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Two phases of calcium requirement during starfish meiotic maturation
Comparative Biochemistry and Physiology, Part A 147 (2007) 432 – 437 www.elsevier.com/locate/cbpa
Two phases of calcium requirement during starfish meiotic maturation Hiroaki Tosuji a,⁎, Yukari Seki a , Keiichiro Kyozuka b a b
Department of Chemistry and Bioscience, Faculty of Science, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan Research Center for Marine Biology, Asamushi, Graduate School of Life Sciences, Tohoku University, Aomori 039-3501, Japan Received 10 October 2006; received in revised form 18 January 2007; accepted 21 January 2007 Available online 30 January 2007
Abstract During meiosis in oocytes of the starfish, Asterina pectinifera, a Ca2+ transient has been observed. To clarify the role of Ca2+ during oocyte maturation in starfish, an intracellular Ca2+ blocker, TMB-8, was applied. The oocyte maturation induced by 1-methyladenine (1-MA) was blocked by 100 μM TMB-8. Reinitiation of meiosis with germinal vesicle breakdown (GVBD) and the following chromosome condensation did not take place. Maturation-promoting factor (MPF) activity did not increase and GVBD and chromosome condensation did not occur. Ca2+ transient observed immediately after 1-MA application in control oocytes was also blocked by TMB-8. When calyculin A, which activate the MPF directly, was applied to the oocytes instead of 1-MA in seawater containing 100 μM TMB-8, GVBD and chromosome condensation were blocked. Cytoplasmic transplantation studies confirmed that MPF was activated, although TMB-8 blocked GVBD. These results show that TMB-8 blocked the increase of MPF activity induced by 1-MA and the process of active MPF inducing GVBD and subsequent chromosome condensation. Together with the above phenomena, it is conceivable that there are two phases of Ca2+ requirement during starfish oocyte maturation. These are the activation of MPF, moreover, GVBD, and the subsequent chromosome condensation. © 2007 Elsevier Inc. All rights reserved. Keywords: Calcium wave; Cdc2; Chromosome condensation; GVBD; Histone kinase; MPF; Oocyte maturation; TMB-8
1. Introduction Starfish oocytes are a useful model for the study of the cell cycle. Fully-grown oocytes are arrested at prophase of the first meiosis and resume meiosis in response to the maturationinducing hormone, 1-methyladenine (1-MA) that acts on a receptor on the egg membrane. During the resumption of meiosis, an increase in intracellular free Ca2+ is involved in many animals (Whitaker and Patel, 1990; Berridge, 1995; Wilding, 1996). When the Ca2+ chelator, BAPTA blocks the increase in intracellular Ca2+, the resumption of meiosis does not occur in mouse oocytes (Kline and Kline, 1992). During oocyte maturation in Xenopus laevis oocytes, a rise in free Ca2+ is involved in triggering oocyte maturation (Cicirelli and Smith, 1987) and cdc2 kinase activation (Lindsay et al., 1995). Furthermore, in the nemertean worm, Cerebratulus lacteus, injecting aqueous extracts from sperm triggers repetitive Ca2+ waves and resumption of meiotic maturation (Stricker, ⁎ Corresponding author. Tel./fax: +81 99 285 8160. E-mail address: [email protected] (H. Tosuji). 1095-6433/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2007.01.018
1997). The role of Ca2+ during starfish oocyte maturation has been reviewed (Meijer and Guerrier, 1984), and the importance of Ca2+ has been well described; however, precise Ca2+ release requesting processes during oocyte maturation are still unclear. Results vary from species to species. In some species, a transient increase in Ca2+ occurred after 1-MA application (Moreau et al., 1978; Picard and Dorée, 1983a,b; Santella and Kyozuka, 1994). While other investigators have found no change in intracellular free Ca2+ in response to the 1-MA in different species (Eisen and Reynolds, 1984; Stricker et al., 1994). As well, buffering cytoplasmic Ca2+ by the microinjecting Ca2+ chelators EGTA or BAPTA do not inhibit 1-MA-induced meiosis reinitiation (Picard and Dorée, 1983b; Witchel and Steinhardt, 1990). Although all investigators have shown that Ca2+ is required in mitosis, the relationship between meiosis and Ca2+ is not clear. TMB-8 {8-(diethylamino) octyl-3,4,5-trimethoxybenzoate hydrochloride}, which blocks Ca2+ release from intracellular Ca2+ stores (Chiou and Malagodi, 1975), was found to inhibit the egg activation of X. laevis (Kline and Nuccitelli, 1985) and sea urchin (Stapleton et al., 1985). In this study, we used TMB8 in place of the Ca2+ chelators (EGTA or BAPTA), which were
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used in most of the above studies. TMB-8 can permeate through the cell membrane, so we were able to confirm that the maturation and development ability recovered perfectly when TMB-8 was washed out. We show here that TMB-8 blocked maturation-promoting factor (MPF) activation, germinal vesicle breakdown (GVBD) and chromosome condensation in Asterina pectinifera oocytes. In addition, it is conceivable that these events are directly related with the intracellular Ca2+ levels. 2. Materials and methods
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(0.25 μg/mL) in buffer A. The sample was examined by Nikon Optiphot microscope equipped with epifluorescence optics using UV filter set, and recorded on Kodak T-max 400 film. 2.4. Microinjection techniques To test MPF activity, the donor cytoplasm from the maturing oocyte was transferred into the immature oocyte (prophase arrested oocyte) by microinjection. The recipient oocyte was put in a holding chamber and microinjected with about 250 pL of donor cytoplasm. The tip of the glass needle used for the injection was approximately 5 μm in diameter.
2.1. Materials 2.5. Assay of the histone H1 kinase activity Starfish (A. pectinifera) were collected during the breeding seasons at Mutsu Bay (Aomori, Japan), Tokyo Bay (Chiba, Japan) and the Yatsushiro Sea (Kagoshima, Japan). They were kept in circulating seawater at 15–16 °C until use. Oocytes containing germinal vesicle were obtained by dissecting the ovaries using fine forceps. To induce the maturation of oocytes, 0.25 μM 1-MA or 5 μM calyculin A (CL-A; a gift from Prof. Fusetani) was applied. All experiments with live oocytes were done at 18–22 °C. To block intracellular Ca2+ release, oocytes were treated with TMB-8 (Aldrich) in Ca2+ -free seawater. The oocytes were pre-incubated in seawater containing 100 μM TMB-8 for 10–15 min before adding 1-MA or CL-A; TMB8 continuously remained in the solution. 2.2. Measurement of intracellular free Ca2+ Intracellular free Ca2+ was measured as previously described (Kyozuka et al., 1998) using Calcium Green (Calcium Green-1 dextran, MW = 10,000, Molecular Probes) as a Ca2+ indicator. Calcium Green was dissolved at 5 mg/mL in 100 mM potassium aspartate, 10 mM Hepes (pH 7.0) and microinjected into immature oocyte cytoplasm. The fluorescence of Calcium Green in the oocyte cytoplasm was detected every 3–5 s by an Olympus IMT-2 inverted fluorescence microscope equipped with Olympus OSP-3, computer-controlled photomultiplier system. The excitation wavelength was 475–490 nm and the emission wavelength was 530 nm. The oocytes were set onto the measurement system using a holding pipette. The fluorescence intensity was measured in a circular area of 70 μm diameter in the oocyte out of 150 μm in diameter (21.8% area of the oocyte). The recorded fluorescence (F) was normalized against the initial value of F (F0) by the equation (F − F0) / F0 (Stricker et al., 1994). 2.3. Fluorescence microscopy To visualize chromosomes, the oocytes were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI). Oocytes were allowed to settle onto protamine coated glass slides. They were fixed with 5% formalin in buffer A (0.1 M KCl, 0.8 M glucose, 5 mM MgCl2 and 5 mM EGTA in 10 mM MOPS buffer, pH 6.7) containing 0.05% Nonidet P-40 for 30 min, then the slides were immersed in the same fixative for 1 h at room temperature. After washing with buffer A, the oocytes were stained with DAPI
Phosphorylation of exogenously added histone H1 was measured as it depends on the MPF activity (Picard et al., 1989). The ATP mixture was reacted with the oocyte homogenates for 5 min at 25 °C. The ATP mixture consisted of 100 μM [γ-32P] ATP (20 Bq/pmol; ICN Biomedicals), 10 mM MgCl2 and 2 mg/mL H1 histone (type III-S; Sigma) in 20 mM Tris–HCl buffer, pH 7.5. After the reaction, we used SDS-PAGE followed by autoradiography instead of a liquid scintillation counter. The reaction was terminated by adding an equal volume of the SDS sample buffer (30% urea, 8% dithiothreitol and 5% SDS in 0.5 M Tris–HCl buffer pH 6.8) to the reacting mixture. Then samples were heated in boiling water for 3 min, and proteins were separated by SDSPAGE (12.5% separating gel). The gel was dried onto Whatman 3 MM paper and autoradiographed on Fuji RX film. 2.6. Immunoblotting (western blotting) MPF from the oocytes were purified using p13suc1-binding Sepharose beads and p34cdc2 was detected by immunoblotting as described previously (Meijer et al., 1989). The eggs were sonicated on ice in the homogenization buffer and a 14,000 ×g supernatant fraction was incubated with p13suc1-Sepharose beads (Oncogene Research Products) for 30 min at 4 °C. The beads were washed three times with the beads buffer. After centrifugation at 10,000 ×g, an SDS sample buffer was added in equivalence to the pellet. Then samples were heated in boiling water for 3 min, and run on SDS-polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose membrane (BA 85; Schleicher and Schüll). The nitrocellulose membrane was incubated with a 1:1000 dilution of the control cdc2 antibody or the phosphospecific cdc2 (Tyr15) antibody (Cell Signaling Technology); antibodies were then visualized using Vectastain ABC kit (Vector Laboratories) with 3-3′-diaminobenzidine + H2O2 as a substrate. 3. Results 3.1. Effect of TMB-8 on oocyte maturation Immature oocytes of A. pectinifera were pretreated with various concentrations of TMB-8 before the addition of 1-MA or CL-A in the same solution. TMB-8 inhibited both 1-MA-and CL-A-induced oocyte maturation as reported (Tosuji et al.,
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1991). In the present study, exposure of the oocytes in more than 50 μM TMB-8 seawater inhibited 1-MA- and CL-Ainduced oocyte maturation completely. The inhibition was reversible. When TMB-8 (100 μM) was removed by washing the oocytes in seawater, they underwent normal GVBD after readdition of 1-MA (mean ± S. E. of GVBD was 97.7 ± 1.6%). 3.2. Ca2+ transients during oocyte maturation Two peaks of Ca2+ release were seen within a few minutes after addition of 1-MA to immature oocyte at the reinitiation of meiosis from prophase {Fig. 1A; this result is shown as a control although same result was reported by Santella and Kyozuka (1994)}. GVBD in this clamped oocyte occurred at around 30 min after the addition of 1-MA. In Ca2+ -free seawater, the same Ca2+ transient was observed (Fig. 1B) and GVBD occurred on time as well. TMB-8 (100 μM) blocked the
Fig. 2. Oocytes staining by DAPI. (A) Immature oocyte. (B) 1-MA-induced ordinary matured oocyte (40 min after the addition of 1-MA). (B′) Oocyte treated with both TMB-8 (100 μM) and 1-MA (40 min after 1-MA addition). (C) CL-A-induced matured oocyte (70 min after CL-A addition). (C′) Oocyte treated with both TMB-8 (100 μM) and CL-A (70 min after CL-A addition). Bar = 50 μm. The darkish circles on A, B′ and C′ correspond to germinal vesicle.
intracellular Ca2+ release completely (Fig. 1C) and GVBD did not occur in these oocytes. The Ca2+ peaks similar to the control immature oocytes took place after 100 μM TMB-8-pretreated oocytes had transferred to seawater containing 1-MA, although the Ca2+ peak was slightly delayed (Fig. 1D). The germinal vesicle breakdown proceeded normally in these oocytes. 3.3. Condensation of chromosomes
Fig. 1. Typical Ca2+ transients in the oocytes. The abscissa shows time from the beginning of measurement. The ordinate shows relative fluorescence intensity {(F − F0) / F0}. (A) 1-methyladenine-induced oocyte maturation in normal seawater (ordinary maturation). (B) 1-methyladenine-induced oocyte maturation in Ca2+ -free seawater. (C) 1-methyladenine-treated oocyte with 100 μM TMB8 in Ca2+ -free seawater. (D) The oocyte was treated with 100 μM TMB-8 in Ca2+ -free seawater for 15 min, then TMB-8 was removed and the oocyte was treated with 1-MA. Figures show the experiments of typical Ca2+ release reactions. 0 min = beginning of treatment with 25 μM 1-MA.
Further process of oocyte maturation in TMB-8 seawater was observed by focusing on chromosomes condensation by staining with DAPI and fluorescent microscopy. In the ordinary oocyte maturation induced by 1-MA, the chromatin began to condense about 10 min after the addition of 1-MA, and they condensed completely in 30 min (Fig. 2B). The 1st polar body formed in 40 min. In CL-A-treated oocytes, the chromatin began to condense about 40 min after the addition and condensed completely within 60 min (Fig. 2C). The 1st polar body formed about 100 min. By comparison, the change of the chromatin form did not observe by time-lapse microscopy during the TMB-8-treatment (100 μM) both 1-MA and CL-Atreated oocytes, the chromosomes did not condense in TMB8 seawater after the treatment in sufficient times (Fig. 2B′,C′).
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Table 1 Determination of MPF activities by cytoplasmic transferring technics Donor
Recipient
GVBD (%)a
1-MA-induced mature oocyte CL-A-induced mature oocyte TMB-8 & 1-MA-treated oocyte TMB-8 & CL-A-treated oocyte 1-MA-induced mature oocyte
immature oocyte immature oocyte immature oocyte immature oocyte TMB-8-treated immature oocyte TMB-8-treated immature oocyte
6/7 (86) 5/6 (83) 0/5 ( 0) 4/6 (67) 0/6 ( 0)
CL-A-induced mature oocyte
0/4 ( 0)
Approximately 250 pL of donor cytoplasm was microinjected into recipient oocyte. The concentration of TMB-8 was 100 μM. aGVBD of the recipient oocyte was counted.
3.4. MPF activities MPF activities of the TMB-8-treated oocytes were examined by cytoplasmic transfer assay. As seen in Table 1, both 1-MAand CL-A-treated oocytes (as controls) had MPF activities. TMB-8 (100 μM) prevented 1-MA-induced MPF activity. However, TMB-8 in CL-A-treated oocytes did have MPF activity, GVBD was not induced in the solution. When cytoplasm from the MPF-activated oocyte was transferred to TMB-8treated immature oocytes, GVBD did not occur in the recipient oocyte.
Fig. 4. Western blotting analysis of cdc2. The nitrocellulose membranes were incubated with an anti-cdc2 (Tyr15) antibody (upper panel) or an anti-cdc2 antibody (lower panel). The antibodies were visualized using Vectastain ABC kit. (Lane1) immature oocyte (IM). (Lane2) 1-MA-treated oocyte (ordinary maturation). (Lane3) TMB-8 and 1-MA-treated oocyte. (Lane4) CL-A-treated oocyte. (Lane5) TMB-8 and CL-A-treated oocyte. Asterisks indicate the band of cdc2 (Tyr15). U, upper band; M, middle band; L, lower band.
3.5. Histone H1 kinase activities MPF activities as histone H1 kinase activities of oocytes during maturation were directly measured. Both 1-MA-and CLA-treated oocytes had high levels of histone H1 kinase activity (Fig. 3A lanes 2,4). TMB-8 (100 μM) in the 1-MA-treated oocyte had a low level of histone kinase activity (Fig. 3A lane 3 and 3B). However, TMB-8 (100 μM) and CL-A-treated oocytes had high levels of the histone kinase activity (Fig. 3A lane 5) in agreement with the cytoplasmic transfer studies. 3.6. Molecular changes of p34cdc2
Fig. 3. Histone H1 kinase activities in oocytes. (A) The oocytes were harvested 40 min after the treatment of 1-MA and 60 min after the treatment of CL-A. (lane 1) immature oocyte (IM). (lane 2) 1-MA-treated oocyte (ordinary maturation). (lane 3) TMB-8 (100 μM) and 1-MA-treated oocyte. (lane 4) CL-A-treated oocyte. (lane 5) TMB-8 (100 μM) and CL-A-treated oocyte. Asterisks indicate the band of histone H1. (B) Time course of phosphorylation of histone H1 during 1-MA-induced oocyte maturation with/without TMB-8. The numbers above the picture are the time (in min) after the treatment of 1-MA.
P34cdc2 was separated with SDS-PAGE and detected by immunoblotting. The bands detected by cdc2 antibody existed in all oocytes (Fig. 4 lower panel). Phosphorylation of Tyr15 on p34cdc2 was observed only in immature oocytes and in TMB-8positive (100 μM) 1-MA-treated oocytes (Fig. 4 upper panel lane 1 and 3). The cdc2 (Tyr15) antibody binds to Tyr15phosphorylated p34cdc2, which is a component of the inactivated MPF. The result indicates MPF in these oocytes were inactive. The band detected by this antibody was not detected in CL-Atreated oocytes (Fig. 4 upper panel lane 4 and 5). A shift to lower bands from the upper band of p34cdc2 was observed in oocytes in which Tyr15 of p34cdc2 was not phosphorylated (Fig. 4 lower panel). These results show that MPF activity induced by CL-A was not affected by TMB-8 treatment. 4. Discussion It has not been firmly established if an increase of intracellular Ca2+ is required for meiotic maturation in starfish oocytes. Ca2+
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transients were observed during 1-MA-induced meiosis reinitiation in Marasthenias glacialis (Moreau et al., 1978; Picard and Dorée, 1983a,b), A. pectinifera (Santella and Kyozuka, 1994). In contrast, other investigators have shown no change in intracellular free Ca2+ in response to 1-MA in Asterias forbesi (Eisen and Reynolds, 1984), Asterina miniata, Orthasterias koehleri and Pisaster ochraceus (Stricker et al., 1994). Ca2+ release may be not a sufficient condition but a necessary condition for starfish oocyte maturation. Oocytes of A. pectinifera showed Ca2+ spikes during oocyte maturation as shown by Santella and Kyozuka (1994). When 1-MA was added by diffusion to oocytes of A. miniata, Ca2+ transients did not occur; however, when 1-MA was added by perfusion, Ca2+ transients occur (Witchel and Steinhardt, 1990). Therefore, Ca2+ transients are shown to be unnecessary for oocyte maturation. The question of whether a transient Ca2+ rise plays a causal role in meiotic maturation has been further confused by the question of whether Ca2+ transients are a response to a hormone. The reversibility for TMB-8 inhibition was confirmed, since it eliminated Ca2+ spikes. This means that GVBD has a close relationship with intracellular Ca2+ release. TMB-8 is a non-specific endocellular Ca2+ release antagonist (Chiou and Malagodi, 1975). It inhibits inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ -release in sea urchin egg homogenate (Clapper and Lee, 1985). As TMB-8 blocked GVBD completely, it is conceivable that the release of free Ca2+ from intracellular Ca2+ stores is necessary for resumption of meiosis in starfish. Calcium transient after 1-MA causes in the cytoplasm and then Ca2+ release in the germinal vesicle followed. The nuclear Ca2+ increase triggers GVBD, because when increase in nuclear Ca2+ is blocked by injecting BAPTA directly into the germinal vesicle, GVBD is blocked after 1-MA application (Santella and Kyozuka, 1994). Increase in intracellular free Ca2+ is necessary during the activation of MPF. Our results support other reports that MPF is regulated by intracellular free Ca2+ levels in amphibian oocytes or eggs (Capony et al., 1986; Bement and Capco, 1991), in ascidian oocytes (Russo et al., 1996), in sea urchin eggs (Patel et al., 1989) and in starfish oocytes (Capony et al., 1986). In the embryonic division of sea urchin and X. laevis, MPF activity also rises through Ca2+ /calmodulin-dependent protein kinase, phosphorylation and activation of cdc25, increase of the phosphorylatedcyclin/cyclin ratio (Whitaker and Patel, 1990; Swanson et al., 1997). In A. pectinifera, 1-MA induces a translocation of calmodulin from the nucleoplasm to the cytoplasm prior to the breakdown of the nuclear envelope, and the injection of anti-calmodulin inhibits the resumption of meiosis (Santella and Kyozuka, 1997a). Additionally, microinjection of InsP3 into A. pectinifera immature oocytes caused Ca2+ release but not maturation (Chiba et al., 1990). In Astropecten auranciacus, the injection of InsP3 or cyclic ADP ribose (cADPr) into the germinal vesicle caused a Ca2+ spike and meiosis resumption in the absence of 1-MA (Santella and Kyozuka, 1997b). The source of intracellular free Ca2+ during starfish oocyte maturation seems to be from the InsP3-sensitive pathway and following cortical Ca2+ flash induced by cADPr (Nusco et al., 2002). Together with our results, it is conceivable that
TMB-8 permeates into germinal vesicle through cytoplasm then it reduces the InsP3-sensitive Ca2+ release. Fluorescence microscopic studies showed that TMB8 inhibited the condensation of chromosomes. TMB-8 inhibits the chromosome condensation although MPF is activated by CL-A. CL-A activates MPF directly by changing the phosphorylation level (Tosuji et al., 1991). In fact, the MPF activity was high in CL-A-treated oocytes regardless of the presence of TMB-8. The TMB-8 positive CL-A-treated oocytes had high MPF activity; however, GVBD did not progress. TMB-8 inhibited the change of phosphorylation levels of cdc2 protein in 1-MA-treated oocytes. During oocyte maturation, the electrophoretic mobility of cdc2 protein was shifted from the upper band to middle and lower bands (Ookata et al., 1992). The upper band is the inactive form of cyclin B-associated cdc2 with presumably phosphorylated Thr14 and Tyr15 residue. The lower band is an active form with presumably dephosphorylated Thr14 and Tyr15 residue. The middle band is present continuously as an intermediate state. In our results, Tyr15 residue of cdc2 was still phosphorylated even though TMB-8-positive oocytes were treated with 1-MA. TMB-8 prevented the dephosphorylation of Tyr15 residue from the viewpoint of molecular change in MPF. Additionally, Table 1 shows that the TMB-8treated oocytes did not mature after they were injected with the activated MPF. The result indicates that GVBD requires intracellular Ca2+ release as well as MPF to be activated. This fact supports the previous reports that EGTA prevents PSTAIR-induced GVBD (Picard et al., 1990) and Ca2+ / calmodulin protein kinase is necessary for nuclear envelope breakdown (Baitinger et al., 1990). Therefore GVBD and chromosome condensation require another release of intracellular free Ca2+ after the activation of MPF. During the cell cycle in sea urchin embryo, Ca 2+ transient is necessary for the chromosome segregation (Groigno and Whitaker, 1998). At the exit of mitosis, the ubiquitin-conjugating enzyme is activated by Ca 2+ /calmodulin-dependent protein kinase (Swanson et al., 1997). Additionally, an injecting of BAPTA blocks the mitosis exit by the increase of the phosphorylated-cyclin/cyclin ratio (Whitaker and Patel, 1990). However, Ca2+ transient seems to be necessary for the chromosome condensation in the case of starfish oocyte maturation (Fig. 2). In conclusion, during the oocyte maturation of the starfish A. pectinifera, it is highly conceivable that condensation of chromosomes and the breakdown of the germinal vesicle are directly related endogenous Ca2+ release after the activation of MPF. In addition, intracellular Ca 2+ release appears necessary in the activating pathway of MPF induced by 1-MA. Acknowledgements We are indebted to the staff of Research Center for Marine Biology, Asamushi, Tohoku University and The Nemoto Lab. of Ochanomizu University for supplying the starfish. We also thank Prof. Nobuhiro Fusetani of Hokkaido University for his kind gift of CL-A.
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Patel, R., Twigg, J., Crossley, I., Golsteyn, R., Whitaker, M., 1989. Calciuminduced chromatin condensation and cyclin phosphorylation during chromatin condensation cycles in ammonia-activated sea urchin eggs. J. Cell Sci., Suppl. 12, 129–144. Picard, A., Dorée, M., 1983a. Hormone-induced parthenogenetic activation of mature starfish oocytes. Exp. Cell Res. 145, 315–323. Picard, A., Dorée, M., 1983b. Is calcium the second messenger of 1-methyladenine in meiosis reinitiation of starfish oocytes? Exp. Cell Res. 145, 325–337. Picard, A., Capony, J.P., Brautigan, D.L., Dorée, M., 1989. Involvement of protein phosphatases 1 and 2A in the control of M phase-promoting factor activity in starfish. J. Cell Biol. 109, 3347–3354. Picard, A., Cavadore, J.C., Lory, P., Bernengo, J.C., Ojeda, C., Dorée, M., 1990. Microinjection of a conserved peptide sequence of p34cdc2 induces a Ca2+ transient in oocytes. Science 247, 327–329. Russo, G.L., Kyozuka, K., Antonazzo, L., Tosti, E., Dale, B., 1996. Maturation promoting factor in ascidian oocytes is regulated by different intracellular signals at meiosis I and II. Development 122, 1995–2003. Santella, L., Kyozuka, K., 1994. Reinitiation of meiosis in starfish oocytes requires an increase in nuclear Ca2+. Biochem. Biophys. Res. Commun. 203, 674–680. Santella, L., Kyozuka, K., 1997a. Association of calmodulin with nuclear structures in starfish oocytes and its role in the resumption of meiosis. Eur. J. Biochem. 246, 602–610. Santella, L., Kyozuka, K., 1997b. Effects of 1-methyladenine on nuclear Ca2+ transients and meiosis resumption in starfish oocytes are mimicked by the nuclear injection of inositol 1,4,5-trisphosphate and cADP-ribose. Cell Calcium 22, 11–20. Stapleton, C.L., Mills, L.L., Chandler, D.E., 1985. Cortical granule exocytosis in sea urchin eggs is inhibited by drugs that alter intracellular calcium stores. J. Exp. Zool. 234, 289–299. Stricker, S.A., 1997. Intracellular injections of a soluble sperm factor trigger calcium oscillations and meiotic maturation in unfertilized oocytes of a marine worm. Dev. Biol. 186, 185–201. Stricker, S.A., Centonze, V.E., Melendez, R.F., 1994. Calcium dynamics during starfish oocyte maturation and fertilization. Dev. Biol. 166, 34–58. Swanson, C.A., Arkin, A.P., Ross, J., 1997. An endogenous calcium oscillator may control early embryonic division. Proc. Natl. Acad. Sci. U. S. A. 94, 1194–1199. Tosuji, H., Kato, Y., Fusetani, N., Nakazawa, T., 1991. Induction of oocyte maturation by calyculin A in starfish. Zool. Sci. 8, 201–205. Whitaker, M., Patel, R., 1990. Calcium and cell cycle control. Development 108, 525–542. Wilding, M., 1996. Calcium and cell cycle control in early embryos. Zygote 4, 1–6. Witchel, H.J., Steinhardt, R.A., 1990. 1-Methyladenine can consistently induce a fura-detectable transient calcium increase which is neither necessary nor sufficient for maturation in oocytes of the starfish Asterina miniata. Dev. Biol. 141, 393–398.
Two phases of calcium requirement during starfish meiotic maturation Hiroaki Tosuji a,⁎, Yukari Seki a , Keiichiro Kyozuka b a b
Department of Chemistry and Bioscience, Faculty of Science, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan Research Center for Marine Biology, Asamushi, Graduate School of Life Sciences, Tohoku University, Aomori 039-3501, Japan Received 10 October 2006; received in revised form 18 January 2007; accepted 21 January 2007 Available online 30 January 2007
Abstract During meiosis in oocytes of the starfish, Asterina pectinifera, a Ca2+ transient has been observed. To clarify the role of Ca2+ during oocyte maturation in starfish, an intracellular Ca2+ blocker, TMB-8, was applied. The oocyte maturation induced by 1-methyladenine (1-MA) was blocked by 100 μM TMB-8. Reinitiation of meiosis with germinal vesicle breakdown (GVBD) and the following chromosome condensation did not take place. Maturation-promoting factor (MPF) activity did not increase and GVBD and chromosome condensation did not occur. Ca2+ transient observed immediately after 1-MA application in control oocytes was also blocked by TMB-8. When calyculin A, which activate the MPF directly, was applied to the oocytes instead of 1-MA in seawater containing 100 μM TMB-8, GVBD and chromosome condensation were blocked. Cytoplasmic transplantation studies confirmed that MPF was activated, although TMB-8 blocked GVBD. These results show that TMB-8 blocked the increase of MPF activity induced by 1-MA and the process of active MPF inducing GVBD and subsequent chromosome condensation. Together with the above phenomena, it is conceivable that there are two phases of Ca2+ requirement during starfish oocyte maturation. These are the activation of MPF, moreover, GVBD, and the subsequent chromosome condensation. © 2007 Elsevier Inc. All rights reserved. Keywords: Calcium wave; Cdc2; Chromosome condensation; GVBD; Histone kinase; MPF; Oocyte maturation; TMB-8
1. Introduction Starfish oocytes are a useful model for the study of the cell cycle. Fully-grown oocytes are arrested at prophase of the first meiosis and resume meiosis in response to the maturationinducing hormone, 1-methyladenine (1-MA) that acts on a receptor on the egg membrane. During the resumption of meiosis, an increase in intracellular free Ca2+ is involved in many animals (Whitaker and Patel, 1990; Berridge, 1995; Wilding, 1996). When the Ca2+ chelator, BAPTA blocks the increase in intracellular Ca2+, the resumption of meiosis does not occur in mouse oocytes (Kline and Kline, 1992). During oocyte maturation in Xenopus laevis oocytes, a rise in free Ca2+ is involved in triggering oocyte maturation (Cicirelli and Smith, 1987) and cdc2 kinase activation (Lindsay et al., 1995). Furthermore, in the nemertean worm, Cerebratulus lacteus, injecting aqueous extracts from sperm triggers repetitive Ca2+ waves and resumption of meiotic maturation (Stricker, ⁎ Corresponding author. Tel./fax: +81 99 285 8160. E-mail address: [email protected] (H. Tosuji). 1095-6433/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2007.01.018
1997). The role of Ca2+ during starfish oocyte maturation has been reviewed (Meijer and Guerrier, 1984), and the importance of Ca2+ has been well described; however, precise Ca2+ release requesting processes during oocyte maturation are still unclear. Results vary from species to species. In some species, a transient increase in Ca2+ occurred after 1-MA application (Moreau et al., 1978; Picard and Dorée, 1983a,b; Santella and Kyozuka, 1994). While other investigators have found no change in intracellular free Ca2+ in response to the 1-MA in different species (Eisen and Reynolds, 1984; Stricker et al., 1994). As well, buffering cytoplasmic Ca2+ by the microinjecting Ca2+ chelators EGTA or BAPTA do not inhibit 1-MA-induced meiosis reinitiation (Picard and Dorée, 1983b; Witchel and Steinhardt, 1990). Although all investigators have shown that Ca2+ is required in mitosis, the relationship between meiosis and Ca2+ is not clear. TMB-8 {8-(diethylamino) octyl-3,4,5-trimethoxybenzoate hydrochloride}, which blocks Ca2+ release from intracellular Ca2+ stores (Chiou and Malagodi, 1975), was found to inhibit the egg activation of X. laevis (Kline and Nuccitelli, 1985) and sea urchin (Stapleton et al., 1985). In this study, we used TMB8 in place of the Ca2+ chelators (EGTA or BAPTA), which were
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used in most of the above studies. TMB-8 can permeate through the cell membrane, so we were able to confirm that the maturation and development ability recovered perfectly when TMB-8 was washed out. We show here that TMB-8 blocked maturation-promoting factor (MPF) activation, germinal vesicle breakdown (GVBD) and chromosome condensation in Asterina pectinifera oocytes. In addition, it is conceivable that these events are directly related with the intracellular Ca2+ levels. 2. Materials and methods
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(0.25 μg/mL) in buffer A. The sample was examined by Nikon Optiphot microscope equipped with epifluorescence optics using UV filter set, and recorded on Kodak T-max 400 film. 2.4. Microinjection techniques To test MPF activity, the donor cytoplasm from the maturing oocyte was transferred into the immature oocyte (prophase arrested oocyte) by microinjection. The recipient oocyte was put in a holding chamber and microinjected with about 250 pL of donor cytoplasm. The tip of the glass needle used for the injection was approximately 5 μm in diameter.
2.1. Materials 2.5. Assay of the histone H1 kinase activity Starfish (A. pectinifera) were collected during the breeding seasons at Mutsu Bay (Aomori, Japan), Tokyo Bay (Chiba, Japan) and the Yatsushiro Sea (Kagoshima, Japan). They were kept in circulating seawater at 15–16 °C until use. Oocytes containing germinal vesicle were obtained by dissecting the ovaries using fine forceps. To induce the maturation of oocytes, 0.25 μM 1-MA or 5 μM calyculin A (CL-A; a gift from Prof. Fusetani) was applied. All experiments with live oocytes were done at 18–22 °C. To block intracellular Ca2+ release, oocytes were treated with TMB-8 (Aldrich) in Ca2+ -free seawater. The oocytes were pre-incubated in seawater containing 100 μM TMB-8 for 10–15 min before adding 1-MA or CL-A; TMB8 continuously remained in the solution. 2.2. Measurement of intracellular free Ca2+ Intracellular free Ca2+ was measured as previously described (Kyozuka et al., 1998) using Calcium Green (Calcium Green-1 dextran, MW = 10,000, Molecular Probes) as a Ca2+ indicator. Calcium Green was dissolved at 5 mg/mL in 100 mM potassium aspartate, 10 mM Hepes (pH 7.0) and microinjected into immature oocyte cytoplasm. The fluorescence of Calcium Green in the oocyte cytoplasm was detected every 3–5 s by an Olympus IMT-2 inverted fluorescence microscope equipped with Olympus OSP-3, computer-controlled photomultiplier system. The excitation wavelength was 475–490 nm and the emission wavelength was 530 nm. The oocytes were set onto the measurement system using a holding pipette. The fluorescence intensity was measured in a circular area of 70 μm diameter in the oocyte out of 150 μm in diameter (21.8% area of the oocyte). The recorded fluorescence (F) was normalized against the initial value of F (F0) by the equation (F − F0) / F0 (Stricker et al., 1994). 2.3. Fluorescence microscopy To visualize chromosomes, the oocytes were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI). Oocytes were allowed to settle onto protamine coated glass slides. They were fixed with 5% formalin in buffer A (0.1 M KCl, 0.8 M glucose, 5 mM MgCl2 and 5 mM EGTA in 10 mM MOPS buffer, pH 6.7) containing 0.05% Nonidet P-40 for 30 min, then the slides were immersed in the same fixative for 1 h at room temperature. After washing with buffer A, the oocytes were stained with DAPI
Phosphorylation of exogenously added histone H1 was measured as it depends on the MPF activity (Picard et al., 1989). The ATP mixture was reacted with the oocyte homogenates for 5 min at 25 °C. The ATP mixture consisted of 100 μM [γ-32P] ATP (20 Bq/pmol; ICN Biomedicals), 10 mM MgCl2 and 2 mg/mL H1 histone (type III-S; Sigma) in 20 mM Tris–HCl buffer, pH 7.5. After the reaction, we used SDS-PAGE followed by autoradiography instead of a liquid scintillation counter. The reaction was terminated by adding an equal volume of the SDS sample buffer (30% urea, 8% dithiothreitol and 5% SDS in 0.5 M Tris–HCl buffer pH 6.8) to the reacting mixture. Then samples were heated in boiling water for 3 min, and proteins were separated by SDSPAGE (12.5% separating gel). The gel was dried onto Whatman 3 MM paper and autoradiographed on Fuji RX film. 2.6. Immunoblotting (western blotting) MPF from the oocytes were purified using p13suc1-binding Sepharose beads and p34cdc2 was detected by immunoblotting as described previously (Meijer et al., 1989). The eggs were sonicated on ice in the homogenization buffer and a 14,000 ×g supernatant fraction was incubated with p13suc1-Sepharose beads (Oncogene Research Products) for 30 min at 4 °C. The beads were washed three times with the beads buffer. After centrifugation at 10,000 ×g, an SDS sample buffer was added in equivalence to the pellet. Then samples were heated in boiling water for 3 min, and run on SDS-polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose membrane (BA 85; Schleicher and Schüll). The nitrocellulose membrane was incubated with a 1:1000 dilution of the control cdc2 antibody or the phosphospecific cdc2 (Tyr15) antibody (Cell Signaling Technology); antibodies were then visualized using Vectastain ABC kit (Vector Laboratories) with 3-3′-diaminobenzidine + H2O2 as a substrate. 3. Results 3.1. Effect of TMB-8 on oocyte maturation Immature oocytes of A. pectinifera were pretreated with various concentrations of TMB-8 before the addition of 1-MA or CL-A in the same solution. TMB-8 inhibited both 1-MA-and CL-A-induced oocyte maturation as reported (Tosuji et al.,
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1991). In the present study, exposure of the oocytes in more than 50 μM TMB-8 seawater inhibited 1-MA- and CL-Ainduced oocyte maturation completely. The inhibition was reversible. When TMB-8 (100 μM) was removed by washing the oocytes in seawater, they underwent normal GVBD after readdition of 1-MA (mean ± S. E. of GVBD was 97.7 ± 1.6%). 3.2. Ca2+ transients during oocyte maturation Two peaks of Ca2+ release were seen within a few minutes after addition of 1-MA to immature oocyte at the reinitiation of meiosis from prophase {Fig. 1A; this result is shown as a control although same result was reported by Santella and Kyozuka (1994)}. GVBD in this clamped oocyte occurred at around 30 min after the addition of 1-MA. In Ca2+ -free seawater, the same Ca2+ transient was observed (Fig. 1B) and GVBD occurred on time as well. TMB-8 (100 μM) blocked the
Fig. 2. Oocytes staining by DAPI. (A) Immature oocyte. (B) 1-MA-induced ordinary matured oocyte (40 min after the addition of 1-MA). (B′) Oocyte treated with both TMB-8 (100 μM) and 1-MA (40 min after 1-MA addition). (C) CL-A-induced matured oocyte (70 min after CL-A addition). (C′) Oocyte treated with both TMB-8 (100 μM) and CL-A (70 min after CL-A addition). Bar = 50 μm. The darkish circles on A, B′ and C′ correspond to germinal vesicle.
intracellular Ca2+ release completely (Fig. 1C) and GVBD did not occur in these oocytes. The Ca2+ peaks similar to the control immature oocytes took place after 100 μM TMB-8-pretreated oocytes had transferred to seawater containing 1-MA, although the Ca2+ peak was slightly delayed (Fig. 1D). The germinal vesicle breakdown proceeded normally in these oocytes. 3.3. Condensation of chromosomes
Fig. 1. Typical Ca2+ transients in the oocytes. The abscissa shows time from the beginning of measurement. The ordinate shows relative fluorescence intensity {(F − F0) / F0}. (A) 1-methyladenine-induced oocyte maturation in normal seawater (ordinary maturation). (B) 1-methyladenine-induced oocyte maturation in Ca2+ -free seawater. (C) 1-methyladenine-treated oocyte with 100 μM TMB8 in Ca2+ -free seawater. (D) The oocyte was treated with 100 μM TMB-8 in Ca2+ -free seawater for 15 min, then TMB-8 was removed and the oocyte was treated with 1-MA. Figures show the experiments of typical Ca2+ release reactions. 0 min = beginning of treatment with 25 μM 1-MA.
Further process of oocyte maturation in TMB-8 seawater was observed by focusing on chromosomes condensation by staining with DAPI and fluorescent microscopy. In the ordinary oocyte maturation induced by 1-MA, the chromatin began to condense about 10 min after the addition of 1-MA, and they condensed completely in 30 min (Fig. 2B). The 1st polar body formed in 40 min. In CL-A-treated oocytes, the chromatin began to condense about 40 min after the addition and condensed completely within 60 min (Fig. 2C). The 1st polar body formed about 100 min. By comparison, the change of the chromatin form did not observe by time-lapse microscopy during the TMB-8-treatment (100 μM) both 1-MA and CL-Atreated oocytes, the chromosomes did not condense in TMB8 seawater after the treatment in sufficient times (Fig. 2B′,C′).
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Table 1 Determination of MPF activities by cytoplasmic transferring technics Donor
Recipient
GVBD (%)a
1-MA-induced mature oocyte CL-A-induced mature oocyte TMB-8 & 1-MA-treated oocyte TMB-8 & CL-A-treated oocyte 1-MA-induced mature oocyte
immature oocyte immature oocyte immature oocyte immature oocyte TMB-8-treated immature oocyte TMB-8-treated immature oocyte
6/7 (86) 5/6 (83) 0/5 ( 0) 4/6 (67) 0/6 ( 0)
CL-A-induced mature oocyte
0/4 ( 0)
Approximately 250 pL of donor cytoplasm was microinjected into recipient oocyte. The concentration of TMB-8 was 100 μM. aGVBD of the recipient oocyte was counted.
3.4. MPF activities MPF activities of the TMB-8-treated oocytes were examined by cytoplasmic transfer assay. As seen in Table 1, both 1-MAand CL-A-treated oocytes (as controls) had MPF activities. TMB-8 (100 μM) prevented 1-MA-induced MPF activity. However, TMB-8 in CL-A-treated oocytes did have MPF activity, GVBD was not induced in the solution. When cytoplasm from the MPF-activated oocyte was transferred to TMB-8treated immature oocytes, GVBD did not occur in the recipient oocyte.
Fig. 4. Western blotting analysis of cdc2. The nitrocellulose membranes were incubated with an anti-cdc2 (Tyr15) antibody (upper panel) or an anti-cdc2 antibody (lower panel). The antibodies were visualized using Vectastain ABC kit. (Lane1) immature oocyte (IM). (Lane2) 1-MA-treated oocyte (ordinary maturation). (Lane3) TMB-8 and 1-MA-treated oocyte. (Lane4) CL-A-treated oocyte. (Lane5) TMB-8 and CL-A-treated oocyte. Asterisks indicate the band of cdc2 (Tyr15). U, upper band; M, middle band; L, lower band.
3.5. Histone H1 kinase activities MPF activities as histone H1 kinase activities of oocytes during maturation were directly measured. Both 1-MA-and CLA-treated oocytes had high levels of histone H1 kinase activity (Fig. 3A lanes 2,4). TMB-8 (100 μM) in the 1-MA-treated oocyte had a low level of histone kinase activity (Fig. 3A lane 3 and 3B). However, TMB-8 (100 μM) and CL-A-treated oocytes had high levels of the histone kinase activity (Fig. 3A lane 5) in agreement with the cytoplasmic transfer studies. 3.6. Molecular changes of p34cdc2
Fig. 3. Histone H1 kinase activities in oocytes. (A) The oocytes were harvested 40 min after the treatment of 1-MA and 60 min after the treatment of CL-A. (lane 1) immature oocyte (IM). (lane 2) 1-MA-treated oocyte (ordinary maturation). (lane 3) TMB-8 (100 μM) and 1-MA-treated oocyte. (lane 4) CL-A-treated oocyte. (lane 5) TMB-8 (100 μM) and CL-A-treated oocyte. Asterisks indicate the band of histone H1. (B) Time course of phosphorylation of histone H1 during 1-MA-induced oocyte maturation with/without TMB-8. The numbers above the picture are the time (in min) after the treatment of 1-MA.
P34cdc2 was separated with SDS-PAGE and detected by immunoblotting. The bands detected by cdc2 antibody existed in all oocytes (Fig. 4 lower panel). Phosphorylation of Tyr15 on p34cdc2 was observed only in immature oocytes and in TMB-8positive (100 μM) 1-MA-treated oocytes (Fig. 4 upper panel lane 1 and 3). The cdc2 (Tyr15) antibody binds to Tyr15phosphorylated p34cdc2, which is a component of the inactivated MPF. The result indicates MPF in these oocytes were inactive. The band detected by this antibody was not detected in CL-Atreated oocytes (Fig. 4 upper panel lane 4 and 5). A shift to lower bands from the upper band of p34cdc2 was observed in oocytes in which Tyr15 of p34cdc2 was not phosphorylated (Fig. 4 lower panel). These results show that MPF activity induced by CL-A was not affected by TMB-8 treatment. 4. Discussion It has not been firmly established if an increase of intracellular Ca2+ is required for meiotic maturation in starfish oocytes. Ca2+
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transients were observed during 1-MA-induced meiosis reinitiation in Marasthenias glacialis (Moreau et al., 1978; Picard and Dorée, 1983a,b), A. pectinifera (Santella and Kyozuka, 1994). In contrast, other investigators have shown no change in intracellular free Ca2+ in response to 1-MA in Asterias forbesi (Eisen and Reynolds, 1984), Asterina miniata, Orthasterias koehleri and Pisaster ochraceus (Stricker et al., 1994). Ca2+ release may be not a sufficient condition but a necessary condition for starfish oocyte maturation. Oocytes of A. pectinifera showed Ca2+ spikes during oocyte maturation as shown by Santella and Kyozuka (1994). When 1-MA was added by diffusion to oocytes of A. miniata, Ca2+ transients did not occur; however, when 1-MA was added by perfusion, Ca2+ transients occur (Witchel and Steinhardt, 1990). Therefore, Ca2+ transients are shown to be unnecessary for oocyte maturation. The question of whether a transient Ca2+ rise plays a causal role in meiotic maturation has been further confused by the question of whether Ca2+ transients are a response to a hormone. The reversibility for TMB-8 inhibition was confirmed, since it eliminated Ca2+ spikes. This means that GVBD has a close relationship with intracellular Ca2+ release. TMB-8 is a non-specific endocellular Ca2+ release antagonist (Chiou and Malagodi, 1975). It inhibits inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ -release in sea urchin egg homogenate (Clapper and Lee, 1985). As TMB-8 blocked GVBD completely, it is conceivable that the release of free Ca2+ from intracellular Ca2+ stores is necessary for resumption of meiosis in starfish. Calcium transient after 1-MA causes in the cytoplasm and then Ca2+ release in the germinal vesicle followed. The nuclear Ca2+ increase triggers GVBD, because when increase in nuclear Ca2+ is blocked by injecting BAPTA directly into the germinal vesicle, GVBD is blocked after 1-MA application (Santella and Kyozuka, 1994). Increase in intracellular free Ca2+ is necessary during the activation of MPF. Our results support other reports that MPF is regulated by intracellular free Ca2+ levels in amphibian oocytes or eggs (Capony et al., 1986; Bement and Capco, 1991), in ascidian oocytes (Russo et al., 1996), in sea urchin eggs (Patel et al., 1989) and in starfish oocytes (Capony et al., 1986). In the embryonic division of sea urchin and X. laevis, MPF activity also rises through Ca2+ /calmodulin-dependent protein kinase, phosphorylation and activation of cdc25, increase of the phosphorylatedcyclin/cyclin ratio (Whitaker and Patel, 1990; Swanson et al., 1997). In A. pectinifera, 1-MA induces a translocation of calmodulin from the nucleoplasm to the cytoplasm prior to the breakdown of the nuclear envelope, and the injection of anti-calmodulin inhibits the resumption of meiosis (Santella and Kyozuka, 1997a). Additionally, microinjection of InsP3 into A. pectinifera immature oocytes caused Ca2+ release but not maturation (Chiba et al., 1990). In Astropecten auranciacus, the injection of InsP3 or cyclic ADP ribose (cADPr) into the germinal vesicle caused a Ca2+ spike and meiosis resumption in the absence of 1-MA (Santella and Kyozuka, 1997b). The source of intracellular free Ca2+ during starfish oocyte maturation seems to be from the InsP3-sensitive pathway and following cortical Ca2+ flash induced by cADPr (Nusco et al., 2002). Together with our results, it is conceivable that
TMB-8 permeates into germinal vesicle through cytoplasm then it reduces the InsP3-sensitive Ca2+ release. Fluorescence microscopic studies showed that TMB8 inhibited the condensation of chromosomes. TMB-8 inhibits the chromosome condensation although MPF is activated by CL-A. CL-A activates MPF directly by changing the phosphorylation level (Tosuji et al., 1991). In fact, the MPF activity was high in CL-A-treated oocytes regardless of the presence of TMB-8. The TMB-8 positive CL-A-treated oocytes had high MPF activity; however, GVBD did not progress. TMB-8 inhibited the change of phosphorylation levels of cdc2 protein in 1-MA-treated oocytes. During oocyte maturation, the electrophoretic mobility of cdc2 protein was shifted from the upper band to middle and lower bands (Ookata et al., 1992). The upper band is the inactive form of cyclin B-associated cdc2 with presumably phosphorylated Thr14 and Tyr15 residue. The lower band is an active form with presumably dephosphorylated Thr14 and Tyr15 residue. The middle band is present continuously as an intermediate state. In our results, Tyr15 residue of cdc2 was still phosphorylated even though TMB-8-positive oocytes were treated with 1-MA. TMB-8 prevented the dephosphorylation of Tyr15 residue from the viewpoint of molecular change in MPF. Additionally, Table 1 shows that the TMB-8treated oocytes did not mature after they were injected with the activated MPF. The result indicates that GVBD requires intracellular Ca2+ release as well as MPF to be activated. This fact supports the previous reports that EGTA prevents PSTAIR-induced GVBD (Picard et al., 1990) and Ca2+ / calmodulin protein kinase is necessary for nuclear envelope breakdown (Baitinger et al., 1990). Therefore GVBD and chromosome condensation require another release of intracellular free Ca2+ after the activation of MPF. During the cell cycle in sea urchin embryo, Ca 2+ transient is necessary for the chromosome segregation (Groigno and Whitaker, 1998). At the exit of mitosis, the ubiquitin-conjugating enzyme is activated by Ca 2+ /calmodulin-dependent protein kinase (Swanson et al., 1997). Additionally, an injecting of BAPTA blocks the mitosis exit by the increase of the phosphorylated-cyclin/cyclin ratio (Whitaker and Patel, 1990). However, Ca2+ transient seems to be necessary for the chromosome condensation in the case of starfish oocyte maturation (Fig. 2). In conclusion, during the oocyte maturation of the starfish A. pectinifera, it is highly conceivable that condensation of chromosomes and the breakdown of the germinal vesicle are directly related endogenous Ca2+ release after the activation of MPF. In addition, intracellular Ca 2+ release appears necessary in the activating pathway of MPF induced by 1-MA. Acknowledgements We are indebted to the staff of Research Center for Marine Biology, Asamushi, Tohoku University and The Nemoto Lab. of Ochanomizu University for supplying the starfish. We also thank Prof. Nobuhiro Fusetani of Hokkaido University for his kind gift of CL-A.
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