inhibitor-containing medium were collected at various time intervals for analysis.
Experiment 3 To examine the roles of CaMKII in post-GVBD events, especially its relationship with spindle assembly and polar body extrusion, pig CEOs were cultured in drug-free medium for 28 h to allow the occurrence of GVBD. Then the oocytes were transferred to medium containing W7 or KN-93 for an additional culture of 16 h. At the end of the culture, oocytes were harvested for nuclear status examination.
Experiment 4 The spatial association of CaM and CaMKII with the events of meiotic maturation was studied by indirect immunostaining and confocal microscopy. Maturing pig oocytes were fixed at various stages of meiotic maturation, and the subcellular localization of CaM and CaMKII protein in the oocytes was observed.
The aim of experiments 5–8 was to investigate the roles of CaMKII in the process of pig oocyte activation.
Experiment 5 A CaMKII activity assay was employed to study the activity changes of CaMKII after egg activation. Mature oocytes were parthenogenetically activated with a single electrical pulse with or without the presence of CaM antagonist W7, CaMKII inhibitor KN-93, or its biologically inactive analog KN-92, with the concentration of 20 µM each. Samples were collected at different times post activation for CaMKII activity assay.
Experiment 6 To determine the necessity of CaMKII activity in porcine egg activation, mature oocytes were parthenogenetically activated with an electrical pulse (physical stimulation) or thimerosal plus DTT (chemical stimulation) with or without the treatment of W7, KN-93, or Ant-AIP-II. Then the nuclear status was examined at 20 h of activation.
Experiment 7 The regulation of CaMKII activity on MPF, MAPK, and p90rsk after egg activation was investigated. Pig oocytes activated with or without CaMKII inhibition were collected at different times post activation. The expression of cyclin B, ERK1/2, and p90rsk as well as the phosphorylation status of ERK1/2 and p90rsk were studied by Western blot analysis.
Experiment 8 To get the structural clue concerning the function of CaM and CaMKII in porcine egg activation, the subcellular localization of these two proteins in MII-arrested pig oocytes, and their translocation after electrical activation were detected by confocal microscopy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The CaMKII specific inhibitor KN-93, its biologically inactive analog KN-92, and cell-permeable CaMKII inhibitory peptide Ant-AIP-II were used to explore the possible participation of CaMKII in regulating meiotic maturation of pig oocytes. As shown in Figure 1A, GVBD occurred in both CEOs (97.7%, n = 105) and denuded oocytes (DOs; 90.05%, n = 85) cultured in drug-free maturation medium for 36 h. When CEOs or DOs were treated with 20 µM KN-93, the GVBD (3.49% in CEOs, n = 127; and 4.17% in DOs, n = 144) was nearly completely inhibited, but 20 µM KN-92 did not affect GVBD in CEOs (97.50%, n = 80) and DOs (89.28%, n = 84). The GV-intact nucleus status of pig oocytes after KN-93 treatment was illustrated in Figure 2A. A cell-permeable CaMKII inhibitory peptide Ant-AIP-II, also inhibited the meiotic resumption of pig CEOs (15.19%, n = 98) and DOs (30.96%, n = 91).
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To activate CaMKII, Ca2+ must first interact with calmodulin. Therefore, if CaMKII participates in regulating the initiation of meiotic maturation, disrupting the interaction between Ca2+ and CaM should prevent the activation of CaMKII, thereby blocking GVBD. This possibility was tested by treating oocytes with plasma membrane–permeable Ca2+ chelator BAPTA-AM (50 µM) and CaM-specific antagonist W7 (20 µM). The GVBD rate of CEOs (96.75%, n = 154) was not affected by the addition of BAPTA-AM in the maturation medium, but the GVBD of DOs (7.62%, n = 118) was significantly inhibited after the same treatment. W7 inhibited the GVBD efficiently in both CEOs (12.10%, n = 126) and DOs (10.43%, n = 115). Ant-AIP-II, KN-93, and W7 with the increasing concentrations were also used to treat the pig CEOs during maturation culture. Each of the three reagents showed a dose-dependent inhibitory effect on GVBD after 36 h of culture, with the maximal inhibition at the concentration of 20 µM (Fig. 1, B–D).
To test the possible involvement of calcium-CaM-CaMKII in the events following GVBD, pig CEOs were cultured in drug-free maturation medium for 28 h to allow the occurrence of GVBD, and then they were transferred into maturation medium containing 50 µM BAPTA-AM, 20 µM W7, or 20 µM KN-93 and further cultured for another 16 h. Most CEOs released their first polar body (PB1) and organized an MII spindle after maturation culture of 44 h in drug-free medium (78.64%, n = 103, illustrated in Fig. 2D). However, after the treatment of oocytes with BAPTA-AM, W7, or KN-93, only 14.50% (n = 89), 12.39% (n = 93), and 10.85% (n = 95) of the oocytes released their PB1, respectively. Results from nucleus status examination indicated that the cell cycle of oocytes was blocked at premetaphase I or MI stage after treatment of BAPTA-AM, W7, or KN-93 (illustrated in Fig. 2, B and C) following GVBD.
Regulation of CaMKII on MAPK and MPF Activity During Meiotic Maturation of Pig Oocytes
The kinetics of ERK2 and p90rsk phosphorylation of cumulus-enclosed pig oocytes cultured in drug-free or drug-containing medium is compared in Figure 3. Phosphorylation of p90rsk was assessed by examining its electrophoretic mobility shift on SDS-PAGE, and phosphorylation of ERK2 was evaluated by both mobility shift and a specific antibody against phospho-ERK1/2. ERK2 and p90rsk existed in an inactive form in oocytes at the GV stage (lane 1). Phosphorylation of both kinases was detected at 20 h (lane 2) and 24 h (lane 3) of maturation culture, and their full phosphorylation was culminated at 36 h of culture (lane 4). However, when the oocytes were cultured in maturation medium containing 20 µM KN-93 (lanes 5 and 6) or 20 µM W7 (lanes 7 and 8) for 20 or 36 h, activation of ERK2 and the full phosphorylation of p90rsk was significantly inhibited, although the partial phosphorylation of p90rsk could still occur.
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As shown in Figure 4, cyclin B, the regulatory subunit of MPF, accumulated gradually in pig oocytes during meiotic maturation from 0–30 h (lanes 1–3). But cyclin B remained undetectable in oocytes cultured in medium containing W7 or KN-93 for 30 h (lanes 4 and 5). ERK2 in the same samples was also detected as a control for the quantity of total proteins in the treated oocytes. Although this kinase failed to be phosphorylated after 30 h of culture with W7 or KN-93 (lanes 4 and 5), its quantity in the oocytes was not significantly different from that of control groups (lanes 1–3). This result indicated that the inhibitory effect of these two inhibitors on cyclin B accumulation in pig oocytes is molecule-specific.
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Subcellular Localization of CaM and CaMKII in Pig Oocytes during Meiotic Maturation
The distribution of CaM and CaMKII during meiotic maturation was compared in Figures 5 and 6. The specimens were stained with PI to visualize the DNA and confirm the stage of meiotic maturation. In pig oocytes at the GV stage, both CaM (Fig. 5A) and CaMKII (Fig. 5C) were localized to the nucleus and the periphery of the cell. In oocytes having just undergone GVBD (approximately 24 h after maturation culture in our system), CaM (Fig. 5B) and CaMKII (Fig. 5D) were concentrated to the periphery of condensed chromosomes. As visualized by the green channel only (insets in Fig. 5, B and D), CaMKII showed a more intense staining than CaM around the chromosomes.
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In the MI oocytes, prominent staining of CaM (Fig. 6C) and CaMKII (Fig. 6I) was observed around the aligned chromosomes, putatively the position of MI spindle, as confirmed by the staining of -tubulin (lower inset in Fig. 6B). However, different localization of CaM and CaMKII on the spindle was visualized by the green channel. Staining of CaM was detected on the whole spindle (Fig. 6A), but the signal of CaMKII expression can be detected only at the spindle poles instead of the equatorial region (Fig. 6G). During the process of PB1 emission, with the transition from metaphase I to anaphase I, CaM (Fig. 6, D and F) and CaMKII (Fig. 6, J and L) were detected at the region between the separating homologues chromosomes, putatively the position of the elongated spindle, as confirmed by the staining of -tubulin (inset in Fig. 6E). Staining of CaM could also be detected in the cytoplasm around the chromosomes of extruding first polar body. But this pattern of localization is absent in the images of CaMKII staining. The insets in Figure 6, B and H, labeled "control" showed the controls for nonspecific binding of the second antibodies when no primary antibodies were applied. All control images shown were collected in the same experiment as their corresponding labeled samples. The level of nonspecific staining is negligible when compared with the images in which the primary antibodies were applied.
Changes in CaMKII Activity During Electrical Activation and Its Effect on Pronuclear Formation
The aim of this part of the study was to determine whether CaMKII is involved in the release of MII arrest after activation of pig oocytes. We monitored CaMKII activity at increasing time intervals up to 2.5 h after electrical activation, when most oocytes had transited into telophase of meiosis II. The relative CaMKII activity was expressed in Figure 7 as the radioactive intensity (cpm) of each sample analyzed by scintillation counting. A basic level of CaMKII activity was detected in the MII-arrested oocytes. Electrical stimulation resulted in an early and steep increase in kinase activity at 15 min. The high activity was kept up to 1 h and then fell progressively until 1.5 h, increased again 2 h after activation, and was still elevated for another 30 min when most oocytes had transited into telophase II. When the mature oocytes were pretreated for 30 min with 20 µM KN-93 or W7 and then were electrically activated with the presence of KN-93 or W7, the prompt increase of CaMKII activity at 15 min of stimulation was significantly inhibited. If the KN-93 was substituted by KN-92 with the same concentration, the electrical pulse-induced increase of CaMKII activity was not affected, compared with the CaMKII activity in the drug-free group at 15 min of stimulation.
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As shown in Figure 8, a high pronucleus (PN) formation rate of pig eggs was obtained after parthenogenetic activation using both physical (electrical pulse) and chemical (thimerosal and DTT treatment) methods in our system. Most eggs formed one or two pronuclei at 20 h after electrical stimulation (86.67%, n = 107) or thimerosal treatment (73.53%, n = 115). When the harvested oocytes were preincubated with 20 µM KN-93, Ant-AIP-II, or W7 for 30 min at 39°C and then parthenogenetically activated and cultured in the media containing KN-93, Ant-AIP-II, or W7 at the same concentration, the PN formation was significantly inhibited (W7, 2.5%, n = 124; Ant-AIP-II, 10.99%, n = 91; KN-93, 0.70%, n = 141) when treated by electrical stimulation. The same inhibitory effect of W7 and KN-93 on PN formation was also observed in eggs activated by thimerosal plus DTT (W7, 1.65%, n = 120; Ant-AIP-II, 7.21%, n = 97; KN-93, 1.67%, n = 95). The activation rate was not influenced by treatment of KN-92, the biologically inactive analog of KN-93, in both electrical pulse-induced (82.10%, n = 80) or thimerosal-induced (70.55%, n = 80) activation.
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Regulation of MPF, MAPK, and p90rsk by CaMKII After Egg Activation
The observation that CaMKII inhibitor prevented interphase transition after egg activation illustrates the possibility that when CaMKII becomes active as a result of egg activation, it may serve to regulate the activity of MPF, MAPK, and MAPK target kinase p90rsk, which are central to the control of meiotic cell cycles. To test this, the activity and/or amount of these kinases were analyzed during normal development and in the presence of CaM or CaMKII inhibitors. As shown in Figure 9, cyclin B, the 60 kDa regulatory subunit of MPF, could be detected in the MII-arrested oocytes (lane 1) by Western blotting. The staining of this protein significantly decreased at 1 h of electrical activation (lane 2) and totally could not be detected at 2 h of activation (lane 3). However, when the CaMKII or CaM was inhibited by KN-93 or W7 during and after electrical stimulation, cyclin B failed to be degraded at 2 h of culture, with a clear band of staining as detected by immunoblotting (lanes 4 and 5).
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Typically, the electrically activated eggs form 1 or 2 PN at 14 h after activation. The MAPK and p90rsk were kept phosphorylated at 3 h of activation but were partly dephosphorylated at 10 h and were completely dephosphorylated at 14 h after activation (Fig. 10A, left panels). The contents of both kinases kept stable during the whole process of electrical activation.
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To investigate the regulation of MAPK/p90rsk by the CaMKII and CaM, MII oocytes were pretreated with KN-93 or W7 and then activated with an electrical pulse. Samples were collected at 15 min, 30 min, 1 h, 2 h, and 3 h post activation and were subject to immunoblotting. Each band was quantified by chemiluminescence densitometry (Fig. 10, B and C). When the CaMKII inhibitor, KN-93 (Fig. 10A, middle panels), or CaM inhibitor W7 (Fig. 10A, right panels) was present from the time of egg activation, dephosphorylation of ERK2 and p90rsk was detected as early as 15 min after activation, and the amount of ERK2 and p90rsk was significantly reduced at 30 min. The phosphorylated ERK2 was completely undetectable 1 h after activation, and the total amount of ERK2 and p90rsk continued to decrease from 1 to 3 h after activation. KN-92 had little effect on phosphorylation of ERK2 and p90rsk as well as the total amount of both kinases (lane 7 in Fig. 10A, middle panels). When treated with thimerosal, ERK2 and p90rsk kept phosphorylated at 6 h post activation and maintained a stable amount until 20 h after activation (Fig. 11A, left panels). The treatment of KN-93 or W7 resulted in an early dephosphorylation of ERK2 and p90rsk at 2 h post activation and a significant amount decrease of both kinases at 6 h after activation, as observed by Western blotting (Fig. 11A, right panels) and further confirmed by densitometry (Fig. 11B). Thus, inhibition of CaMKII not only quickly inactivates of ERK2 and p90rsk but also decreases the amount of both kinases.
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Localization of CaM and CaMKII in Pig Oocytes During Electrical Activation
The subcellular distribution of CaM and CaMKII in the MII-arrested and electrically activated pig oocytes is shown in Figure 12. In pig oocytes with chromosomes on the metaphase plate and extruded first polar body (arrow) 44 h after maturation culture, CaM existed in the whole spindle area (A), and CaMKII was localized on either side of the chromosomes (C), with no detectable signal at the middle plate as observed by green channel (inset in C). The distribution of both proteins in the MII-arrested oocytes was similar to that observed in oocytes at the MI stage. At 2 h after electrical activation, the second meiosis transited into anaphase (B) or telophase (D), and the CaM and CaMKII were repositioned. CaM was localized around the chromosomes destined to be extruded with the second polar body (PB2) and the cortical region from where PB2 will be emitted (C). CaMKII distributed between the two sets of chromosomes supposed to be occupied by spindle microtubules at anaphase II (data not shown). In activated eggs at telophase II, accumulation of CaMKII could be detected at the cytoplasmic connection between the extruding PB2 and the egg (D), which is also a subcellular area occupied by microtubule bundles, as shown in Figure 12D (inset). No specific localization of CaM and CaMKII was detected after pronuclear formation (data not shown).
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DISCUSSION |
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The initiation of meiotic maturation was significantly inhibited by CaM antagonist W7, CaMKII inhibitory peptide Ant-AIP-II, or CaMKII inhibitor KN-93 in both cumulus-enclosed and denuded oocytes, and the inhibitory effect of each reagent on the meiotic resumption of pig CEOs is dose dependent. It was reported that an intracellular calcium transient prior to GVBD is the prerequisite for spontaneous maturation of pig oocytes [26]. Here we provided the evidence that this GVBD-initiating Ca2+ signal might be mediated by CaM and CaMKII. However, only the gonadotropin-induced maturation, instead of spontaneous meiotic resumption, was prevented by KN-93 in mouse oocytes [23]. The same authors failed to observe the inhibitory effect of W7 on GVBD in both FSH-induced and spontaneous models. It seems that CaM or CaMKII inhibition is more efficient in blocking GVBD in pig oocytes than in mouse oocytes. As Carroll and Swann [44] pointed out, the time required for GVBD in mouse oocytes is shorter, compared with that in large mammals, supporting the possibility that certain calcium-dependent steps have already occurred in mouse oocytes in response to spontaneous calcium oscillations prior to their removal from the follicles in these experiments. According to our results, only the GVBD of DOs was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. There may exist a redundant mechanism of meiotic initiation, and signals could be transported into oocytes by the gap junctions connecting the oocytes with the cumulus cells [45]. We also cannot exclude the possibility that the Ca2+ in cumulus-oocyte complexes was not thoroughly chelated by BAPTA-AM.
The functions of CaMKII in the initiation of meiotic maturation are still unknown. Results from confocal microscopy revealed that CaM and CaMKII accumulated in the nucleus before GVBD. In interphase rat embryo fibroblast 3Y1 cells, CaMKII was also localized in the nucleus [46]. CaMKII may transduce some of the Ca2+ signals into the nucleus [47]. In maturing pig oocytes, MPF was activated before GVBD with the increasing synthesis of cyclin B, and MAPK and p90rsk were activated by phosphorylation at the time around GVBD [16, 48]. We showed in this study that CaM or CaMKII inhibitor suppressed the accumulation of cyclin B as well as the phosphorylation of MAPK and p90rsk at the same time of blocking GVBD, suggesting that calcium-dependent mechanisms may also participate in the initial activation of MPF and MAPK cascade during meiotic maturation, which has never been inferred before. However, the conclusion that CaMKII facilitates GVBD by activating MAPK cannot be reached because there is evidence that MAPK activity is not a prerequisite for spontaneous GVBD in both mouse and pig oocytes [8, 9]. CaMKII may also be involved in the regulation of MPF activity in term of p34cdc2 dephosphorylation, which is more important than MAPK in controlling the meiotic initiation of mammalian oocytes [49].
It is well recognized that extracellular calcium is necessary for first polar body emission in mouse and pig oocytes following GVBD [7]. We proved here in pig oocytes that the function of inflexed Ca2+ is mediated by CaM and CaMKII, which is also the case in mouse oocytes [23]. CaM and CaMKII may also have a key role in spindle formation or chromosome separation because they are associated with the meiotic apparatus, especially in the polar region and midbody of the spindle. In both mitotic and meiotic cell cycles, a large number of proteins are specifically phosphorylated. These phosphoproteins are associated with microtubule organizing centers (MTOCs) such as centrosomes, kinetochores, and midbodies [50]. Ca2+/calmodulin-dependent phosphorylation of a 62-kDa protein induces microtubule depolymerization in sea urchin mitotic apparatus [51]. During mitosis of rat 3Y1 cells, rat glioma cells, and human epidermatoid carcinoma KB cells, CaMKII was found to be a dynamic component of the mitotic apparatus, particularly present at MTOCs [36]. In extracts of Xenopus oocytes arrested at S phase, CaMKII activity is required for the initiation of centrosome duplication [52]. Our results, together with previous reports, suggest CaM/CaMKII may play important roles in spindle organization and polar body extrusion in the two meiosis.
Both methods of parthenogenetic activation employed in our experiment release the pig oocytes from MII arrest by mimicking transient intracytoplasmic Ca2+ elevation occurring after fertilization [42]. A quick increase in CaMKII activity was observed in pig oocytes after electrical activation. CaMKII inhibitor KN-93 and CaM antagonist W7 efficiently prevented the CaMKII activity elevation and interphase transition of pig oocytes after activation, suggesting that CaM and CaMKII are the direct targets of calcium signal in inducing meiotic resumption at the MII stage. Former studies showed that CaMKII inhibition blocked the release of mouse oocytes from MII arrest induced by ethanol [32], calcium ionophore A23187 [31], or sperm penetration [33]. In Xenopus oocytes, a constitutively active mutant of CaMKII stimulated meiotic resumption and cyclin degradation even in the absence of Ca2+ [30]. Because intracytoplasmic calcium transient is the initiating signal of egg activation in all animals studied, a similar conserved mechanism involving CaMKII activation may also be employed in response to calcium signal throughout animal kingdom.
The MII arrest of mammalian oocytes is maintained by the activity of both MPF and Mos/MEK/MAPK/p90rsk cascade [53]. After egg activation, MPF was quickly inactivated by a calcium-induced and anaphase-promoting complex/cyclosome-dependent degradation pathway, which is the prerequisite for metaphase/anaphase transition [54]. However, the MAPK phosphorylation level and the amount of this kinase remain stable after egg activation until the initiation of pronuclear formation. It is not fully known by what kind of mechanism that MPF was inactivated by calcium transients and how the MAPK activity was sustained after egg activation. We report here that the parthenogenetic stimulus-induced degradation of cyclin B, the regulatory subunit of MPF, was significantly inhibited by both KN-93 and W7, suggesting that CaMKII, after activated by calcium and CaM, may facilitate anaphase transition by activating cyclin B proteolysis mechanism. ERK2 and p90rsk were dephosphorylated as early as 15 min post activation and their amount decreased dramatically shortly after. The same phenomenon was reported in mouse oocytes activated with Ca2+ ionophore A23187 [40]. Colocalization of MAPK, p90rsk, and CaMKII in mouse [40] and pig ([16] and this report) oocytes suggests that these kinases could directly interact in specialized area in the cell. CaMKII could serve to potentiate MAPK and p90rsk activity after egg activation because the primary sequence of ERK2 indicates consensus phosphorylation site for CaMKII at Thr92 (GeneBank Accession #X58712) [40]. In combining these results, we hypothesized that CaMKII might play dual roles in mediating calcium signal after egg activation: on one hand, it inactivates MPF and releases the oocytes from MII arrest by stimulating a cyclin B degradation pathway; but on the other hand, CaMKII maintains the phosphorylated state and amount level of MAPK/p90rsk through an unknown mechanism. The two key protein kinases controlling meiotic cell cycles may be differently regulated by the same calcium modulator, CaMKII.
In this study, only the involvement of CaMKII in parthenogenetic activation of pig oocytes was examined. However, the physiological stimulus of oocyte activation is sperm penetration. The dynamics of CaMKII and the effect of CaMKII inhibitors are different between fertilized and parthenogenetically activated mouse oocytes. In mouse oocytes activated with ethanol, a peak of CaMKII activity was detected as early as 6.5 min after activation, and then its activity decreased soon. But in fertilized eggs CaMKII was stimulated further by the succeeding calcium oscillations after initial transient activation [33]. CaMKII inhibitor myristoylated-AIP prevented the interphase transition in ethanol-activated mouse eggs but not in fertilized eggs [33]. However, the evaluation of the roles of CaMKII in fertilized pig eggs is proved difficult. The time of sperm penetration after insemination is highly asynchronous in pig eggs, ranging from 3 to 5 h post insemination [55]. Only by employing the parthenogenetic activation model could we get a large number of eggs activated at a precise time point for biochemical analysis. Although we observed that the pronuclear formation was also inhibited in pig eggs after fertilization in vitro in the presence of CaMKII inhibitor KN-93 (data not shown), we could not rule out the possibility that this effect is due to the inhibition of KN-93 on sperm capacitation or acrosome reaction. Further study is needed to reveal the roles of CaMKII in pig eggs under physiological conditions.
Egg activation includes both early events such as cortical granule exocytosis and zona inhibition of polyspermy, and late events such as chromosome segregation, second polar body emission, pronuclear formation, and syngamy. Evidence from a variety of species has shown that Ca2+ acts more like a switch activating a plethora of calcium-dependent intracellular signals that then mediate individual structural changes. Other reports and this study provide concrete proofs that CaMKII is a mediator of Ca2+ effect. Another molecule downstream to calcium signal in egg activation is protein kinase C (PKC). There is also evidence that activation of PKC after fertilization is required for remodeling the mouse egg into the zygote. Oocytes pretreated with PKC inhibitors were not capable of forming a second polar body, and their pronuclear formation was significantly inhibited following fertilization [56]. PKC activity is responsible for not only MPF inactivation [57] but also cortical granule (CG) exocytosis in mouse oocytes [58, 59] and rat oocytes [60]. Here an interesting question arose: Do CaMKII and PKC function in a parallel manner, or do they cooperate in mediating calcium-induced events? In mouse oocytes, CG exocytosis induced by sperm penetration and PKC activators can also be prevented by CaMKII inhibitor KN-93 [61]. Furthermore, as discussed above, both PKC and CaMKII are indispensable for the release of MII arrest. We reported in pig oocytes that, although PKC activity is necessary for the induction of CG exocytosis, PKC activators failed to induce the MII arrest release and interphase transition [62, 63]. In combination of these results, we hypothesized that calcium-induced activation events is mediated by the cooperation of CaMKII and PKC in mouse oocytes. However, in pig oocytes, further study is necessary before the relationship between these two calcium target kinases can be determined because the effects of CaMKII inhibition on CG exocytosis and PKC inhibition on interphase transition have not been tested.
In conclusion, CaMKII is involved in multiple steps of meiotic maturation and activation of pig oocytes, such as the initiation of spontaneous meiotic resumption, emission of polar bodies, release of MII arrest, and formation of pronucleus. In response to calcium signal, CaMKII may play dual roles after egg activation (i.e., the induction of cyclin B degradation and the maintenance of MAPK/p90rsk activity). The localization of CaM and CaMKII in meiotic cell cycle progression is tightly associated with meiotic apparatus, suggesting their roles in cytoskeleton organization and cell division. Thus, CaMKII may be a key mediator of Ca2+ signal in meiosis and fertilization of pig oocytes.
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FOOTNOTES |
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2 Correspondence: Qing-Yuan Sun, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China. FAX: 8610 6256 5689; sunqy1{at}yahoo.com
Received: 13 March 2003.
First decision: 5 April 2003.
Accepted: 10 June 2003.
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REFERENCES |
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-CaM kinase isozyme. FEBS Lett 1995 373:71-75[CrossRef][Medline]
