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Originally published In Press as doi:10.1074/jbc.M301851200 on July 16, 2003

J. Biol. Chem., Vol. 278, Issue 43, 42505-42514, October 24, 2003
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The M-phase-promoting Factor Modulates the Sensitivity of the Ca2+ Stores to Inositol 1,4,5-Trisphosphate via the Actin Cytoskeleton*

Dmitri Lim{ddagger}, Emanuela Ercolano{ddagger}, Keiichiro Kyozuka§, Gilda A. Nusco{ddagger}, Francesco Moccia{ddagger}, Klaus Lange¶, and Luigia Santella{ddagger}||

From the {ddagger}Laboratory of Cell Biology, Stazione Zoologica A. Dohrn, I-80121 Naples, Italy, the §Asamushi Marine Biological Station, Aomori 039-3501, Japan, and Kladower Damn 25b, 14089 Berlin, Germany

Received for publication, February 21, 2003 , and in revised form, June 18, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The resumption of the meiotic cycle (maturation) induced by 1-methyladenine in prophase-arrested starfish oocytes is indicated by the breakdown of the germinal vesicle and is characterized by the increased sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate (InsP3) to InsP3 starting at the animal hemisphere (where the germinal vesicle was originally located) and propagating along the animal/vegetal axis of the oocyte. This initiates Ca2+ signals around the germinal vesicle before nuclear envelope breakdown. Previous studies have suggested that the final activation of the maturation-promoting factor (MPF), a cyclin-dependent kinase, which is the major element controlling the entry of eukaryotic cells into the M phase, occurs in the nucleus. MPF is then exported to the cytoplasm where its activity is autocatalytically amplified following a similar animal/vegetal spatial pattern. We have investigated whether activated MPF was involved in the increased sensitivity of the Ca2+ response to InsP3. We have found that the development of increased sensitivity of the Ca2+ stores to InsP3 receptors together with the Ca2+ signals in the perinuclear region was blocked in oocytes treated with the specific MPF inhibitor roscovitine. That the nuclear MPF activation is indeed required for changes of the InsP3 receptors sensitivity was shown by enucleating or by dissecting oocytes into vegetal and animal hemispheres prior to the addition of 1-MA. MPF activity 50 min after 1-methyladenine addition was much lower in the enucleated oocytes and in the vegetal hemisphere, which did not contain the germinal vesicle, as compared with the animal hemisphere, which did contain it. The Ca2+ increase induced by InsP3 under these experimental conditions correlated with the changes in actin cytoskeleton induced by MPF.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Starfish oocytes acquire the ability to successfully respond to the sperm following activation by 1-methyladenine (1-MA)1 (1). The resumption of meiotic cycle is characterized by the breakdown of the envelope of the large germinal vesicle (nucleus) (GVBD), which occurs about 20 min after 1-MA application. Earlier work in our laboratory has detected an increase in both cytoplasmic and nuclear calcium 2 min after the addition of 1-MA. The Ca2+ rise in the germinal vesicle was essential to the resumption of meiosis, because the nuclear injection of BAPTA blocked it (2). The progression of meiotic cell cycle is controlled by the M phase/maturation-promoting factor (MPF) (3-4), which is a Ser/Thr kinase composed of a regulatory cyclin B subunit and of a catalytic CDK1 subunit (5). During the prophase of the first meiotic division, CDK1 becomes complexed to the cyclin B subunit. Although phosphorylation on Thr-161 is a prerequisite for the activation of CDK1/cyclin B (6), it is activated after dephosphorylation on Thr-14 and Tyr-15 by cdc25 dual-specificity phosphatase (7-9). The latter is in turn activated by a phosphorylation kinase through a calcium/calmodulin-dependent step (10). The translocation of MPF in the germinal vesicle along with cdc25 is thought to be essential for final MPF activation (11-13). Studies in a number of cell types have shown that the perinuclear/centrosomal region also accumulates MPF and many of its regulators (14, 15). The initial activation of MPF in Asterina pectinifera, however, has been suggested to occur in the cytoplasm via the Akt (protein kinase B)-dependent inactivation of Myt1 (a member of Wee1 family) that phosphorylates CDK1 on Thr-14 and Tyr-15 (16). The response of Ca2+ stores to InsP3 has been shown to increase during oocyte maturation, so that more calcium is released when InsP3 is injected into a mature oocyte (17). This is not due to either redistribution or an increase in the expression of InsP3 receptors (18). The significance for the increase is not clear, because, at variance with the fertilization, the role of InsP3-dependent calcium release during meiosis resumption is controversial (18).

The spatiotemporal dynamics of Ca2+ release during maturation have been explored in the present work by photoliberating InsP3 at selected time intervals after the addition of 1-MA. The increase in the sensitivity of the InsP3 receptors has been confirmed, but found to be spatially inhomogeneous; i.e., it starts in the perinuclear area at the animal pole and propagates to the entire oocyte along the animal-vegetal axis. The increase in the sensitivity to InsP3 spatially and temporally coincides with a Ca2+ increase induced by 1-MA around the germinal vesicle a few minutes before GVBD. The finding that Ca2+ signaling initiates in the perinuclear area suggested the involvement of nuclear-produced factors, in line with the finding that the response of oocytes to InsP3 prior to GVBD was strongly affected by the removal of the germinal vesicle (19). Recent reports showing that MPF controls calcium signals at fertilization in ascidian oocytes (20) and sea urchin eggs (21) had indicated that the nuclear factor was MPF. The experiments with MPF inhibitor roscovitine described here support the indication and suggest that the increased sensitivity of the Ca2+ stores to InsP3 is mediated by actin cytoskeleton changes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Oocytes—Japanese starfishes (A. pectinifera) were collected during the breeding seasons in May through June and September through October in the Mutsu Bay (Aomori, Japan) and kept in running natural seawater (16 °C). Astropecten auranciacus starfishes were collected during the breeding season (January through June) in the Gulf of Naples. Immature oocytes (containing the germinal vesicle) were dissected manually from the gonads and kept in artificial filtrated seawater (460 mM NaCl, 10.1 mM KCl, 9.2 mM CaCl2, 35.9 mM MgCl2, 17.5 mM MgSO4, 2.5 mM NaHCO3, pH 8.0) for 30 min prior use. Oocytes in which the breakdown of the germinal vesicle (GVBD) occurred spontaneously were discarded. Maturation was promoted by adding the hormone 1-methyladenine (Sigma Chemical Co., St. Louis, MO) at a final concentration of 1 µM for A. pectinifera oocytes and 10 µM for A. auranciacus oocytes. Experiments were performed at 20 °C. The germinal vesicle from immature oocytes was manually removed using fine steel needles. To separate animal and vegetal hemispheres of the oocytes they were cut using fine glass needles as described elsewhere (22). Only oocytes with an equal volume of animal and vegetal contents were used for the experiments. The two separate hemispheres were kept for 40-60 min at 16 °C prior to performing experiments.

Photolysis of Caged InsP3 during Maturation and Ca2+ Imaging—To perform photolysis experiments of caged InsP3 during the maturation process, the calcium fluorescent dye, Oregon Green 488 BAPTA-1, coupled to a 70-kDa dextran (OGBD, Molecular Probes, Eugene, OR) was injected at a concentration of 5 mg/ml in the injection buffer (450 mM potassium chloride, 10 mM HEPES, pH 7.0) together with 5 µM of caged InsP3 (Calbiochem, La Jolla, CA) into the cytoplasm of immature or mature oocytes. The volume of the injected dye and of the caged InsP3 corresponded to 1-2% of the total cell volume. Thus, the final concentrations of injected substances in the cellular environment were 50-100 nM for InsP3 and 50-100 µg/ml for OGBD. Immature oocytes were injected with a mixture of caged InsP3 and OGBD 5-10 min prior to 1-MA application. During the re-initiation of the meiotic cycle induced by the addition of 1-MA, the photo liberation of InsP3 was performed using different oocytes from the same animal, which were irradiated at 2- to 3-min intervals. Cytosolic Ca2+ changes were measured using a cooled CCD camera (MicroMax, Princeton Instruments, Inc., Trenton, NJ) mounted on a Zeiss Axiovert 200 microscope with a Plan-Neofluar 20x/0.50 objective. Photoliberation of caged InsP3 was performed using a computer-controlled shutter (Lambda 10-2, Sutter Instruments, Co., Novato, CA) by irradiating with a wavelength of 330 nm for 0.5 s several times and acquiring images between irradiation. Fluorescence images were processed with MetaMorph Imaging System software (Universal Imaging Corp., West Chester, PA). To exclude variations of fluorescent intensity, the signals were corrected for variations in dye concentration by normalizing fluorescence (F) against baseline fluorescence (F0). The region of interest used to measure the fluorescence level was positioned as shown in the figures. In experiments with the separated animal and vegetal hemispheres, the mixture of caged InsP3 and OGBD was injected before the dissection of the oocyte to obtain an equal amount of the caged compound in the two separated halves of the oocyte. For the detection of the local Ca2+ changes that preceded GVBD, the ratiometric fluorescence image observation was applied according to the procedures previously described (23) with the slight modifications of fluorescent dyes from calcium green dextran and rhodamine to OGBD and rhodamine dextran 70,000 (RHD). Briefly, a mixture of OGBD (5 mg/ml) and RHD (3 mg/ml) was introduced into the cytoplasm or germinal vesicle of immature oocytes. After the oocytes were set in the measuring chamber under a fluorescent microscope with the cooled CCD camera system described above, 1-MA (1 µM final) was applied to the chamber. Two sets of fluorescence images were collected with the excitation filters of 485 and 555 nm and the emission filters of 535 and >565 nm, respectively. The ratio of the fluorescence images was calculated in a pixel-to-pixel manner using MetaMorph analysis system. Fluorescence images were collected every 1-5 s until the breakdown of the nuclear envelope was completed.

Inhibition of MPF—Roscovitine (Sigma), the specific MPF inhibitor (24) was prepared as a 50 mM stock solution in Me2SO, aliquoted, and kept at -20 °C. Because A. pectinifera starfish oocytes incubated with roscovitine resumed meiosis following the addition of the hormone, although with a delay (19), injection of the MPF inhibitor was used. For injection the stock solution was dissolved (500 µM) in injection buffer, and 1-2% of the oocyte volume was directly injected into the cytoplasm or the germinal vesicle of immature oocytes (final concentration, 5-10 µM). Control oocytes were injected with equal amount of Me2SO dissolved in injection buffer. Injection of Me2SO had no effect on GVBD. The actin-depolymerizing drug latrunculin-A (Lat-A) was purchased from Molecular Probes. A 3 mM stock solution in Me2SO was prepared and kept frozen. Filtered seawater containing 6 µM Lat-A was prepared just before the experiment. Immature oocytes were gently transferred to seawater containing Lat-A and 1-MA and kept in a free position during the Ca2+ measurement acquisition.

Cdc2 Kinase Activity Assay—The cdc2-kinase assay has been performed using a commercially available kit from Promega (SignaTect® Cdc2 protein kinase assay system) that utilizes the peptide PKTPKKAKKL as a specific substrate for cdc2. The "threonine" phosphorylated by cdc2 is embedded in the canonical consensus site for cdc2 phosphorylation (PKTPK). The peptide is specific substrate for cdc2 and is not phosphorylated by other kinases, e.g. mitogen-activated protein kinases, allowing the determination of cdc2 kinase activity directly on a total cell lysate.

Preparation of Samples and in Vitro Kinase Assay—Immature entire, enucleated oocytes, the animal and vegetal halves from cut oocytes prior to the addition of the hormone and the same samples 50 min after 1-MA application, were lysed with 15 µl of lysis buffer LB-150 containing 50 mM Tris, pH 7.6, 150 mM NaCl 0.5 mM EDTA, 1% Nonidet P-40, 0.5 mM dithiothreitol, 100 µg/ml phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors (Roche Applied Science, Ingelheim, Germany). The protein concentration of the total cell lysate was estimated with the Bradford Microassay (Bio-Rad, Mississauga, Ontario, Canada), and 5 µl of lysate (about 4 µg or 5 µg) was used for the cdc2 kinase assay.

100 µg of lysates of starfish oocytes were incubated in 30 µl of kinase buffer (50 mM Hepes, pH 7.5, 5 mM MgCl2, 100 mM NaCl, 5 mM MnCl2, 1 mM dithiothreitol, 20 mM {beta}-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 2 µg/ml pepstatin A, 2 µg/ml aprotinin, 1 µg/ml lysozyme, 1 µM okadaic acid) containing 50 mM ATP, 5 µCi [{gamma}-32P]ATP (3000 Ci/mmol, Amersham Biosciences, Baie d'Urfè, Quebec, Canada), and 2 µl of MPF (CDK1/cyclin B kinase, kindly provided by Dr. L. Meijer) or 0.5 µl of human recombinant p34cdk1/cyclin B (Calbiochem, La Jolla, CA). After incubation for 30 min at 30 °C the reaction mixture was solubilized in SDS-sample buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 1 mM dithiothreitol, 1% SDS, 0,002% bromphenol blue) for 5 min at 95 °C, and separated on SDS-PAGE, using 8% gels. Proteins were transferred onto polyvinylidene difluoride membrane (Amersham Biosciences), and the membrane was subjected to autoradiography to detect 32P incorporation.

Immunoblot Analysis—Samples were diluted in two volumes of SDS-sample buffer, boiled for 3-5 min, and run on a SDS-polyacrylamide gel. Proteins were stained with Coomassie Brilliant Blue to verify that each lane was loaded with approximately the same amount of samples. Proteins were electrophoretically transferred to Hybond-P polyvinylidene difluoride membrane membranes. The membranes were then blocked with nonfat dry milk in Tris-buffered saline containing 1% Tween 20 (TBS-T) (pH 7.6) and were then incubated with affinity-purified polyclonal antibodies against starfish InsP3 receptors (10 µg/ml) kindly provided by Dr. K. Mikoshiba. The membranes were incubated overnight at 4 °C in a orbital shaker, after which they were washed three times with TBS-T and probed with horseradish peroxidase secondary antibody conjugates (Amersham Biosciences). Labeled proteins were visualized using the ECL plus system and Hyperfilm ECL (Amersham Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Calcium Release Promoted by the Uncaging of Injected InsP3 in Immature and Mature Oocytes—The amount of InsP3 necessary to release the same amount of Ca2+ in mature starfish oocytes is 20- to 50-fold lower than in immature oocytes. The sensitivity of InsP3 receptors evidently increases during the maturation process (17). It has been recently shown that the increased response to InsP3 was not linked to the up-regulation and/or to the redistribution of the receptors in starfish oocytes (18). The experiment in Fig. 1A further shows that the difference between the two maturation stages is not due to a larger Ca2+ load in the ER, because higher concentrations of InsP3 (1.2 mM in the pipette) liberates the same amount of Ca2+ in immature (1.22 ± 0.14, n = 7) and mature (1.22 ± 0.08, n = 4) oocytes.



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  		FIG. 1.
Ca2+ increase induced by the photoliberation of caged InsP3 in an immature and a mature oocyte. A, Ca2+ increase induced by the photoactivation of increasing concentrations of caged InsP3 injected into an immature (open circles) and mature (filled squares) oocyte. The curves show a dependence of the Ca2+ response on the amount of photoliberated InsP3, where each point represents mean ± S.D. of four to seven oocytes. B, photoliberation of InsP3 (arrow, 5 µM in the pipette) in an immature oocyte induces a Ca2+ increase in both the cortical region of the oocyte (blue line) and in the center (red line); C, Ca2+ increase induced by the uncaging of 5 µM InsP3 in a maturing oocyte exposed for 13 min to 1-MA. The Ca2+ response was much higher in the perinuclear region (blue line) than in the center of the oocyte (red line); D, the uncaging of 5 µM InsP3 shows a higher sensitivity of the InsP3 receptors in both the cortical (blue line) and the central region (red line) of a mature oocyte. Regions of interest were positioned as shown in the phase-contrast images. Bar = 45 µm.

 

The final concentration of InsP3 liberated in the cytoplasm by the uncaging reaction cannot be estimated with absolute precision, but a concentration of 5 µM caged InsP3 in the pipette elicits a Ca2+ response of 0.3 ± 0.1 (n = 12) in immature oocytes, while producing a near maximal response in mature cells (0.98 ± 0.09; n = 12). The pattern of development of the increased sensitivity to InsP3 was thus studied in oocytes during the maturation process. The graph of Fig. 1B shows the CCD camera analysis of the Ca2+ response induced by the photo liberation of caged InsP3 (5 µM in the pipette) in an oocyte not exposed to the hormone. 10-30 min after the injection, the caged compound was presumably diffused throughout the entire cytoplasm and could thus initiate a uniformly distributed Ca2+ increase following the irradiation. The graph in panel B shows the fluorescence increase in the perinuclear area beneath the plasma membrane (blue line) and in the center of the oocyte (red line). In the cortical region (blue circle) the Ca2+ increase reached a peak of 0.38 ± 0.06, (n = 12), which decayed to the baseline in about 60 s. The increase in the center of the oocyte peaked at value of 0.26 ± 0.09 (n = 12) and decayed to the baseline 60 s later.

Fig. 1C shows the Ca2+ response to the uncaging of InsP3 13 min after the addition of 1-MA. The Ca2+ increase in the perinuclear area (blue circle) reached a peak of 0.88 ± 0.12 (n = 10) 4 s after the photoliberation reaction and decayed to the baseline level in about 60 s. The Ca2+ response in this restricted region at the animal hemisphere (blue line) was much higher than in the center of the oocyte, where the Ca2+ increase peaked at 0.45 ± 0.1 (n = 10) 15-17 s after irradiation (red line). Because the irradiation was performed after the caged InsP3 was presumably distributed to the entire oocyte, at this stage of maturation the InsP3 receptors were evidently more sensitive to InsP3 in the animal hemisphere. Injection of non-caged InsP3 in the center of the oocyte 13 min after 1-MA addition induced the same pattern of Ca2+ response (data not shown).

The photoliberation of 5 µM InsP3 (in the pipette) 50 min after the application of 1-MA, i.e. when the oocytes were fully mature (Fig. 1D), caused a rapid release of calcium in both the cortical region and in the center of the cell, which was followed by the elevation of the fertilization envelope 3 min later (arrow in the phase-contrast image). The graph of the relative fluorescence shows a peak amplitude of 1.0 ± 0.09 (n = 12) in the cortex (blue line) of the oocyte and of 0.97 ± 0.09 (n = 12) in the center (red line).

Development of the Increased InsP3 Response during the Process of Oocyte Maturation—Caged InsP3 was injected into immature oocytes (not treated with 1-MA) loaded with a mixture of the calcium dye and caged InsP3. In a first series of experiments, InsP3 was liberated 15 min after the injection and the Ca2+ response was continuously monitored after UV irradiation. Fig. 2 shows that no Ca2+ increase (first and second relative fluorescence images in panel a) was detected following the photolysis of InsP3. Only 3.9 s after the irradiation a Ca2+ increase (n = 10) was measured in the narrow region between the plasma membrane and the germinal vesicle (see the first overlay image and the third relative fluorescence image in panel a). The increase formed a "ring" of Ca2+ beneath the plasma membrane (see images 4.5 and 7.0 s after irradiation), which subsequently propagated to the center of the oocyte. Panel b shows an experiment in which another oocyte from the same animal has been irradiated 10 min after the addition of 1-MA. The slightly higher Ca2+ response was observed with the same pattern of propagation seen in the previous experiment on the immature oocyte. When InsP3 was uncaged between 12 and 14 min after the application of the hormone, an elevation of Ca2+ was detected in the animal region 0.6 s after the InsP3 photoliberation (second relative fluorescence images in panels c and d) indicating that the development of the increased sensitivity of the InsP3 receptors to InsP3 was initiated. The indication was supported by the higher release of Ca2+ measured at the cortical level (see also the graph of Fig. 1C). 22 min after 1-MA addition the Ca2+ response still remained polarized but now completely enveloped the germinal vesicle in the animal hemisphere (see also the overlay image of panel f). The signal further propagated along the animal-vegetal axis expanding to the entire oocyte only when InsP3 was photoactivated 30 min after hormonal stimulation (panel g).



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  		FIG. 2.
Spatiotemporal pattern of the InsP3 induced Ca2+ increase during oocyte maturation. a, the overlay and relative fluorescence images show that the Ca2+ increase following the InsP3 photorelease was detected 3.9 s after UV irradiation (third image) in the animal hemisphere of an immature oocyte not treated with 1-MA. b, the slightly higher Ca2+ response was detected in another oocyte irradiated 10 min after 1-MA exposure. c and d, two oocytes 12 and 14 min after 1-MA addition, respectively. At this time the animal region of the oocytes was much more sensitive to InsP3, because a Ca2+ response was already detected 0.6 s after UV irradiation. The area of the higher sensitivity to InsP3 spread toward the vegetal hemisphere (e and f) and became global 30 min after 1-MA treatment (g). The figure shows one of more than ten representative series of experiments. Bar = 45 µm.

 

Calcium Increase in the Perinuclear Region Just before Meiosis Entry—The sensitization of InsP3 receptors in the perinuclear area was interesting, because it spatially and temporally coincided with a perinuclear increase in calcium concentration detected before GVBD. Fig. 3A shows the ratiometric images of the perinuclear Ca2+ increase observed 12 min after the addition of 1-MA. The Ca2+ changes in panel A are the ratio between the fluorescence of the Ca2+-sensitive dye OGBD shown in panel B and that of the Ca2+-insensitive dye RHD (panel C), which have been co-injected prior to the addition of 1-MA. The images in panel B show the fluorescence Ca2+ changes revealed by OGBD, whereas panel C shows the baseline fluorescence of RHD, which remained constant until GVBD. The ratio between the two fluorescent dyes allowed the evaluation of the fluorescent changes only linked to the changes in the concentration of Ca2+, eliminating nonspecific contributions, as those provoked by contraction movements that may occur before GVBD. The Ca2+ increase starts at the animal side of the perinuclear area 12 min after 1-MA addition (second image in panel A), envelopes the germinal vesicle about 1 min later (fourth ratiometric image of panel A) and decays 14 min after hormonal treatment (sixth image of panel A) before the GVBD commenced. The preference of the Ca2+ elevation for the perinuclear area was emphasized by the fact that both dyes were conjugated to 70,000 Mr dextran and could not cross the intact nuclear envelope. The final images show that dye entry into the germinal vesicle following the breakdown of the nuclear envelope only occurred after calcium was increased around the germinal vesicle. The graph of Fig. 3D shows that the Ca2+ increase (indicated by the circle) started 12 min after the application of 1-MA, reached the peak 1 min later, and decayed to the baseline at the time of the nuclear envelope breakdown (GVBD), as indicated by the entry of the dye into the germinal vesicle (see the arrows in the fluorescence images of panels B and C). The graphs of Fig. 3E show the fluorescence intensity of the Ca2+ response revealed by OGBD (green line) and RHD (red line).



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  		FIG. 3.
Ratiometric measurement of the perinuclear Ca2+ increase. A, ratiometric images show that the perinuclear Ca2+ increase occurred 12 min after 1-MA addition and preceded the GVBD, which occurred at 16 min and 45 s as indicated by the nuclear entry of the dyes previously injected into the cytoplasm (see the arrows in the last images of B and C). The graph in D shows a numerical representation of the ratio of OGBD (green line in E) and RHD fluorescence (red line in E). The perinuclear Ca2+ increase (see the dotted circle in D and E) preceded GVBD (arrows in B and C). Bar = 45 µm.

 

Ca2+ Responses to InsP3 in Enucleated Oocytes and in the Animal and Vegetal Halves—To investigate whether the increased sensitivity of the receptors to InsP3 in the animal hemisphere prior to GVBD was dependent on the action of components that were activated in the germinal vesicle, immature oocytes pre-injected with caged InsP3 were manually enucleated prior to hormonal treatment. Uncaging of InsP3 50 min after 1-MA addition showed a Ca2+ release in enucleated oocytes lower than that measured in control mature oocytes but higher than in control immature oocytes (see Fig. 4B). Additionally, immature oocytes were manually separated with a fine glass needle into animal and vegetal halves (see the light microscope images of Fig. 4A). InsP3 was then uncaged 50 min after the application of the maturing hormone in the two separated halves. The fourth and fifth columns in Fig. 4B show that the uncaging of InsP3 induced strikingly different Ca2+ responses in the two separated halves of the oocyte. The Ca2+ increase in the animal half peaked at a value of 1.36 ± 0.07 (n = 11) and at a much lower value in the vegetal half (0.66 ± 0.04, n = 11).



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  		FIG. 4.
Ca2+ increase induced by the photoliberation of caged InsP3 correlates with MPF activity. Immature oocytes were manually enucleated or cut into the animal and vegetal halves by a fine glass needle (A). B, the enucleated oocytes and the separated halves together with the control entire oocytes were exposed to 1-MA for 50 min. Uncaging of InsP3 (5 µM in the pipette) revealed higher Ca2+ response in whole oocytes and animal halves containing the nucleus (second and fourth columns). The third and fifth columns show the Ca2+ rise measured in enucleated oocytes and vegetal halves not containing the nucleus. The lowest Ca2+ response is detected in immature control oocytes. C, the same samples used for the Ca2+ measurement shown in B were subjected to the in vitro cdc2 kinase activity assay as described under "Experimental Procedures." The histogram shows that MPF activity strongly correlates with the InsP3-induced Ca2+ increase. The data are representative of three independent experiments.

 

MPF Activity in Enucleated Oocytes and in Separated Animal and Vegetal Halves—The results shown above are in line with the hypothesis that a factor of nuclear origin played a role in the increased sensitivity of InsP3 receptors. One likely candidate was MPF, because it controls the Ca2+ signals during the fertilization of ascidian oocytes (20) and sea urchin eggs (21). It was thus decided to assess whether MPF was involved in changes of sensitivity of InsP3 receptors in starfish oocytes. Because the final activation of MPF in these cells has been suggested to occur in the germinal vesicle (13), oocytes were manually enucleated prior to the addition of 1-MA. MPF activity was estimated in enucleated oocytes 50 min after hormonal treatment. Fig. 4C shows that MPF activity was very low in immature oocytes not treated with 1-MA (first column) but much higher in the oocytes exposed to 1-MA for 50 min (second column). Interestingly, MPF activity was nearly twice in nucleated (control) as compared with enucleated oocytes (59%, third column of Fig. 4C). The activity was also measured in the separated animal and vegetal halves of the oocytes exposed to the hormone for 50 min. MPF activity in the animal and in the vegetal halves of the oocyte amounted to 77 and 38%, respectively, of that of the intact mature oocytes, but to 38% of it in the vegetal hemisphere (fourth and fifth columns of Fig. 4C). The comparison of the histograms shown in panels B and C of Fig. 4 revealed a strong correlation between InsP3-induced Ca2+ release (panel B) and the activity of MPF (panel C) measured in enucleated oocytes and in animal and vegetal halves.

The Increase of Sensitivity of the InsP3 Receptors Is Prevented by the MPF Blocker Roscovitine—Indications that MPF could be involved in the development of the increased sensitivity of InsP3 receptors to InsP3 derived from experiments on p13suc1, a protein binding to MPF in yeast and Xenopus. Depletion of the Xenopus homologue of p13suc1 (Xep-9) has been found to prevent MPF activation (25). The role of MPF in modulating the response to InsP3 in the perinuclear area was assessed by using the specific MPF inhibitor roscovitine. Because we had previously observed that oocytes incubated with roscovitine resumed meiosis with a significant delay (19), we have investigated whether the GVBD delay was paralleled by an effect of roscovitine on the development of the increased sensitivity of Ca2+ stores to InsP3 around the germinal vesicle. The experiments have indeed shown that the increased sensitivity to InsP3 in the perinuclear area (closed circles) was delayed by about 30 min in oocytes incubated for 30 min with roscovitine (Fig. 5A, open triangles). When roscovitine was injected either into the cytoplasm or the germinal vesicle of the oocytes, the increased perinuclear Ca2+ responses to InsP3 and GVBD were abolished during the period under investigation (up to 3 h, open rhombs). Experiments were also performed to assess the role of p13suc1 in the development of the increased sensitivity of Ca2+ stores to InsP3. However, the injection of the protein (1 mg/ml in the pipette, 5% of the oocyte volume) into immature oocytes only induced a 10-min delay in the breakdown of the nuclear envelope. The Ca2+ increase around the germinal vesicle induced by the InsP3 uncaging, which is normally detected 10-12 min after the application of 1-MA, was delayed by about the same time by the pre-injection of p13suc1 (data not shown).



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  		FIG. 5.
Effect of MPF on the increased sensitivity of InsP3 receptors during the maturation process. A, the delay of GVBD of more than 30 min was observed when oocytes are preincubated for 30 min with 50 µM roscovitine prior to 1-MA addition (open triangle and arrow) compared with the control oocyte (closed circles and arrow). The increase of sensitivity of InsP3 receptors was delayed of more than 20 min (open triangles). When roscovitine was directly injected at a final concentration of 5-10 µM, either into the nucleus or into the cytoplasm of the oocyte 30 min prior to 1-MA addition, both the increased sensitivity of InsP3 receptors and GVBD were inhibited (open rhomb). All experiments were performed three times, and representative data are shown. B, active MPF injected into immature oocytes (5-7% of oocyte volume) induced GVBD in 48% of injected oocytes (15/31 in two independent experiments). Oocytes that have undergone GVBD exhibited much higher Ca2+ response than those in which GVBD did not occur or than in immature control oocytes.

 

Injection of MPF into Immature Oocytes Increases the Sensitivity to InsP3—To add support to the suggestion that the activity of MPF correlated with the increased sensitivity of Ca2+ stores to InsP3, active MPF was injected in the cytoplasm of immature oocytes (the injected volume of 350-500 pl corresponded to 5-7% of the volume of the cell) (Fig. 5B). 1 h after the injection about 50% of the oocytes underwent GVBD without hormonal treatment (10 out of 20 and 5 out of 11 in two independent experiments). Control oocytes were injected with the same volume of a buffer solution, mimicking the intracellular milieu, the calcium dye OGBD 70 kDa, or a mouse antibody IgG as control. In no case meiosis resumption was observed. The photoliberation of previously injected caged InsP3 induced a Ca2+ response, which was about 5-fold higher in oocytes that underwent GVBD (1.38 ± 0.35, n = 5) as compared with untreated immature oocytes. MPF-injected oocytes that failed to resume meiosis showed a Ca2+ response following InsP3 uncaging that only reached a peak of 0.37 ± 0.08 (n = 3), i.e. of the same range of that measured in immature oocyte not treated with 1-MA (Fig. 5B).

MPF Does Not Phosphorylate InsP3 Receptors—The results above compellingly showed that MPF was responsible for the development of increased sensitivity of the Ca2+ stores to InsP3. The possibility that MPF phosphorylated the InsP3-sensitive Ca2+ stores, increasing Ca2+ efflux from ER, was thus explored. The kinase assay was performed on starfish oocyte lysates using a preparation of active starfish MPF (the p34cdk1/cyclin B, kindly donated by Dr. L. Meijer, Roscoff, France). The autoradiogram of Fig. 6 shows that no proteins corresponding to the molecular mass of the InsP3 receptors (~240 kDa), as revealed by immunoblotting with specific anti-starfish InsP3 receptor antibodies, became phosphorylated in starfish lysates. The same result was obtained when the experiment was repeated using human recombinant MPF (data not shown). Therefore, the increased sensitivity to InsP3 was not due to the direct phosphorylation of the InsP3 receptors by MPF. The activity of the InsP3 receptors is modulated by a number of serine/threonine and tyrosine kinases. In principle, MPF could phosphorylate and activate other kinases, thus indirectly increasing the sensitivity of the InsP3 receptors to InsP3. This point was thus explored in experiments using kinase inhibitors. A potent inhibitor of calmodulin-dependent kinase II, the autocamtide 2-related inhibitory peptide, had no effect on the sensitization of the InsP3-dependent Ca2+ pool during maturation (data not shown). Experiments were also performed by uncaging InsP3 in maturing oocytes incubated with KT5823, an inhibitor of the cGMP-dependent protein kinase, or with 5-24, an inhibitor of the protein kinase A (data not shown). In both cases, the experiments gave negative results, i.e. no effects were recorded on the sensitization of the Ca2+ stores to InsP3. In hindsight, this was perhaps expected, because a significant decrease in the cAMP and cGMP levels has been shown to occur in starfish oocytes within 5 min after exposure to 1-MA, with a minimum level being reached shortly before GVBD (26, 27).



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  		FIG. 6.
Starfish MPF does not directly phosphorylate InsP3 receptors from starfish oocyte lysates. The lysate of starfish oocytes was subjected to an in vitro kinase reaction using active MPF from starfish. No bands corresponding to the molecular mass of the InsP3 receptors were phosphorylated by MPF. The same membrane was analyzed by immunoblotting with specific starfish antibodies that recognized the InsP3 receptors from starfish lysates.

 

Modulation of the Actin Cytoskeleton by MPF Is Responsible for the Sensitization of the Ca2+ Stores to InsP3—Because the increased sensitivity of the Ca2+ pool to InsP3 receptors during maturation could not be traced back to the direct (or indirect) phosphorylation of the InsP3 receptors by MPF, other MPF targets that could be involved in the process of InsP3 sensitization were sought.

Recent work has indicated a role of actin filaments in facilitating calcium release from InsP3-and ryanodine-sensitive ER stores in hippocampal neurons (28). Along with similar lines, independent experiments in our laboratory had shown that the actin cytoskeleton played a role in regulating the InsP3-dependent Ca2+ release in the A. auranciacus species (29). It was found that the addition of the depolymerizing drug latrunculin-A (Lat-A) induced a massive release of intracellular Ca2+ in oocytes treated for 1 h with the maturing hormone 1-MA. The experiments were repeated in this contribution to show that Lat-A, which induced a large Ca2+ release in mature oocytes (29), was ineffective in oocytes not treated with 1-MA (not shown). Thus F-actin is apparently stabilized in immature cells against the depolymerizing action of Lat-A, so that the Lat-A sensitivity of the Ca2+ stores depends on the maturation process. The experiment in Fig. 7A correlates the sensitivity of the actin cytoskeleton to Lat-A with the Ca2+ response induced by InsP3. In the immature oocytes (not treated with 1-MA), Lat-A failed to affect the InsP3-mediated emptying of the Ca2+ stores. Interestingly, when the maturation process was induced by 1-MA in the presence of Lat-A, the photoliberation of InsP3 generated a Ca2+ response, which was strongly inhibited. Thus, these data strongly support the suggestion that changes in the actin cytoskeleton play a role in modulating the Ca2+ response to InsP3.



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  		FIG. 7.
Lat-A-induced Ca2+ increase during maturation of starfish oocytes. A, Lat-A treatment inhibited the Ca2+ increase following InsP3 uncaging in mature, but not in immature oocytes. B, Lat-A, added 10 min after 1-MA stimulation (arrow), elicited a Ca2+ increase 18-19 min after the addition of the hormone (black line). The Ca2+ increase induced by Lat-A mimicked the Ca2+ response following the photoliberation of InsP3 in maturing oocytes (blue circles). The Lat A-induced Ca2+ liberation was blocked both by the actin-polymerizing drug jasplakinolide (red line) and by roscovitine (green line). C, relative fluorescence images of the Ca2+ increase induced by Lat-A in a maturing oocyte. The Ca2+ increase began 18.8 min after hormonal application (second image) at the animal pole of the oocyte (the position of the germinal vesicle is indicated in the first image of C, upper row). D, the Ca2+ response in oocytes following InsP3 uncaging performed at different times during the maturation process. The overlay and relative fluorescence images show the same spatiotemporal pattern in the Ca2+ response induced by InsP3 and by Lat-A, respectively (C).

 

The spatiotemporal dynamics of the onset of the Ca2+ response induced by Lat-A was investigated next. Fig. 7C shows that the sensitivity of the Ca2+ stores to the drug became evident between 18 and 20 min after the addition of the hormone in the oocytes of A. auranciacus, used for this set of experiments. The Lat-A-induced Ca2+ response originated in the animal hemisphere also in A. auranciacus and led to the same spatial pattern of increased Ca2+ response following InsP3 uncaging as shown in panel D. The graph of panel B shows the relative fluorescence analyses of the Ca2+ increase induced by the addition of Lat-A to an oocyte exposed to 1-MA for 18-19 min (black line). In addition, the F-actin-stabilizing agent jasplakinolide completely inhibited the Lat-induced Ca2+ response in mature oocytes (red line) confirming the F-actin specificity of the Lat-A effect. Interestingly, Ca2+ liberation induced by the depolymerizing drug started at the animal hemisphere at the time at which the increased sensitivity to InsP3 was established as indicated by blue circles.

At this point it was logical to explore whether the rearrangement of the actin cytoskeleton was linked to phosphorylation events mediated by MPF. For this purpose the experiments described in panel B were repeated, and the actin depolymerizing drug and the hormone were added to oocytes pre-treated with roscovitine at a concentration known to inhibit MPF activity. The treatment with roscovitine completely blocked the Lat-A-induced Ca2+ release (green line). Thus, during the maturation process, the sensitivity of the actin cytoskeleton to Lat-A appears to be dependent on rearrangements of actin modulated by MPF.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A calcium increase in the perinuclear area where a variety of Ca2+ stores are present (30) is required to trigger entry into the mitosis in early sea urchin embryos (31, 32). Activation of calmodulin (CaM) occurs near the nuclear envelope just before mitosis entry (33). Ca2+ transients are also associated with the progression of the meiotic cycle (34). For instance, meiosis progression in starfish oocytes was blocked by the microinjection of BAPTA directly into the germinal vesicle (2). Nuclear injection of antibodies against CaM and of a peptide calmodulin inhibitor corresponding to the CaM-binding domain of myosin light-chain kinase completely inhibited meiosis resumption (35). In the present contribution, by using two different species of starfish as experimental models, we have detected for the first time a perinuclear Ca2+ signal that precedes the germinal vesicle breakdown and that coincides spatially and temporally with the increased sensitivity of intracellular Ca2+ stores to InsP3.

In starfish oocytes, InsP3 has been shown to release significantly more Ca2+ at the end of the maturation process (17). The effect could be traced back to: (a) the increase in the amount of Ca2+ trapped in the ER as shown in mouse oocytes (36); (b) the increase in the expression and/or redistribution of the InsP3 receptors during the maturation process as previously described in Xenopus and mouse oocytes (36, 37); and (c) changes in the InsP3 receptors that increase their response to InsP3. Western blot analysis and immunohistochemistry have recently shown that the expression and the distribution of the InsP3 receptors in A. pectinifera starfish oocytes did not change during the meiotic process (18). The higher sensitivity to InsP3 was not linked to a larger Ca2+ load in the ER, because increased concentrations of InsP3 injected into an immature oocyte released the same amount of Ca2+ that became mobilized in oocytes at the end of the maturation process (17). This result was confirmed in this contribution by using the caged variant of InsP3. We have examined the spatiotemporal pattern of the increased responsiveness to InsP3 during maturation and found that the increased Ca2+ response to InsP3 starts at the animal pole of the oocyte, where the germinal vesicle is located, and propagates along the animal/vegetal axis. The same pattern of propagation was observed in the absence of external Ca2+ (data not shown) indicating that the increase in the Ca2+ response was due to the increase in the sensitivity of intracellular Ca2+ stores to InsP3.

At this point it was logical to involve the nucleus in the process. The working hypothesis was that nuclear components could be required for the onset of the increased Ca2+ response to InsP3 in the perinuclear region. This was suggested by experiments in which the nucleoplasm of oocytes treated for 8 min with 1-MA, which is the period during which the presence of the hormone in the surrounding medium is required for GVBD to eventually occur (38), was injected into the germinal vesicle of immature oocytes not treated with the hormone (data not shown). When the maturing nucleoplasm was injected into the nucleus of the immature oocytes, it always induced the increased Ca2+ response to InsP3 in the perinuclear region and re-initiation of meiosis. By contrast, the transplantation of the same amount of maturing nucleoplasm into the cytoplasm of receiving oocytes failed to promote GVBD (data not shown). Evidently, it was essential that the maturing nucleoplasm could act within the nucleus still existing.

Because protein phosphorylation is the most frequently used tool to modulate cell reactions, it was assessed whether kinase-mediated phosphorylation could modulate the activity of the InsP3 receptors. The rationale for focusing on MPF as the candidate kinase has been outlined in the introduction and under "Results." Moreover, the activation of MPF during Xenopus oocyte maturation follows an inhomogeneous pattern, occurring first in the animal hemisphere and then in the vegetal one (14, 39). Experiments in enucleated oocytes and in the two oocyte hemispheres were linked to the finding that nuclear amplification of MPF could modulate the development of the increased sensitivity to InsP3 along the animal/vegetal axis. In fact, photoliberation of InsP3 50 min after hormonal treatment in the separated animal and vegetal halves revealed a Ca2+ response that was much higher in the animal half compared with the vegetal and even higher than that measured in entire oocytes. However, the Ca2+ response induced by InsP3, as well as MPF activity, in oocytes from which the germinal vesicle had been removed prior to 1-MA addition, and in vegetal halves, which do not contain the germinal vesicle, was higher than that measured in control not treated oocytes and lower than that in entire oocytes and in the animal halves. These results are consistent with the hypothesis that the initial activation of MPF occurs in the cytoplasm (16) through a positive feedback, followed by a final and full activation in the germinal vesicle (12, 13). The nuclear translocation of cdc25 depends on Plk phosphorylation (40) and is suggested to be essential for meiotic competence of goat oocytes (41). Activated MPF is then exported from the nucleus to directly phosphorylate several targets or to cause their indirect phosphorylation (42). In starfish oocytes the finding that the increased sensitivity of the InsP3 receptors at the animal pole occurs several minutes before the breakdown of the nuclear envelope suggests that MPF itself or a downstream component activated by MPF exported from the germinal vesicle to induce the sensitization of InsP3 receptors. The finding that InsP3 receptors were not phosphorylated directly or indirectly by MPF has prompted experiments aimed at showing whether MPF was involved in the rearrangement of actin cytoskeleton during the maturation process, which in the end could regulate the release of Ca2+ from InsP3-sensitive stores. These experiments have shown that the increased sensitivity of the Ca2+ stores to InsP3 depends on the dynamic state of F-actin. Future investigations will hopefully identify the components that link phosphorylation to the sensitivity of F-actin to Lat-A.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed. Tel.: 39-081-5833-289; Fax: 39-081-7641-355; E-mail: santella{at}szn.it.

1 The abbreviations used are: 1-MA, 1-methyladenine; GVBD, germinal vesicle breakdown; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis; MPF, maturation-promoting factor; InsP3, inositol 1,4,5-trisphosphate; OGBD, Oregon Green 488 BAPTA-1, coupled to a 70-kDa dextran; CCD, charge-coupled device; RHD, rhodamine dextran 70,000; Lat-A, latrunculin-A; TBS, Tris-buffered saline; ER, endoplasmic reticulum; CaM, calmodulin. Back


   ACKNOWLEDGMENTS
 
We are grateful to E. Carafoli for helpful suggestions and discussions. We are indebted to Dr. L. Meijer for the kind donation of the starfish p34cdk1/cyclin B, Dr. T. Kishimoto for p13suc1, and K. Mikoshiba for the starfish InsP3 receptors antibodies. We are grateful to G. Gragnaniello for his help in the preparation of the figures and to the Marine Resources Service of the Stazione Zoologica di Napoli for maintaining the starfish.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
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 RESULTS
 DISCUSSION
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