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Research Article |
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic
ABSTRACT
Aurora kinase B (AURKB) is a chromosomal passenger protein that is essential for a number of processes during mitosis. Its activity is regulated by association with two other passenger proteins, INCENP and Survivin, and by phosphorylation on Thr 232. In this study, we examine expression and phosphorylation on Thr-232 of AURKB during meiotic maturation of pig oocytes in correlation with histone H3 phosphorylation and chromosome condensation. We show that histone H3 phosphorylation on Ser-10, but not on Ser-28, correlates with progressive chromosome condensation during oocyte maturation; Ser-10 phosphorylation starts around the time of the breakdown of the nuclear envelope, with the maximal activity in metaphase I, whereas Ser-28 phosphorylation does not significantly change in maturing oocytes. Treatment of oocytes with 50 µM butyrolactone I (BL-I), an inhibitor of cyclin-dependent kinases, or cycloheximide (10 µg/ml), inhibitor of proteosynthesis, results in a block of oocytes in the germinal vesicle stage, when nuclear membrane remains intact; however, condensed chromosome fibers or highly condensed chromosome bivalents can be seen in the nucleoplasm of BL-I- or cycloheximide-treated oocytes, respectively. In these treated oocytes, no or only very weak AURKB activity and phosphorylation of histone H3 on Ser-10 can be detected after 27 h of treatment, whereas phosphorylation on Ser-28 is not influenced. These results suggest that AURKB activity and Ser-10 phosphorylation of histone H3 are not required for chromosome condensation in pig oocytes, but might be required for further processing of chromosomes during meiosis.
gamete biology, kinases, meiosis, oocyte development
INTRODUCTION
Fully-grown pig oocytes remain arrested at the diplotene stage of the first meiotic prophase, defined as the germinal vesicle (GV) stage. Removing these oocytes from their follicles results in the spontaneous resumption of meiosis, which is characterized by several morphological changes that include chromosome condensation, breakdown of the germinal vesicle (GVBD), and rearrangement of the microtubule network during meiosis I, followed by the extrusion of the first polar body and block of the oocytes in metaphase of the second meiosis (the so-called metaphase II [MII] block).
The meiotic resumption is controlled by a network of protein kinases and phosphatases. A large body of evidence has demonstrated a key role for the M-phase promoting factor (MPF) or CDC2 kinase, which in pig oocytes becomes activated approximately at the time of GVBD. It is believed to be responsible for the major morphological changes occurring during meiosis I and also during the MII block (see above) [1–6].
Chromosome condensation, which is the first visible process occurring at the beginning of maturation, is essential for the correct packaging of chromatin fibers into chromosomes and their proper segregation during meiotic maturation. The different stages of chromatin condensation, which in pig oocytes takes place within the intact GV, have been described in detail by Motlik and Fulka [7]. Freshly isolated pig oocytes are in the GVI stage, which is characterized by a condensed heterochromatin ring around the nucleolus. During the next 16–20 h, chromatin gradually forms characteristic fibers (GVII and GVIII), which are further compacted into fully condensed chromosomes in the GVIV stage, which is then followed by GVBD and resumption of meiosis [7].
The process of chromatin condensation is not yet fully understood in either mitotic or meiotic cells. Although it has long been believed that the major role in this process is played by CDC2 kinase and that hyperphosphorylation of the linker histone H1 during mitosis (probably mediated by CDC2 kinase) is causally linked to mitotic chromosome condensation [8], mounting evidence has shown that chromosome condensation can occur in the absence of H1 or H1 phosphorylation in vitro [9–11] and in vivo [12, 13]. It has also been shown recently that the process of chromosome condensation during meiotic maturation of pig oocytes does not require an active CDC2 kinase [5].
Apart from linker histone H1, two of the nucleosome core histones become specifically phosphorylated during mitosis: histones H2A and H3 [14]. Phosphorylation of histone H3 on serine 10 (Ser-10) is linked with chromosome condensation of mitotic cells and transcriptional activation during interphase [15, 16]. In mammalian cells, phosphorylation of histone H3 initiates at the centromeric heterochromatin in late G2 phase before spreading throughout the condensing chromatin during mitosis. In general, a precise spatial and temporal correlation between H3 phosphorylation and initial stages of chromatin condensation has been observed [17]. In Tetrahymena, Ser-10 phosphorylation of histone H3 is required for normal chromosome segregation during meiosis and mitosis [18]. It has further been suggested that this histone H3 modification is required for the initiation but not for the maintenance of the chromosomes' condensed state [19]. In mammalian cell lines, a coincidence between chromosome condensation and phosphorylation of histone H3 on another residue, serine 28 (Ser-28), has been also documented [20, 21]. Finally, Bui et al. [22] report a correlation between chromosome condensation and histone H3 phosphorylation (Ser-10) during meiotic maturation of pig oocytes.
On the other hand, it has been shown that during meiosis in maize meiocytes, histone H3 phosphorylation does not play a role in the early steps of chromosome condensation [23]. Similar results have been observed during meiosis in murine spermatocytes [24] and during Xenopus oocyte maturation [25], and these results demonstrate that histone H3 phosphorylation cannot be solely responsible for condensation of meiotic chromosomes.
A mitotic kinase responsible for histone H3 phosphorylation belongs to a conserved family of mitotic Aurora/Ipl 1p kinases. In Drosophila, it has been documented that one of its members, Aurora kinase B (AURKB) is required for histone H3 phosphorylation [26], and recently, X AURKB has been identified as kinase, which phosphorylates directly histone H3 during mitosis in Xenopus egg cell-free extracts [27]. AURKB has been demonstrated to be responsible for histone H3 phosphorylation on Ser-10 and Ser-28 in mammalian cells during mitosis [21]. Giet and Glover [26] also suggest that AURKB-mediated histone H3 phosphorylation is required for recruitment of condensin to chromosomes. Condensin, a five-subunit protein complex, has been shown previously to play a central role in both assembly and maintenance of mitotic chromosome structure [28]. A direct link between histone H3 phosphorylation and condensin recruitment onto chromosomes has recently also been suggested by the colocalization of members of the condensin complex with phosphorylated histone H3 during the early stages of mitotic chromosome condensation [29].
In this study, we examined the correlation between chromosome condensation and phosphorylation of histone H3 (Ser-10) during in vitro maturation of pig oocytes both in control samples and also using inhibitors of proteosynthesis cyclin-dependent kinases, or specifically Aurora kinases—cycloheximide, butyrolactone I, and ZM447439, respectively. We report here that neither AURKB activity nor histone H3 phosphorylation is required for chromosome condensation in pig oocytes and that these changes are rather concomitant events, which might be required for further processing of chromosomes during meiosis.
Materials and methods
Oocyte Collection, Culture, and Evaluation
Ovaries, collected from slaughtered noncycling gilts, were transported in physiological saline at 20°C to the laboratory. The ovaries were briefly washed in physiological saline. Cumulus-oocyte complexes were aspirated from 2- to 5-mm follicles using a 21-gauge needle attached to a syringe. Only oocytes surrounded by compact cumuli were used for the culture. Isolated oocytes were cultured in M 199 medium (Sigma) supplemented with 10% fetal bovine serum (Sigma), 0.68 mM glutamine, 100 mM sodium pyruvate, 200 mM cysteamine, 100 IU/ml penicillin, 0.1 mg/ml streptomycin (Sigma), and 1 IU/ml FSH (Bioveta). In the experiments examining the effect of the inhibitors, the culture medium was supplemented with butyrolactone I (BL-I; 50 µM final concentration; Calbiochem), cycloheximide (10 µg/ml final concentration; Sigma), or ZM447439 (5–10 µM final concentration; AstraZeneca), and the oocytes were cultured in these supplemented media for 27 h. Stock solutions of inhibitors were prepared in DMSO. All cultures were performed in a humidified atmosphere of 5% CO2 at 38.5°C. At the end of culture the cumulus of oocytes were removed by vortexing for 2 min. Denuded oocytes were then washed by physiological saline, and after the last wash, the oocytes were stored at –80°C until use in immunoblotting or kinase assay experiments.
For checking the current stage of maturation at the time of sample collection, an aliquot of 15–20 oocytes was fixed and stained according to the following procedure: cumulus cells were removed by vortexing for 2 min (after cultivation) or 10 min (immediately after isolation) in narrow glass tubes. Denuded oocytes were mounted on microscope slides with Vaseline (Sigma), covered with cover glass, and fixed in solution ethanol:acetic acid (3:1, v/v) for 48 h. Staining was performed with 2% orcein in 50% aqueous-acetic acid and 1% sodium citrate. The slides were then placed in 40% acetic acid and observed with a phase-contrast NU Zeiss microscope.
Immunoblotting of Oocyte Extracts
All the reagents were obtained from Sigma unless specifically stated.
Oocytes (100 oocytes per extract) were subjected to SDS-PAGE gel (15% acrylamide, 0.75 mm thick) [30] and proteins were transferred from gels to Immobilon P membrane (Millipore Corporation) using a semidry blotting system (Whatman Biometra GmbH) for 30 min at 5 mA/cm2. The blocking of the membrane was performed in 5% nonfat milk in TBS-Tween buffer (TBS-T; 20 mM Tris, pH 7.4, 137 mM NaCl, and 0.5% Tween 20) for 1 h, and after three washes for 10 min in TBS-T buffer the membrane was incubated overnight with rabbit polyclonal antiphospho-Ser-10 histone H3 antibody (1:500 dilution; Cell Signaling Technology) in 3% BSA/TBS-T, rabbit polyclonal antiphospho-Ser-28 histone H3 (1:1000 dilution; Upstate) in 5% nonfat milk/TBS-T, polyclonal rabbit antiphospho-Thr-32 AURKB antibody (1:500 dilution; Cell Signaling Technology) in 3% BSA/TBS-T, or mouse monoclonal anti-AURKB antibody (1:1000 dilution; BD Transduction Laboratories). Then the membranes were washed three times for 10 min in TBS-T and incubated with horseradish peroxidase-conjugated donkey anti-rabbit or anti-mouse IgG antibodies (1:7500 dilution; Jackson Immuno Research) in 5% nonfat milk/TBS-T for 1 h at room temperature. Proteins or phosphorylated forms of proteins were visualized by the ECL-PLUS detection system (Amersham Biosciences) according to manufacturer instructions.
Histone H3 Kinase Assay
All reagents were obtained from Sigma unless specifically stated.
Histone H3 kinase activity was measured in oocytes by their capacity to phosphorylate histone H3 as an external substrate. At each time interval during the culture, 10 oocytes per sample were collected, washed 4 times in PBS, and transferred in 3 Ml of PBS into Eppendorf tubes. Samples were immediately frozen on dry ice and stored at –80°C until assays were performed. Immediately before kinase assay, 5 µl of homogenization buffer (50 mM Hepes-kalium hydroxide (KOH), pH 7.4, 100 mM KCl, 20 mM MgCl2 and 8 mM MnCl2, 20 mM para-nitrophenyl phosphate, 40 mM beta-glycerophosphate, 40 mM NaF, 0.2 mM EDTA, 2 mM dithiothreitol (DTT), 0.2 mM Na3VO4, 2 mM benzamidine, 40 µg/ml leupeptin, 40 µg/ml aprotinin, and 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, Pefablock SC [Roche Diagnostics]) were added to each sample and the samples were subjected to three rounds of freezing and thawing on dry ice. After the final thawing, the tubes were briefly vortexed and centrifuged at 10000 x g for 15 sec. The kinase reaction was initiated by addition of 5 µl kinase buffer (25 mM Hepes-KOH, pH 7.4, 50 mM KCl, 10 mM MgCl2, and 4 mM MnCl2 with inhibitors of phosphatases), containing 10 mg/ml histone H3, together with 2 µCi/sample [
-32P]ATP (10 mCi/ml; Amersham). The reaction was conducted for 30 min at 30°C and terminated by adding 10 µl 3x concentrated SDS-PAGE sample buffer and boiling for 3 min. After electrophoresis on 15% SDS-PAGE gel [30], the gels were stained with Coomassie Blue R250, destained overnight, dried, and autoradiographed using AR Kodak films (Sigma).
Immunofluorescent Microscopy
After culture, the zona pellucida was removed from oocytes using 0.25% pronase diluted in PBS containing 0.3% (w/v) BSA (Roche) at 37°C. After being washed twice in PBS-BSA, the oocytes were fixed for 1 h in PBS containing 4% (w/v) para-formaldehyde at 4°C. The fixed oocytes were washed three times with PBS-BSA, permeabilized with 0.3% (v/v) Triton-X 100 in PBS-BSA for 10 min, and quenched with 50 mM NH4Cl in PBS-BSA for 20 min. The oocytes room temperature and then incubated with rabbit polyclonal antiphospho-Ser-10 histone H3 antibody (1:200 dilution; Cell Signaling Technology) or with rabbit polyclonal antiphospho-Ser-28 histone H3 antibody (1:500 dilution; Upstate) at 4°C overnight. After being washed three times in PBS-BSA-0.05% Tween for 15 min each, the oocytes were incubated with Alexa Fluor 594-labeled goat anti-rabbit IgG (1:1000 dilution; Molecular Probes) for 30 min at room temperature; for the next 30 min, Hoechst 33342 was added to reach a final concentration of 4 µg/ml (Sigma). After being washed three times in PBS-BSA-Tween, the oocytes were mounted on slides with mounting medium (0.1M Tris-HCl at pH 8.5, 96 mg/ml polyvinyl alcohol, 240 mg/ml glycerol, 25 mg/ml 1,4-Diazabicyclo[2.2.2]octane (Sigma), and 0.25 µg/ml Hoechst 33342) and observed using fluorescent microscopy (Olympus IX70).
RESULTS
Histone H3 Phosphorylation During In Vitro Maturation of Pig Oocytes
To investigate the level of histone H3 phosphorylation in pig oocytes, we have used three different methods: kinase assay to determine overall histone H3 phosphorylation, immunoblotting with antiphospho-Ser-10 and antiphospho-Ser-28 histone H3 antibodies for detection of specific phosphorylation changes, and immunofluorescent analyses using the above-mentioned antibodies for localization of phosphorylated histone H3.
The results of the kinase assay, in which purified histone H3 was used as an external substrate, show that overall histone H3 phosphorylation gradually increases during pig oocyte maturation, starting just before GVBD, with a maximum in the metaphase I (MI) stage and partial decrease in the MII stage (Fig. 1A). A similar pattern of phosphorylation changes was obtained using antiphospho-Ser-10 histone H3 antibodies (Fig. 1B). Phosphorylation of histone H3 on this residue tightly correlates with the gradual condensation of chromatin during the first meiotic division. On the other hand, immunoblotting with antiphospho-Ser-28 histone H3 antibodies has shown that Ser-28 is phosphorylated already in GV stage oocytes and its phosphorylation does not significantly change during the whole period of maturation (Fig. 1C).
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In GV stage, no histone H3 phosphorylated on Ser-10 has been detected using immunofluorescent analysis (Fig. 2B), whereas histone H3 phosphorylated on Ser-28 is present mostly in the germinal vesicle (Fig. 2B'). Both phosphorylated forms of histone H3 colocalize with condensed chromosomes in the MI and MII stages, respectively (Fig. 2, D, F, D', and F').
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Aurora Kinase B Expression and Activation During In Vitro Maturation of Pig Oocytes
AURKB has been shown previously to be at least partly responsible for histone H3 phosphorylation in Drosophila, Xenopus egg cell-free extracts, and mammalian somatic cells [21, 26, 27]. We document for the first time the presence of AURKB in mammalian oocytes. Using immunoblotting analysis with anti-AURKB antibodies, we show that AURKB is expressed in pig oocytes and that its level does not significantly change during maturation (Fig. 1E).
Recently, Yasui et al. [31] have described autophosphorylation of AURKB on Thr-232, and they also have proved that AURKB phosphorylated on this residue represents an active form of the kinase. Using antiphospho-Thr-232 AURKB antibodies, we show that Thr-232 phosphorylation and thereby activation of AURKB precedes phosphorylation of histone H3 on Ser-10; the level of phosphorylation on Thr 232 is at a maximum in MI and declines in MII, similarly to the phosphorylation of histone H3 on Ser-10 (Fig. 1D). This temporal correlation suggests that histone H3 could be phosphorylated on Ser-10 by AURKB in maturing pig oocytes.
The Effects of Cycloheximide and Butyrolactone I on Aurora Kinase B Activity, Histone H3 Phosphorylation, and Chromosome Condensation
To elucidate further the role of histone H3 phosphorylation in the process of chromosome condensation, we have used inhibitors of proteosynthesis and cdk-kinases, cycloheximide and BL-I, respectively. We have shown previously that both inhibitors are able to block the oocytes in the GV stage;however, the chromosomes become condensed within an intact germinal vesicle [4, 6]. The effect of the inhibitors on oocyte morphology is summarized in Table 1. Figure 3 represents oocytes treated with BL-I for 27 h, GVIII stage with partially condensed chromosome bivalents (Fig. 3A); with cycloheximide for 27 h, GVIV stage with fully condensed bivalents (Fig. 3B); and finally, control oocytes cultured for 27 h representing the MI stage (Fig. 3C).
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No or only very weak AURKB activity (measured as phosphorylation on Thr-232 by immunoblotting) was detected in oocytes treated with BL-I or cycloheximide for 27 h (Fig. 4D). Similarly, both inhibitors prevent phosphorylation of histone H3 on Ser-10 (Fig. 4B). On the other hand, Ser-28 phosphorylation of histone H3 is not influenced by BL-I or cycloheximide treatment (Fig. 4C). For immunofluorescent analysis, we have used oocytes treated for 27 h with cycloheximide, which possess fully condensed chromosome bivalents in an intact GV. No histone H3 phosphorylated on Ser-10 was detected in these treated oocytes (Fig. 2H), whereas Ser-28 phosphorylated histone H3 can be seen within the GV in cycloheximide-treated oocytes (Fig. 2H').
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The Effects of ZM447439, a Dual Inhibitor of Aurora Kinase Family, on Histone H3 Phosphorylation and Chromosome Condensation
To confirm our above mentioned findings showing that AURKB is responsible for histone H3 phosphorylation on Ser-10 in maturing pig oocytes and that this histone H3 modification is not essential for chromatin condensation, we used a dual inhibitor of Aurora kinases, ZM447439. The effect of the inhibitor on oocyte morphology is summarized in Table 2. Figure 3, D, and E, shows that the treatment of the oocytes with ZM447439 does not impair the process of chromatin condensation. A lower concentration of the inhibitor (2–5 µM) does not prevent the breakdown of the nuclear membrane; however, the metaphase plate does not form properly and the oocytes remain blocked in this late diakinesis-like stage—a compact clump of condensed chromosomes—and do not progress further (Fig. 3D). Oocytes subjected to treatment with higher concentration of the inhibitor (7–10 µM) remain blocked in the GV stage with condensed chromosomes visible in the ooplasm (Fig. 3E).
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The activity of AURKB (measured as phosphorylation on Thr-232 by immunoblotting) was largely diminished in oocytes treated with ZM447439 (in final concentrations of 5 µM and higher) for 27 h (Fig. 5A); lower concentrations of the inhibitor do not influence its activity (data not shown). Similarly, ZM447439 prevents phosphorylation of histone H3 on Ser-10 (Fig. 5B). On the other hand, Ser-28 phosphorylation of histone H3 is not influenced by ZM447439 treatment (Fig. 5C).
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DISCUSSION
In this study we show that AURKB becomes gradually phosphorylated on Thr-232 and thereby activated during pig oocyte maturation (it starts to be activated just before GVBD), with maximum activity in MI and partial decline in MII. Under control conditions, AURKB activity tightly correlates with phosphorylation of histone H3 on Ser-10, as well as with gradual condensation of chromatin. On the other hand, our results demonstrate that histone H3 is already phosphorylated on Ser-28 at the beginning of maturation (GVI stage), and this phosphorylation does not significantly change in maturing oocytes. We further show that the treatment of oocytes with inhibitors of proteosynthesis or cdk-kinases, cycloheximide, or BL-I, respectively, results in the block of oocytes in the GV stage; the nuclear membrane remains intact, but condensed chromosome fibers or highly condensed chromosome bivalents can be seen in the nucleoplasm (GVIV or GVIII, respectively). In these treated oocytes, no or only very weak AURKB activity, as well as phosphorylation of histone H3 on Ser-10, is detected even after 27 h of treatment, whereas the phosphorylation on Ser-28 is not influenced.
M-phase-specific histone H3 phosphorylation on Ser-10 has been documented previously in a number of different organisms (for a review, see [15]). Our results showing the changes of histone H3 Ser-10 phosphorylation during maturation of pig oocytes are in quite good agreement with those published previously by Bui et al. [22], except for the later stages of maturation. Contrary to the results of Bui et al. [22], who describe stable high levels of histone H3 phosphorylation between MI and MII stage, our results show a partial decrease of histone H3 Ser-10 phosphorylation in MII. These partial discrepancies are probably caused by the different methods of histone H3 phosphorylation measurements used by us and Bui et al. [22]. Whereas the quantitative results of Bui et al. [22] are based solely on histone H3 kinase assay measurements, our data from kinase assay have also been confirmed by immunoblotting analyses using specific antibodies against phosphorylated Ser-10 of histone H3.
In C. elegans and Drosophila, RNA interference experiments have shown clearly that Ser-10 phosphorylation of histone H3 is mediated by a mitotic kinase called AURKB [26, 32, 33], and similar conclusions have been also obtained in Xenopus egg cell-free extracts [27]. Goto et al. [21] suggest that in mammalian cells, AURKB is responsible for phosphorylation of histone H3 not only on Ser-10 but also on Ser-28. AURKB belongs to a family of protein kinases, which are conserved from yeasts to mammals. Although in yeasts only one Aurora kinase exists, at least three of them have been identified in mammalian cells [34–36]. AURKB has specific spatiotemporal behavior during mitosis; it has been shown to be associated with chromosomes in the inner centromere region at metaphase, then is relocalized to the spindle midzone during early anaphase, and then colocalizes with the spindle midbody in cytokinesis (for reviews see [37, 38]). In mammalian cells AURKB is activated after Aurora kinase A and before CDC2 kinase during prophase, and remains active during mitosis [39]. Similarly, AURKB activity has been detected in mitotic extracts from Xenopus eggs [27].
Our results demonstrate for the first time that AURKB also functions during mammalian meiosis, showing the activation of AURKB (measured by its phosphorylation on Thr-232) during meiotic maturation of pig oocytes shortly before GVBD, with a maximum in MI and a partial decline in MII, while its expression remains stable during the whole maturation period. Furthermore, we show that the activation of AURKB is in tight temporal correlation with histone H3 phosphorylation on Ser-10, suggesting that AURKB might function as an M-phase-specific histone H3 kinase in pig oocytes. This suggestion is further supported by the results of our experiments, in which we have used ZM 447439, an inhibitor of Aurora kinases [40]. ZM 447439 inhibits both Aurora kinase A and AURKB; however, Yang et al. [41] have shown that inactivation of AURKB bypasses Aurora kinase A in mitosis. It has also been shown that ZM447439 inhibits mitotic phosphorylation of histone H3, and this fact does not affect chromosome condensation [33, 40]. Our results are in good agreement with those obtained on mitotic cells and show that the treatment of oocytes with ZM447439 for 27 h results in the decrease of both AURKB activity and histone H3 phosphorylation on Ser-10, whereas the process of chromosome condensation is not influenced by this treatment. The fact that the condensed chromosomes are clumped and do not form a proper metaphase plate (after treatment with a lower concentration of the inhibitor) could possibly be explained by the inhibition of AURKB function monitoring the correct bi-orientation of the chromosomes on the M-phase spindle.
On the other hand, our results differ from those of Goto et al. [21], in that histone H3 phosphorylation on Ser-28 does not correlate with AURKB activity; instead, phosphorylation of Ser-28 remains on a stable level during the whole period of pig oocyte maturation. Furthermore, treatment of oocytes with ZM447439 does not induce any changes in the level of histone H3 phosphorylation on Ser-28. These differences between our results and those of Goto et al. [21] could possibly be explained by the different regulation of histone H3 Ser-28 phosphorylation in meiotic and mitotic cells.
All the above-mentioned findings, together with the fact that histone H3 phosphorylation and AURKB activation have been observed exclusively in connection with mitotic chromosomes, lead some authors to suggest that these events are involved in regulation of chromosome condensation. However, the results of experiments studying the function of H3 phosphorylation by disruption of AURKB function are somewhat controversial. In Drosophila, C. elegans, or fission yeast, the mutants or Aurora-depleted cells show defects in chromosome condensation together with defects in chromosome segregation [33, 42–44]. H3 phosphorylation is also required for chromosome condensation in Tetrahymena [45] and human cultured cells [21]. On the other hand, in Xenopus egg extracts, neither condensin targeting nor chromosome compaction is compromised when H3 phosphorylation is largely diminished by depletion of AURKB [27, 46, 47]. Further, it has been also shown that the affinity of one member of human condensin, CAP-D2, for histone H3 is not affected by histone H3 phosphorylation [48].
To address the question of possible H3 phosphorylation importance for chromosome condensation, we have set up a model using pig oocytes cultured with inhibitors of protein synthesis or cdk-kinases, cycloheximide and BL-I respectively. We have shown previously that these inhibitors block maturation of pig oocytes in the GV stage; they also impair the activation of CDC2 kinase and MAPK3/MAPK1 kinases, but they do not interfere with the process of chromosome condensation [4, 5]. In these treated oocytes we have measured histone H3 phosphorylation on Ser-10 and Ser-28, as well as the activity of AURKB. The results show that although the chromosomes become condensed in inhibitor-treated oocytes, AURKB is not activated and histone H3 Ser-10 phosphorylation is substantially decreased compared to control MI oocytes, whereas Ser-28 phosphorylation is not influenced.
Although Bui et al. [22] reported recently that histone H3 phosphorylation on Ser-10 is involved in chromosome condensation during maturation of pig oocytes, our results suggest rather the opposite, i.e., that neither AURKB activity nor histone H3 phosphorylation are essential for this process in maturing pig oocytes. However, we believe that our results and those of Bui et al. [22] are not, in fact, in contradiction. Their conclusion is based on experiments with inhibitors of protein phosphatases 1/2A, calyculin-A, and okadaic acid, which induce premature chromosome condensation in pig oocytes as well as histone H3 phosphorylation. We believe that these results and those showing the increasing histone H3 phosphorylation in control maturing oocytes might rather suggest that both activation of AURKB and increasing Ser-10 phosphorylation are events concomitant to the process of chromosome condensation and might be required for further processing of chromosomes during later stages of meiosis. This hypothesis is also a subject of our further studies.
ACKNOWLEDGMENTS
The authors are indebted to Mrs. K. Uhlirova, Mrs. P. Jandurova, Mrs. L. Travnickova, and S. Hladky for their skillful technical assistance. Prochazka and Vasa from Roudnice n/L are appreciated for donation of the porcine ovaries. We are most grateful to Nicholas Keen and AstraZeneca Pharmaceuticals for providing ZM447439.
FOOTNOTES
1 Supported by the Institutional Research Plan of the Institute of Animal Physiology & Genetics no. AVOZ 50450515 and by grant no. 204/03/0816 of Grant Agency of the Czech Republic. 
2 Correspondence: Michal Kubelka, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Rumburska 89, 277 21 Libechov, Czech Republic. FAX: 420 315 639510; kubelka{at}iapg.cas.cz
Received: 26 September 2005.
First decision: 13 October 2005.
Accepted: 31 January 2006.
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