Biol Reprod Lalor Postdoctoral Fellowships -- Application Deadline January 15, 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print February 11, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026070
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
70/6/1843    most recent
biolreprod.103.026070v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bui, H.-T.
Right arrow Articles by Miyano, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bui, H.-T.
Right arrow Articles by Miyano, T.
Agricola
Right arrow Articles by Bui, H.-T.
Right arrow Articles by Miyano, T.
BIOLOGY OF REPRODUCTION 70, 1843–1851 (2004)
DOI: 10.1095/biolreprod.103.026070
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Involvement of Histone H3 (Ser10) Phosphorylation in Chromosome Condensation Without Cdc2 Kinase and Mitogen-Activated Protein Kinase Activation in Pig Oocytes1

Hong-Thuy Bui, Emi Yamaoka, and Takashi Miyano2

Department of Life Science, Graduate School of Science and Technology, Kobe University, Rokkodai-cho Nada-ku, Kobe 657-8501, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
When oocytes resume meiosis, chromosomes start to condense and Cdc2 kinase becomes activated. However, recent findings show that the chromosome condensation does not always correlate with the Cdc2 kinase activity in pig oocytes. The objectives of this study were to examine 1) the correlation between chromosome condensation and histone H3 phosphorylation at serine 10 (Ser10) during the meiotic maturation of pig oocytes and 2) the effects of protein phosphatase 1/2A (PP1/ PP2A) inhibitors on the chromosome condensation and the involvement of Cdc2 kinase, MAP kinase, and histone H3 kinase in this process. The phosphorylation of histone H3 (Ser10) was first detected in the clump of condensed chromosomes at the diakinesis stage and was maintained until metaphase II. The kinase assay showed that histone H3 kinase activity was low in oocytes at the germinal vesicle stage (GV) and increased at the diakinesis stage and that high activity was maintained until metaphase II. Treatment of GV-oocytes with okadaic acid (OA) or calyculin-A (CL-A), the PP1/PP2A inhibitors, induced rapid chromosome condensation with histone H3 (Ser10) phosphorylation after 2 h. Both histone H3 kinase and MAP kinase were activated in the treated oocytes, although Cdc2 kinase was not activated. In the oocytes treated with CL-A and the MEK inhibitor U0126, neither Cdc2 kinase nor MAP kinase were activated and no oocytes underwent germinal vesicle breakdown (GVBD), although histone H3 kinase was still activated and the chromosomes condensed with histone H3 (Ser10) phosphorylation. These results suggest that the phosphorylation of histone H3 (Ser10) occurs in condensed chromosomes during maturation in pig oocytes. Furthermore, the chromosome condensation is correlated with histone H3 kinase activity but not with Cdc2 kinase and MAP kinase activities.

gamete biology, kinases, meiosis, ovary, phosphatases


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The meiotic resumption of oocytes is controlled by protein kinase MPF (maturation-promoting factor or M-phase promoting factor), which is now also known as Cdc2 kinase. Cdc2 kinase is activated around the germinal vesicle breakdown (GVBD) of oocytes and has been thought to be the key regulator of such major morphological changes during oocyte maturation as chromosome condensation and GVBD and spindle formation. Mitogen-activated protein (MAP) kinase, also termed extracellular signal-regulated kinase (ERK1/ERK2 in mammals), is another kinase known to become activated during oocyte maturation in a number of species, including Xenopus [1], clams [2], and also in mammalian species, such as mice [3], pigs [4], goats [5], cattle [6], and humans [7]. The role of MAP kinase during oocyte maturation has not been firmly established, although it is known that its activation modulates microtubule dynamics.

Chromosome condensation is the first step of oocyte maturation. It is a unique process characterized by a dramatic change in chromosome morphology and is required for the correct segregation of chromosomes during oocyte maturation. The precise change in the nucleus in pig oocytes during maturation was described by Motlik and Fulka [8]. Fully grown pig oocytes have a condensed heterochromatin ring around the nucleolus in the germinal vesicle (GV). When the oocytes resume meiosis in vitro and in vivo, chromosomes start to condense before GVBD. Although the chromosome condensation has been thought to be induced by Cdc2 kinase, recent findings show that the chromosome condensation does not always correlate with Cdc2 kinase activity and that MAP kinase substitutes for the role of Cdc2 kinase [9]. However, the cause of the chromosome condensation has not been completely understood.

The chromatin fiber is composed of repetitive units, the nucleosomes, which are comprised of an octamer of two each of the core histones H2A, H2B, H3, and H4, around which 200 base pairs of DNA are wrapped [10]. Each histone contains an N-terminal tail domain, which is involved in the stabilization of the chromatin fiber [11]. The phosphorylation of histone H3 is thought to be involved in transcriptional activation [12] and chromosome condensation during mitotic cell division [13]. A strong correlation between the initial chromosome condensation and histone H3 phosphorylation has been observed in mammalian cells [14], and its phosphorylation at Ser10 has been suggested to play a key role in mitotic chromosome condensation [15]. The spatiotemporal distribution of histone H3 phosphorylation is different among the cell types so far examined. However, there is no evidence about histone H3 phosphorylation in meiotic chromosome condensation in mammalian oocytes.

Both Ipl1/aurora-B kinase and its genetically interacting protein phosphatase Glc7/PP1 has been shown to be required for H3 phosphorylation during mitosis in Saccharomyces cerevisiae and Caenorhabditis elegans [16, 17]. It is suggested in somatic cells that the balance of histone H3 kinase and PP1 acting on Ser10 of histone H3 regulates chromosome condensation. Inhibitors of the protein serine/ threonine phosphatases PP1 and PP2A, such as okadaic acid (OA) and calyculin A (CL-A), induce rapid chromosome condensation in oocytes [18]. However, it remains to be elucidated whether the effect of PP1/PP2A inhibitors on the chromosome condensation correlates with Cdc2 kinase, MAP kinase, or histone H3 kinase.

In this study, we investigated the changes in histone H3 phosphorylation at Ser10 during meiotic maturation and rapid chromosome condensation induced by the inhibition of PP1/PP2A in pig oocytes. Histone H3 phosphorylation was detected in the clump of condensed chromosomes at the diakinesis stage and maintained during maturation of the oocytes. Histone H3 was also phosphorylated in condensed chromosomes in the inhibitor-treated oocytes. Furthermore, the chromosome condensation occurred without GVBD in the absence of the activation of both Cdc2 kinase and MAP kinase but was accompanied by histone H3 kinase activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture of Pig Oocytes

Pig ovaries were obtained from prepubertal gilts at a local slaughterhouse. The ovaries were washed once with 0.2% (w/v) cetyltrimethylammonium bromide and twice with Dulbecco phosphate-buffered saline (PBS) containing 0.1% (w/v) polyvinyl alcohol (PBS-PVA; Sigma Chemical Co., St. Louis, MO). Antral follicles 4–6 mm in diameter were dissected in PBS-PVA from ovaries following the technique described by Moor and Trounson [19]. After being opened in 25 mM HEPES buffered TCM-199 (Earl salt; Nissui Pharmaceutical, Tokyo, Japan), oocytes-cumulus complexes with a piece of parietal granulosa tissue (oocyte-cumulus-granulosa cell complexes; OCGCs) were isolated from the follicles. Following two washes in HEPES buffered TCM-199, the OCGCs were cultured in 2 ml of maturation medium. The maturation medium was bicarbonate-buffered TCM-199 supplemented with 10% (v/v) heat-treated fetal calf serum (Biocell Inc., Carson, CA), 0.1 mg/ml sodium pyruvate, 0.08 mg/ml kanamycin sulphate (Sigma Chemical Co.), 2.2 mg/ml sodium bicarbonate, and 0.1 IU/ml human menopausal gonadotropin (Pergonal; Teikoku Zoki, Tokyo, Japan), and two everted theca shells with gentle agitation in an atmosphere of 5% CO2 in humidified air at 38.5°C. The OCGCs were cultured for various times to obtain oocytes at the germinal vesicle (0 h), diakinesis (18 h), metaphase I (24–27 h), anaphase I and telophase I (30–33 h), and metaphase II (42 h) stages.

To examine the effects of the inhibition of PP1/PP2A on chromosome condensation, oocytes-cumulus complexes (OCCs) were used. The OCCs were cultured in maturation medium containing 50 nM calyculin A (CL- A; Sigma-Aldrich, Germany) or 2.5 µM okadaic acid (OA; sodium salt; Sigma-Aldrich) for 0, 0.5, 1, 2, 3, 4, and 6 h. To inhibit the activation of MAP kinase during the PP1/PP2A inhibition, the OCCs were cultured in maturation medium containing 50 nM CL-A and an MEK inhibitor, 0.1 mM U0126 (Promega, Madison, WI).

After culturing, the oocytes were denuded completely by pipetting and used for immunostaining or kinase activity assay.

Immunofluorescent Microscopy

After being washed twice in PBS-PVA, the denuded oocytes were fixed in PBS-PVA containing 4% (w/v) paraformaldehyde and 0.2% (v/v) Triton X-100 for 40 min. The fixed oocytes were washed twice in PBS- PVA for 15 min each and stored overnight in 1% (w/v) bovine serum albumin (BSA; International Regents Corporation, Kobe, Japan) supplemented with PBS-PVA (BSA-PBS-PVA) at 4°C. The oocytes were blocked with 10% (v/v) goat serum (DakoCytomation A/S, Glostrup, Denmark) in BSA-PBS-PVA for 45 min and then incubated for double labeling in a mixture of rabbit polyclonal anti-phospho-histone H3 at Ser10 (1: 100 dilution; #9701; Cell Signaling Technology Inc., Beverly, MA) and mouse monoclonal anti-lamin A/C (1:100 dilution; sc-7292, Santa Cruz Biotechnology Inc., Santa Cruz, CA) antibodies at 4°C overnight. After being washed three times in BSA-PBS-PVA for 15 min each, the oocytes were incubated in the mixture of Alexa Fluor 488 labeled goat-anti rabbit IgG (1:400 dilution; Molecular Probes Inc., Eugene, OR) and Alexa Fluor 350-labeled goat-anti mouse IgG (1:400 dilution; Molecular Probes Inc.) as the conjugated second antibodies for 40 min at room temperature. After being washed three times in BSA-PBS-PVA for 15 min each, the chromosomes were stained with propidium iodide (400 µg/ml; Sigma Chemical Co.). Following complete washing, the oocytes were mounted on slides by Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA) and observed under a fluorescent microscope (U- LH100HGAPO; Olympus Optical Co., Tokyo, Japan). Determination of the meiotic stages was based on the chromosome configuration described by Motlik and Fulka [8]. In the negative control, the oocytes were reacted with nonimmune rabbit serum instead of rabbit polyclonal anti-phospho- histone H3 at Ser10 antibody or with mouse IgG instead of mouse monoclonal anti-lamin A/C antibody.

Kinase Assay

After denudation and three washes in PBS-PVA, each group of oocytes was transferred into an Eppendorf tube with 1 µl of PBS-PVA for double kinase assay following the technique described by Kanayama et al. [20]. Each sample contained two oocytes for double assay of Cdc2 kinase and MAP kinase and five oocytes for double assay of Cdc2 kinase and histone H3 kinase. Thereafter, 4 µl of ice cold extraction buffer was added, and the samples were frozen at –80°C before the assay. The extraction buffer was composed of 80 mM ß-glycerophosphate, 25 mM HEPES (pH 7.2), 20 mM EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM APMSF, 0.1 mM Na3VO4, 1 µg/ml leupeptin (Sigma Chemical Co.) and 1 µg/ml aprotinin (Sigma Chemical Co.). To examine the kinase activities during maturation, the meiotic stage of the oocytes was determined before they were transferred into the Eppendorf tubes by staining with 12 µg/ml Hoechst 33342 (Polysciences Inc., Warrington, PA) for 20 min followed by observation under a fluorescent microscope. After being thawed, the oocytes were centrifuged at 13 000 x g for 10 min, added with 5 µl of kinase buffer and 5 µl of substrate solution, and incubated for 20 min at 37°C. The kinase buffer was composed of 75 mM HEPES (pH 7.2), 75 mM ß- glycerophosphate, 75 mM MgCl2, 6 mM DTT, 10 mM EGTA, 60 µM ATP, 15 µM cAMP-dependent protein kinase inhibitor peptide (Sigma Chemical Co.) and 0.3 µCi/µl [{gamma}-32P]ATP (250 µCi/25 µl; Amersham Pharmacia Biotech, Buckinghamshire, UK). The substrate solution for the double assay of the Cdc2 kinase and MAP kinase was composed of 4.25 µl of histone H1 (5 mg/ml, from calf thymus; Boehringer, Tokyo, Japan) and 0.75 µl of myelin basic protein (5 mg/ml, MBP from bovine brain; Sigma Chemical Co.). The substrate solution for the double assay of Cdc2 kinase and histone H3 kinase was composed of 2.5 µl of histone H1 and 2.5 µl of histone H3 (1 mg/ml, from calf thymus; Boehringer). The reaction was terminated by the addition of 5 µl of 4x SDS sample buffer [21] and boiling for 5 min. The samples were loaded onto a 15% gel for separation of labeled myelin basic protein and histone H1, or histone H3 and histone H1. After running, the gels were dried and autoradiographed. The autoradiograms were scanned with an image analysis system with Image Master ID Elite software Version 3.00 (Amersham Pharmacia Biotech). The kinase activity in the oocytes at metaphase I was arbitrarily set to 100%, and the other bands were expressed relative to that as a mean percentage ± SEM.

At least 30 immunostained oocytes were examined by immunofluorescent microscopy in each group. Each kinase assay experiment was performed in at least three replicates. Statistical differences in the activities of the kinases were analyzed by one-way analysis of variance (F1-test) followed by the Tukey multiple range test. Other values were analyzed using the chi-square test. P values less than 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phosphorylation of Histone H3 Takes Place During Oocyte Maturation

Phosphorylated histone H3 at Ser10 was detected in pig maturing oocytes using an antibody against histone H3 with a single phosphorylated Ser10. Histone H3 phosphorylation was not detected in any oocytes at the GV stage before culture (0%, 0/41 oocytes; Fig. 1A''). Phosphorylation of histone H3 was first detected in the clump of condensed chromosomes (green color; Fig. 1B'') in all of the examined oocytes (100%, 38/38 oocytes) at the diakinesis stage after 18 h of culture and thereafter it was maintained during oocyte maturation (Fig. 1, C''–F''). The integrity of the nuclear envelope was monitored by immunostaining with an anti- lamin A/C antibody. At the GV stage, the lamin A/C antibody recognized the entire contour of the nuclear envelope (Fig. 1A). At the diakinesis stage, chromosomes were frequently in contact with the envelope and lamin A/C staining was closely associated with the chromosome (Fig. 1, B and B'). The nuclear membrane breakdown initiated during the diakinesis stage and the nuclear membrane was completely disassembled when the oocytes entered metaphase I (Fig. 1, C–F). The control oocytes did not show any fluorescence at any stages.



View larger version (69K):
[in this window]
[in a new window]
  		FIG. 1. Immunofluorescent localization of phosphorylated histone H3 (Ser10) during maturation of pig oocytes. Anti-lamin A/C- and Alexa 350-labeled antibodies stained the nuclear membrane (blue), PI stained the chromosome (red), and anti-phospho-histone H3- (Ser10) and Alexa 488-labeled antibodies stained phosphorylated histone H3 (green) in the same oocyte. The meiotic stages are (A) germinal vesicle stage (GV), (B) diakinesis (D), (C) metaphase I (MI), (D) anaphase I (AI), (E) telophase I (TI), (F) metaphase II (MII). Scale bar = 10 µm.

The changes in the activities of Cdc2 and the histone H3 kinases during oocyte maturation were measured after determination of the meiotic stage of each oocyte by Hoechst 33342 (Fig. 2). Histone H3 kinase activity was lowest in the GV-stage oocytes, increased at the diakinesis stage, reached a peak at metaphase I, and was maintained until metaphase II (Fig. 2C). Cdc2 kinase was inactive at the GV and diakinesis stages. Thereafter, the activity increased and reached a peak at metaphase I. The activity fell during anaphase I and telophase I and increased again at metaphase II (Fig. 2B).



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 2. A) An autoradiograph showing the changes in Cdc2 kinase and histone H3 kinase activities during maturation of pig oocytes. Histone H1 (H1) and histone H3 (H3) were used for the substrates of Cdc2 kinase and histone H3 kinase, respectively. B, C) The intensity of each phosphorylated histone H1 and histone H3 were measured by densitometry analysis of blots. The data are represented as the mean ± SEM. The lower figures of the graphs show the meiotic stages of oocytes as presented in the legend for Figure 1. The experiments were conducted three times. Values with different superscripts were significantly different (P < 0.05).

PP1/PP2A Inhibitors Induce Rapid Chromosome Condensation with Histone H3 Phosphorylation

The effects of CL-A and OA on histone H3 phosphorylation, chromosome condensation, and nuclear membrane morphology were examined. Both CL-A (Fig. 3 and Table 1) and OA (Fig. 4 and Table 2) induced rapid chromosome condensation in a similar manner. Chromosomes started to condense after 2 h of treatment, but most of the chromosome configuration was still at the GV I-IV stages, which were described by Motlik and Fulka for normal maturation of pig oocytes [8]. Some oocytes had a chromosome configuration that looked like GV III or GV IV, but the chromosomes were much more condensed (GV') (Figs. 3C' and 4C'). Chromosome condensation progressed throughout the treatment. After 6 h, in most of the oocytes, the condensation of the chromosomes was like that at metaphase I (81% in the CL-A and 71% in the OA-treated oocytes). Histone H3 started to be phosphorylated after 2 h in 56% and 93% of the CL-A- and OA-treated oocytes, respectively. After 3 h, most of the treated oocytes showed histone H3 phosphorylation, which was maintained during the course of treatment. In the nontreated oocytes, the chromosomes stayed in the GV and no histone H3 phosphorylation was observed during the culture. In all of the CL- A- and OA-treated oocytes, an intact nuclear membrane was observed around the condensed chromosomes until 4 h. At 6 h, 66% of the CL-A-treated oocytes underwent GVBD, while no OA-treated oocytes underwent GVBD (Tables 1 and 2). However, 100% of the OA-treated oocytes underwent GVBD after 8 h (data not shown).



View larger version (76K):
[in this window]
[in a new window]
  		FIG. 3. Effect of calyculin A (CL-A) on histone H3 phosphorylation, chromosome configuration, and nuclear membrane morphology of pig oocytes. Each treated oocyte with different times was stained with anti-lamin A/C- and Alexa 350-labeled antibodies for nuclear membrane (blue), PI for chromosome (red), and anti-phospho-histone H3- (Ser10) and Alexa 488-labeled antibodies for phosphorylated histone H3 (green). A, B) germinal vesicle stage (GV). C) Chromosomes started to condense with phosphorylated histone H3 (GV'). D) Early diakinesis stage (ED). E) Late diakinesis stage (LD). F) Chromosomes condensed like in metaphase I (MI). Scale bar = 10 µm.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Effects of calyculin A on chromosome condensation in pig oocytes.*



View larger version (86K):
[in this window]
[in a new window]
  		FIG. 4. Effect of okadaic acid (OA) on histone H3 phosphorylation, chromosome configuration, and nuclear membrane morphology of pig oocytes. Each treated oocyte with different times was stained with anti-lamin A/C- and Alexa 350-labeled antibodies for nuclear membrane (blue), PI for chromosome (red), and anti-phospho-histone H3- (Ser10) and Alexa 488-labeled antibodies for phosphorylated histone H3 (green). Panels show the identical stages of oocytes as presented in the legend for Figure 3. Scale bar = 10;gmm.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Effects of okadaic acid on chromosome condensation in pig oocytes.*

Change in The Activities of Cdc2 Kinase, MAP Kinase, and Histone H3 Kinase During Chromosome Condensation in CL-A- and OA-Treated Oocytes

When the oocytes were treated with CL-A, both histone H3 kinase (shown by phosphorylation of histone H3 in Fig. 5A) and MAP kinase (shown by phosphorylation of MBP in Fig. 5A) were activated after 2 h, and the activities increased during the time of treatment. Cdc2 kinase (shown by phosphorylation of H1 in Fig. 5A) was not activated until 6 h. In the control nontreated oocytes, Cdc2 kinase, MAP kinase, and histone H3 kinase were not activated. The oocytes treated with OA also showed a similar result to the CL-A-treated oocytes (data not shown). These results indicate that the PP1/PP2A inhibitors induce activation of both MAP kinase and histone H3 kinase, although they have no effect on Cdc2 kinase. To investigate whether MAP kinase activation is required for the phosphorylation of histone H3, the oocytes were treated with CL-A in the presence of the MAP kinase inhibitor U0126. In the oocytes, MAP kinase was not activated, but the histone H3 kinase was still activated after 2 h of treatment (Fig. 5A). There were no differences between the histone H3 kinase levels of oocytes treated with CL-A and CL-A+U0126 (Fig. 5B). Histone H3 phosphorylation and chromosome condensation in the oocytes were also examined after 6 h of treatment (Table 3). The histone H3 phosphorylation and chromosome condensation in the oocytes treated with CL-A and CL-A+U0126 was similar. However, no GVBD was observed in the CL-A+U0126-treated oocytes.



View larger version (62K):
[in this window]
[in a new window]
  		FIG. 5. A) Autoradiographs showing effects of calyculin A (CL-A) and MAP kinase inhibitor U0126 on the activities of Cdc2 kinase, MAP kinase, and histone H3 kinase in pig oocytes. Histone H1 (H1), myelin basic protein (MBP), and histone H3 (H3) were used for the substrates of Cdc2 kinase, MAP kinase, and histone H3 kinase, respectively. Samples were subjected to a double kinase assay of H1/MBP or H1/H3. Change in the activities of Cdc2 kinase, MAP kinase, and histone H3 kinase in oocytes treated with (+) or without (–) the CL-A and CL-A and MAP kinase inhibitor U0126. B) The intensity of phosphorylated histone H3 was measured by densitometry analysis of blots. The activities of histone H3 kinase in MI oocytes were set to 100%. The data represent the histone H3 kinase activities as the mean ± SEM. Values with different superscripts are significantly different (P < 0.05). The experiments were conducted three times with similar results.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Effects of MAP kinase inhibitor, U0126 on chromosome condensation induced by calyculin A in pig oocytes.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Pig oocytes undergo GVBD in a similar time course required to activate the Cdc2 kinase [22]. Following the gonadotropic stimulation, pig oocytes start to accumulate cyclin B1 at 21 h, and Cdc2 kinase activity gradually increases around GVBD at 24 h and reaches a maximal level at the metaphase I at 27 h in our culture conditions [23]. Pig oocytes have MAP kinase in the inactive form before the resumption of meiosis, and the MAP kinase becomes phosphorylated and activated after GVBD [24, 25]. It has been reported that the phosphorylation of histone H3 in the nucleosome is closely linked to mitotic chromosome condensation and occurs at the Ser10 position in diverse organisms [26, 27]. We determined the phosphorylation of histone H3 (Ser10) in maturing pig oocytes. There was no phosphorylation of histone H3 in the oocytes at the GV stage (G2 phase in cell cycle), and phosphorylation started in condensed chromosomes at the diakinesis stage. This observation differs from that of mitotic chromosomes in somatic cells, where phosphorylated histone H3 (Ser10) is first detected in pericentromeric heterochromatin in the late G2 phase, spreads throughout the chromosomes, completes in late prophase and is maintained through metaphase [28]. Thus, the meiotic phosphorylation of histone H3 (Ser10) appears late at the time at which the chromosomes have become highly condensed. During meiosis in maize, the phosphorylation of histone H3 (Ser10) also takes place at the diakinesis-metaphase I transition [29]. In mitosis in animal cells and meiosis in maize, histone H3 (Ser10) phosphorylation is diminished in anaphase (anaphase I in meiosis) and disappears at telophase (telophase I) [28, 29]. On the other hand, the phosphorylation of histone H3 (Ser10) in pig oocytes was maintained until metaphase II, even in anaphase I and telophase I. The activity of histone H3 kinase in pig oocytes showed a change similar to the phosphorylation of histone H3 (Ser10) in the present study. It increased the activity at the diakinesis stage and maintained high activity until metaphase II. This indicates the active histone H3 kinase perhaps phosphorylates histone H3 at the Ser10 residue in condensed chromosomes in pig oocytes during maturation.

PP1/PP2A is implicated in regulating meiosis in starfish and Xenopus oocytes [30, 31]. PP1/PP2A has recently been shown to exist in mouse oocytes, and their relocation and activity change are detected during oocyte maturation [32]. PP1/PP2A has also been suggested to play a role in regulating chromosome condensation in mouse oocytes [33] and mouse mammary cells [34]. Histone H3 phosphorylation at both Ser10 and Ser28 are governed by aurora-B kinase and PP1 in mammalian somatic cells [17, 35]. In somatic cells, PP1 was reported to be phosphorylated and inactivated by Cdc2 kinase [36]. Goto et al. also suggested that Cdc2 kinase may play central roles in mitosis-specific chromosome condensation through the regulation of histone H3 Ser28 phosphorylation by the inactivation of PP1 [35]. The present study showed that activation of Cdc2 kinase was not required for either chromosome condensation or histone H3 phosphorylation in pig oocytes because CL-A and OA induced chromosome condensation and histone H3 phosphorylation without the activation of Cdc2 kinase. This finding is consistent with a recent report showing that Cdc2 kinase is not required for chromosome condensation in pig oocytes treated with OA and butyrolactone I (an inhibitor of Cdks) [9].

It has been suggested that the MAP kinase cascade induces histone H3 phosphorylation (Ser10) [12]. It has been shown that the ERKs-activated Rsk-2 kinase is directly involved in histone H3 phosphorylation in mouse [37] and human fibroblasts [12]. However, the involvement of MAP kinase in chromosome condensation is controversial. In mouse spermatocyte experiments, it was suggested that ERK1 is specifically activated during G2/M transition and is essential for chromosome condensation [38]. In Xenopus oocyte experiments, it was suggested that histone H3 kinase activation, which concerned chromosome condensation during oocyte meiotic maturation, did not require Cdc2 kinase activation but rather depended on activation of the MAPK/p90Rsk pathway [39]. Experiments in pig oocytes suggested that the inhibition of protein phosphatase promptly activated MAP kinase, which in turn induced premature chromosome condensation [40]. In disagreement with these results, a recent study suggested that activation of the ERK1/p90Rsk2 pathway was not necessary for the phosphorylation of H3 in vivo in mouse spermatocytes [41]. In fact, PP1/PP2A inhibitors induce rapid chromosome condensation concomitantly with MAP kinase activation in mouse and pig oocytes [18, 40]. Numerous observations suggest that PP2A plays an important role in the downregulation of the Ras/MAP kinase pathway [42]. PP2A dephosphorylates and inactivates MAP kinase kinase (MEK) and MAP kinase in vitro [43]. Therefore, OA or CL-A perhaps inhibited PP2A and induced MAP kinase activation in our experiments. In a similar time course, CL- A- and OA-treated pig oocytes showed chromosome condensation accompanied by the activation of both MAP kinase and histone H3 kinase. However, even after the activity of MAP kinase was inhibited in the oocytes, the chromosomes condensed, histone H3 (Ser10) was phosphorylated, and histone H3 kinase was activated. These results suggest that MAP kinase is not required for the chromosome condensation.

The disassembly of nuclear lamina is an essential prerequisite for GVBD [44]. Active Cdc2 kinase phosphorylates lamins on specific serine residues and causes nuclear lamina disassembly [45]. It is thought that oocyte GVBD is induced by active Cdc2 kinase. When pig oocytes were treated with PP1/PP2A inhibitors in our experiment, the oocytes underwent GVBD after 6–8 h. Cdc2 kinase was not activated during the culture period, but MAP kinase was activated in the oocytes after 2 h of treatment. Because MAP kinase has a weak activity to phosphorylate nuclear lamins [46], MAP kinase is supposed to substitute for the role of Cdc2 kinase in GVBD in the oocytes. Actually, when oocytes were treated with an MEK inhibitor and CL- A, MAP kinase activation was completely inhibited. In the oocytes, CL-A did not induce GVBD at all, while chromosome condensation occurred in the GV. Under our normal maturational conditions, the Cdc2 kinase of oocytes was still inactive at the diakinesis stage, and these oocytes had a nuclear membrane (Fig. 1B). However, the chromosomes were highly condensed with their histone H3 phosphorylation, and histone H3 kinase was activated (Fig. 2). Together with the above results, these data suggest that the activation of Cdc2 kinase is required for nuclear membrane breakdown but not for chromosome condensation in the pig oocytes.

In the present study, the change in histone H3 kinase activity accurately corresponded with histone H3 phosphorylation (Ser10) in pig oocytes and chromosome condensation. Although we did not identify histone H3 kinase in pig oocytes, accumulating data reveal that aurora-B is required for mitotic histone H3 (Ser10) phosphorylation in Drosophila and mammals [47, 48], and aurora-B is a mitotic histone H3 kinase in humans [49]. Goto et al. reported that aurora-B phosphorylated histone H3 at both Ser10 and Ser28 during mitosis in mammalian somatic cells [35]. In both yeasts and nematodes, the reduction of PP1 activity is responsible for histone H3 phosphorylation interacting with aurora kinase [16]. Sugiyama et al. have suggested that human aurora-B is a mitotic histone H3 kinase that associated protein phosphatases (PP1 or PP2A) as negative regulators of kinase activation [49]. Moreover, chromatin-associated PP1 regulates aurora-B activity and histone H3 phosphorylation [17]. Based on these findings and our present results, we propose that a balance of histone H3 kinase and PP1/PP2A activities regulates the meiotic phosphorylation of histone H3 and that the PP1/PP2A inhibition induces histone H3 phosphorylation, leading to chromosome condensation in pig oocytes (Fig. 6).



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 6. A hypothetical model showing how PP1/PP2A inhibition induces histone H3 phosphorylation leading to chromosome condensation in pig oocytes.


   ACKNOWLEDGMENTS
 
We thank the staff of the Kobe Meat Inspection Office for supplying the pig ovaries.


   FOOTNOTES
 
1 Supported, in part, by a Grant-in-Aid for Creative Scientific Research (13GS0008) and a Grant-in-Aid (14360170) to T.M., and the 21st Century COE Program to H.-T.B. from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Back

2 Correspondence. FAX: +81 78 803 5807; miyano{at}kobe-u.ac.jp Back

Received: 2 December 2003.

First decision: 17 December 2003.

Accepted: 5 February 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Haccard O, Jessus C, Cayla X, Goris J, Merlevede W, Ozon R. In vivo activation of a microtubule-associated protein kinase during meiotic maturation of the Xenopus oocyte. Eur J Biochem 1990 192:633-642[Medline]
  2. Shibuya EK, Boulton TG, Cobb MH, Ruderman JV. Activation of p42 MAP kinase and the release of oocytes from cell cycle arrest. EMBO J 1992 11:3963-3975[Medline]
  3. Sobajima T, Aoki F, Kohmoto K. Activation of mitogen-activated protein kinase during meiotic maturation in mouse oocytes. J Reprod Fertil 1993 97:389-394[Abstract/Free Full Text]
  4. Inoue M, Naito K, Aoki F, Toyoda Y, Sato E. Activation of mitogen- activated protein kinase during meiotic maturation in porcine oocytes. Zygote 1995 3:265-271[Medline]
  5. Dedieu T, Gall L, Crozet N, Sevellec C, Ruffini S. Mitogen-activated protein kinase activity during goat oocyte maturation and the acquisition of meiotic competence. Mol Reprod Dev 1996 45:351-358[CrossRef][Medline]
  6. Fissore RA, He CL, Vande Woude GF. Potential role of mitogen- activated protein kinase during meiosis resumption in bovine oocytes. Biol Reprod 1996 55:1261-1270[Abstract]
  7. Sun QY, Blumenfeld Z, Rubinstein S, Goldman S, Gonen Y, Breitbart H. Mitogen-activated protein kinase in human eggs. Zygote 1999 7:181-185[CrossRef][Medline]
  8. Motlik J, Fulka J. Breakdown of the germinal vesicle in pig oocytes in vivo and in vitro. J Exp Zool 1976 198:155-162[CrossRef][Medline]
  9. Kubelka M, Anger M, Kalous J, Schultz RM, Motlik J. Chromosome condensation in pig oocytes: lack of a requirement for either cdc2 kinase or MAP kinase activity. Mol Reprod Dev 2002 63:110-118[CrossRef][Medline]
  10. Arents G, Burlingame RW, Wang BC, Love WE, Moudrianakis EN. The nucleosomal core histone octamer at 3.1 A resolution: a tripartite protein assembly and a left-handed superhelix. Proc Natl Acad Sci U S A 1991 88:10148-10152[Abstract/Free Full Text]
  11. Tse C, Hansen JC. Hybrid trypsinized nucleosomal arrays: identification of multiple functional roles of the H2A/H2B and H3/H4 N- termini in chromatin fiber compaction. Biochemistry 1997 36:11381-11388[CrossRef][Medline]
  12. Sassone-Corsi P, Mizzen CA, Cheung P, Crosio C, Monaco L, Jacquot S, Hanauer A, Allis CD. Requirement of Rsk-2 for epidermal growth factor-activated phosphorylation of histone H3. Science 1999 285:886-891[Abstract/Free Full Text]
  13. Wei Y, Yu L, Bowen J, Gorovsky MA, Allis CD. Phosphorylation of histone H3 is required for proper chromosome condensation and segregation. Cell 1999 97:99-109[CrossRef][Medline]
  14. Van Hooser A, Goodrich DW, Allis CD, Brinkley BR, Mancini MA. Histone H3 phosphorylation is required for the initiation, but not maintenance, of mammalian chromosome condensation. J Cell Sci 1998 111:3497-3506[Abstract]
  15. Wei Y, Mizzen CA, Cook RG, Gorovsky MA, Allis CD. Phosphorylation of histone H3 at serine 10 is correlated with chromosome condensation during mitosis and meiosis in Tetrahymena. Proc Natl Acad Sci U S A 1998 95:7480-7484[Abstract/Free Full Text]
  16. Hsu JY, Sun ZW, Li X, Reuben M, Tatchell K, Bishop DK, Grushcow JM, Brame CJ, Caldwell JA, Hunt DF, Lin R, Smith MM, Allis CD. Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes. Cell 2000 102:279-291[CrossRef][Medline]
  17. Murnion ME, Adams RR, Callister DM, Allis CD, Earnshaw WC, Swedlow JR. Chromatin-associated protein phosphatase 1 regulates aurora-B and histone H3 phosphorylation. J Biol Chem 2001 276:26656-26665[Abstract/Free Full Text]
  18. Lu Q, Dunn RL, Angeles R, Smith GD. Regulation of spindle formation by active mitogen-activated protein kinase and protein phosphatase 2A during mouse oocyte meiosis. Biol Reprod 2002 66:29-37[Abstract/Free Full Text]
  19. Moor RM, Trounson AO. Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J Reprod Fertil 1977 49:101-109[Abstract/Free Full Text]
  20. Kanayama N, Miyano T, Lee J. Acquisition of meiotic competence in growing pig oocytes correlates with their ability to activate Cdc2 kinase and MAP kinase. Zygote 2002 10:261-270[CrossRef][Medline]
  21. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970 227:680-685[CrossRef][Medline]
  22. Naito K, Toyoda Y. Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes. J Reprod Fertil 1991 93:467-473[Abstract/Free Full Text]
  23. Miyano T, Lee J. Change in cyclin B1 and MAP kinase molecules during maturation and fertilization of pig oocytes. In: Miyamoto H, Manabe N (eds.), Reproductive Biotechnology. Kyoto, Japan: Hokuto Shobo; 2001:9–19
  24. Inoue M, Naito K, Nakayama T, Sato E. Mitogen-activated protein kinase translocates into the germinal vesicle and induces germinal vesicle breakdown in porcine oocytes. Biol Reprod 1998 58:130-136[Abstract/Free Full Text]
  25. Lee J, Miyano T, Moor RM. Localisation of phosphorylated MAP kinase during the transition from meiosis I to meiosis II in pig oocytes. Zygote 2000 8:119-125[CrossRef][Medline]
  26. Strahl BD, Allis CD. The language of covalent histone modifications. Nature 2000 403:41-45[CrossRef][Medline]
  27. Hans F, Dimitrov S. Histone H3 phosphorylation and cell division. Oncogene 2001 20:3021-3027[CrossRef][Medline]
  28. Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, Bazett-Jones DP, Allis CD. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 1997 106:348-360[CrossRef][Medline]
  29. Kaszas E, Cande WZ. Phosphorylation of histone H3 is correlated with changes in the maintenance of sister chromatid cohesion during meiosis in maize, rather than the condensation of the chromatin. J Cell Sci 2000 113:3217-3226[Abstract]
  30. Picard A, Capony JP, Brautigan DL, Doree M. Involvement of protein phosphatases 1 and 2A in the control of M phase-promoting factor activity in starfish. Cell Biol 1989 109:3347-3354
  31. Rime H, Huchon D, Jessus C, Goris J, Merlevede W, Ozon R. Characterization of MPF activation by okadaic acid in Xenopus oocyte. Cell Differ Dev 1990 29:47-58[CrossRef][Medline]
  32. Smith GD, Sadhu A, Mathies S, Wolf DP. Characterization of protein phosphatases in mouse oocytes. Dev Biol 1998 204:537-549[CrossRef][Medline]
  33. Gavin AC, Tsukitani Y, Schorderet-Slatkine S. Induction of M-phase entry of prophase-blocked mouse oocytes through microinjection of okadaic acid, a specific phosphatase inhibitor. Exp Cell Res 1991 192:75-81[CrossRef][Medline]
  34. Guo XW, Th'ng JPH, Swank RA, Anderson HJ, Tudan C, Bradbury EM, Roberge M. Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation. EMBO J 1995 14:976-985[Medline]
  35. Goto H, Yasui Y, Nigg EA, Inagaki M. Aurora-B phosphorylates histone H3 at serine28 with regard to the mitotic chromosome condensation. Genes Cells 2002 7:11-17[Abstract]
  36. Kwon YG, Lee SY, Choi Y, Greengard P, Nairn AC. Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by Cdc2 kinase. Proc Natl Acad Sci U S A 1997 94:2168-2173[Abstract/Free Full Text]
  37. Chadee DN, Hendzel MJ, Tylipski CP, Allis CD, Bazett-Jones DP, Wright JA, Davie JR. Increased Ser-10 phosphorylation of histone H3 in mitogen-stimulated and oncogene-transformed mouse fibroblasts. J Biol Chem 1999 274:24914-24920[Abstract/Free Full Text]
  38. Sette C, Barchi M, Bianchini A, Conti M, Rossi P, Geremia R. Activation of the mitogen-activated protein kinase ERK1 during meiotic progression of mouse pachytene spermatocytes. J Biol Chem 1999 274:33571-33579[Abstract/Free Full Text]
  39. Schmitt A, Gutierrez GJ, Lénárt P, Ellenberg J, Nebreda AR. Histone H3 phosphorylation during Xenopus oocyte maturation: regulation by the MAP kinase/p90Rsk pathway and uncoupling from DNA condensation. FEBS Lett 2002 518:23-28[CrossRef][Medline]
  40. Sun QY, Wu GM, Lai L, Bonk A, Cabot R, Park KW, Day BN, Prather RS, Schatten H. Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro. Biol Reprod 2002 66:580-588[Abstract/Free Full Text]
  41. Di Agostino S, Rossi P, Geremia R, Sette C. The MAPK pathway triggers activation of Nek2 during chromosome condensation in mouse spermatocytes. Development 2002 129:1715-1727[Abstract/Free Full Text]
  42. Millward TA, Zolnierowicz S, Hemmings BA. Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem Sci 1999 24:186-191[CrossRef][Medline]
  43. Sontag E, Fedorov S, Kamibayashi C, Robbins D, Cobb M, Mumby M. The interaction of SV40 small tumor antigen with protein phosphatase 2A stimulates the map kinase pathway and induces cell proliferation. Cell 1993 75:887-897[CrossRef][Medline]
  44. Gerace L, Burke B. Functional organization of the nuclear envelope. Annu Rev Cell Biol 1988 4:335-374[CrossRef][Medline]
  45. Peter M, Nakagawa J, Doree M, Labbe JC, Nigg EA. In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase. Cell 1990 61:591-602[CrossRef][Medline]
  46. Peter M, Sanghera JS, Pelech SL, Nigg EA. Mitogen-activated protein kinases phosphorylate nuclear lamins and display sequence specificity overlapping that of mitotic protein kinase p34cdc2. Eur J Biochem 1992 205:287-294[Medline]
  47. Giet R, Glover DM. Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis. J Cell Biol 2001 152:669-682[Abstract/Free Full Text]
  48. Crosio C, Fimia GM, Loury R, Kimura M, Okano Y, Zhou H, Sen S, Allis CD, Sassone-Corsi P. Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian aurora kinases. Mol Cell Biol 2002 22:874-885[Abstract/Free Full Text]
  49. Sugiyama K, Sugiura K, Hara T, Sugimoto K, Shima H, Honda K, Furukawa K, Yamashita S, Urano T. Aurora-B associated protein phosphatases as negative regulators of kinase activation. Oncogene 2002 21:3103-3111[CrossRef][Medline]



This article has been cited by other articles:


Home page
 DevelopmentHome page
H.-T. Bui, S. Wakayama, S. Kishigami, J.-H. Kim, N. Van Thuan, and T. Wakayama
The cytoplasm of mouse germinal vesicle stage oocytes can enhance somatic cell nuclear reprogramming
Development, December 1, 2008; 135(23): 3935 - 3945.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
J. E. Swain, J. Ding, J. Wu, and G. D. Smith
Regulation of spindle and chromatin dynamics during early and late stages of oocyte maturation by aurora kinases
Mol. Hum. Reprod., May 1, 2008; 14(5): 291 - 299.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
J. E. Swain, J. Ding, D. L. Brautigan, E. Villa-Moruzzi, and G. D. Smith
Proper Chromatin Condensation and Maintenance of Histone H3 Phosphorylation During Mouse Oocyte Meiosis Requires Protein Phosphatase Activity
Biol Reprod, April 1, 2007; 76(4): 628 - 638.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
H.-T. Bui, N. Van Thuan, S. Kishigami, S. Wakayama, T. Hikichi, H. Ohta, E. Mizutani, E. Yamaoka, T. Wakayama, and T. Miyano
Regulation of chromatin and chromosome morphology by histone H3 modifications in pig oocytes
Reproduction, February 1, 2007; 133(2): 371 - 382.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
H.-T. Bui, N. Van Thuan, T. Wakayama, and T. Miyano
Chromatin remodeling in somatic cells injected into mature pig oocytes.
Reproduction, June 1, 2006; 131(6): 1037 - 1049.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
L. Jelinkova and M. Kubelka
Neither Aurora B Activity nor Histone H3 Phosphorylation Is Essential for Chromosome Condensation During Meiotic Maturation of Porcine Oocytes
Biol Reprod, May 1, 2006; 74(5): 905 - 912.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
K. J. McManus and M. J. Hendzel
ATM-dependent DNA Damage-independent Mitotic Phosphorylation of H2AX in Normally Growing Mammalian Cells
Mol. Biol. Cell, October 1, 2005; 16(10): 5013 - 5025.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
70/6/1843    most recent
biolreprod.103.026070v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bui, H.-T.
Right arrow Articles by Miyano, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bui, H.-T.
Right arrow Articles by Miyano, T.
Agricola
Right arrow Articles by Bui, H.-T.
Right arrow Articles by Miyano, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS