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BOR - Papers in Press, published online ahead of print February 27, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.067397
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BIOLOGY OF REPRODUCTION 78, 1073–1080 (2008)
DOI: 10.1095/biolreprod.107.067397
© 2008 by the Society for the Study of Reproduction, Inc.

Gamete Biology; 

Nuclear Histone Deacetylases Are Not Required for Global Histone Deacetylation During Meiotic Maturation in Porcine Oocytes1

Tsutomu Endo , Kiyoshi Kano , and Kunihiko Naito  2

Laboratory of Applied Genetics, Graduate School of Agriculture and Life Science, University of Tokyo, Tokyo 113-8657, Japan

ABSTRACT

Histone acetylation plays an important role in the regulation of chromatin structure and gene function. In mammalian oocytes, histones H3 and H4 are highly acetylated during the germinal vesicle (GV) stage, and global histone deacetylation takes place via a histone deacetylase (HDAC)-dependent mechanism after GV breakdown (GVBD). The presence of HDACs in the GVs of mammalian oocytes in spite of the high acetylation states of nuclear histones indicates that the HDACs in the nucleus are inactive but become activated after GVBD. However, the fluctuation pattern, the localization of HDAC activity during meiotic maturation and, moreover, the responsibility of nuclear HDACs for global histone deacetylation are still unknown. Here, we demonstrated using porcine oocytes that total HDAC activity was maintained throughout meiotic maturation, and high HDAC activity was observed in both the nucleus and the cytoplasm at the GV stage. The experiments with valproic acid (VPA), a specific class I HDAC inhibitor, revealed that the HDACs in GVs were class I, and those in the cytoplasm were other than class I. Interestingly, VPA had no effect on global histone deacetylation after GVBD, indicating that nuclear HDACs were not required for global histone deacetylation. To confirm this possibility, we removed the nuclei from immature oocytes, injected somatic cell nuclei into the enucleated oocytes, and showed that injected somatic cell nuclei were dramatically deacetylated after nuclear envelope breakdown. These results revealed that nuclear contents, including class I HDACs, are not required for the global histone deacetylation during meiosis, and that cytoplasmic HDACs other than class I are responsible for this process.

gamete biology, HDAC, kinases, meiosis, oocyte development

INTRODUCTION

Nuclear core histones, histone H3 (H3) and histone H4 (H4), undergo various modifications, such as phosphorylation, acetylation, and methylation, on their N-terminal tails [1]. These posttranslational modifications play crucial roles in the regulation of chromatin structure [1, 2] and transcription [3, 4]. In mammalian somatic cells, almost histone acetylation patterns of H3 and H4 are maintained throughout mitosis [5], and the maintained histone acetylations have been thought to work as cell memories that generate cell type-specific gene expression patterns [6, 7]. On the other hand, highly acetylated H3 and H4 in mammalian germinal vesicle (GV)-stage oocytes were globally deacetylated after GV breakdown (GVBD) [8, 9]. The inhibition of meiotic histone deacetylation induces aneuploidy in fertilized oocytes and results in early embryonic death, indicating the importance of this oocyte-specific deacetylation for the construction of meiotic chromosomes [10].

Not just meiotic chromosomes in oocytes, but all acetylations on H3 and H4 in somatic cell nuclei were also removed after transfer into mature oocytes [8, 11–13], suggesting that global histone deacetylation is attributable to the unique deacetylation activity in maturing mammalian oocytes rather than the chromatin structure of meiotic chromosomes. The molecules responsible for this global histone deacetylation have not yet been identified. The treatment of mouse and porcine oocytes with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, prevented the global histone deacetylation observed after GVBD, showing the involvement of HDAC activity in the process of meiosis-specific deacetylation [8, 9]. Thus far, more than 17 isoforms have been identified in mammalian HDACs, and they are generally classified into three groups: class I (HDACs 1, 2, 3, and 8), class II (HDACs 4, 5, 6, 7, 9, and 10), and class III (SIRTs 1, 2, 3, 4, 5, 6, and 7) [14, 15]. The mRNAs of Hdacs 1, 2, 3, 4, 6, 8, 9, and Sirt1 are present during meiotic maturation in mouse oocytes [13], and those of HDACs 1, 2, 3, and 7 are present in bovine oocytes [16]. At the protein level, the presence of HDACs 1, 2, 4, and 6 in mouse oocytes [13, 17, 18]; HDACs 1, 2, and 3 in bovine oocytes [19]; and HDAC1 in porcine oocytes [20] has been reported. Immunocytochemical analysis revealed the localization of HDACs 1 and 2 in the nuclei of mouse oocytes [8, 13]. Recently, we and others reported the presence of HDAC1 in the nuclei of porcine oocytes [20, 21]. In Xenopus oocytes, HDACm, a homolog of HDAC1, accumulates primarily in the nucleus but rarely in the cytoplasm [22].

Considering the findings of previous reports demonstrating the presence of class I HDACs in the nucleus, together with the fact that both H3 and H4 are highly acetylated in the nucleus, it is imaginable that these class I HDACs in the nucleus are inactive during the GV stage and that their activation is induced by cytoplasmic factors after GVBD. In a previous report, we demonstrated using porcine oocytes that global histone deacetylation could be induced, even in GV-stage oocytes, by the artificial destruction of the nuclear membrane [20]. This finding suggests that global histone deacetylation does not depend on meiotic resumption, but instead is correlated with the mixing of nuclear contents and cytoplasmic factors (e.g., an activator of HDACs or cofactors for recruiting HDACs to the chromatin) after the breakdown of the nuclear membrane. Alternatively, it is also possible that the HDACs in the nucleus are not involved in global histone deacetylation, and other HDACs present in the cytoplasm are instead responsible for this meiosis-specific deacetylation. However, the fluctuation pattern and the localization of HDAC activities during meiotic maturation in mammalian oocytes, particularly the responsibility of class I HDACs present in the nucleus for global histone deacetylation, have not yet been investigated.

In the present study, we attempted to address these questions using porcine oocytes maturing in vitro. We assayed total HDAC activity during meiotic maturation, and then separately assayed HDAC activity in the nucleus and the cytoplasm. The involvement of class I HDACs was analyzed by treatment of samples with valproic acid (VPA), a class I-specific HDAC inhibitor. Finally, to confirm the involvement of the nucleic materials in global histone deacetylation, we removed the nuclei from immature porcine oocytes and injected somatic cell nuclei into the enucleated oocytes, and then the acetylation states of somatic cell nuclei were examined by immunostaining.

MATERIALS AND METHODS

Collection and Maturation of Porcine Oocytes

Porcine oocytes were obtained as described previously [9]. Briefly, porcine cumulus-oocyte complexes (COCs) were aspirated from the follicles (2–5 mm in diameter) of ovaries obtained from animals at a slaughterhouse. Groups of 10~20 COCs with intact, unexpanded cumulus cells were cultured in 0.1-ml drops of culture medium for up to 48 h at 37°C, 100% humidity, and 5% CO2 in air. The culture medium consisted of modified Krebs-Ringer bicarbonate solution (TYH) [23], 1.0 IU/ml eCG (Sankyo, Tokyo, Japan), 3.2 mg/ml BSA (Sigma), and 20% porcine follicular fluid collected as described previously [24]. For the treatment with inhibitors, 0.5 µM trichostatin A (TSA; Wako Pure Chemical, Osaka, Japan) or 1 mM VPA (Alexis Biochemicals, Lausen, Switzerland) was added to the culture medium.

Enucleation of Porcine Oocytes

Prior to micromanipulation, the noncultured oocytes were denuded with 150 IU/ml hyaluronidase (type IV; Sigma) and gentle pipetting. Enucleation was performed as described in previous reports [25, 26]. Briefly, denuded oocytes were centrifuged at 20 000 x g for 5 min at room temperature to localize cytoplasmic lipid droplets and visualize the nuclei. The centrifuged oocytes were incubated for 5 min in culture medium supplemented with 15 µg/ml cytochalasin B (Sigma). Each oocyte was held with a holding pipette, and the zona immediately above the nucleus was cut with an injection pipette without damaging the oolemma. The zona-cut oocyte was aspirated with the holding pipette such that the nucleus, together with a small amount of cytoplasm, was pushed out of the zona through the slit. Karyoplasts were separated from the encleated oocytes by pipetting. The time at the end of manipulation was defined as 0 h of culture.

HDAC Activity Assay

Histone deacetylase activity was examined using the CycLex HDAC Deacetylase Fluorometric Assay Kit (CY-1150; CycLex Co., Nagano, Japan) according to the manufacturer's instructions. Briefly, 100 denuded oocytes were collected in 2 µl assay buffer (20 mM Tris-HCl, pH 8.0; 125 mM NaCl, 1% glycerol) and, if necessary, the sample was stored at –80°C until use. Then, 1 µl lysis buffer (5% NP-40 in assay buffer) was added to 2 µl of each sample, and the samples were kept on ice for a period of 30 min. The lysates (3 µl) were then transferred into each well of 96-well microplates (436110; Nunc, Roskilde, Denmark) and were incubated with a mixture of fluorescence-labeled acetylated substrates according to the kit manufacturer's instructions. As a positive control of HDAC activity, crude HDACs purified from MCF4 cells were used. Thus, the reaction was initiated at 37°C, and fluorescence intensity was measured in 30-min intervals up to 120 min using a microplate fluorometer (Fluoroskan Ascent FL; Thermo Fisher Scientific Inc., Waltham, MA) with excitation at 350–380 nm and emission at 440–460 nm. For the treatment with inhibitors, 0.5 µM TSA or 0.4, 1.0, or 4.0 mM VPA was added to the lysate before the reaction.

Cumulus Cell Culture

Cumulus cells were isolated from the COCs with gentle pipetting in TYH medium, and the cells were collected into 1.5-ml tubes. The cell suspensions were centrifuged at 800 x g for 5 min and resuspended with Dulbecco modified Eagle medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum. The cells were cultured in the DMEM for 2–5 days at 37°C, 100% humidity, and 5% CO2 in air. At the end of the culture period, the cells were subjected to immunocytochemistry.

Immunocytochemistry

Fixation, permeabilization, and blocking of denuded oocytes were performed as described previously [9]. The oocytes were then treated with anti-K8 acetylated H4 antibody (06–760; Upstate Biotechnology, Lake Placid, NY) or anti-K14 acetylated H3 antibody (06–911; Upstate Biotechnology) overnight. After being washed, the oocytes were incubated in a mixture of fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin G (IgG; 1:100 dilution with PBS; DAKO, Glostrup, Denmark) for 30 min. The oocytes were counterstained with Hoechst 33342 to visualize the chromosomes. Examination of the oocytes was carried out using a confocal laser scanning microscope (LSM510-V2.01 Axioplan MOT; Carl Zeiss, Oberkochen, Germany).

The cultured cumulus cells were fixed and permeabilized with methanol for 20 min at –20°C. After being washed with PBS, the cells were blocked for 10 min in PBS containing 10% (v/v) swine serum (DAKO). Thereafter, the cells were treated with one of the antibodies described above for 2 h. After three washes with PBS, the cells were incubated in a mixture of FITC-conjugated anti-rabbit IgG (1:100 dilution with PBS; DAKO) for 1 h. The cells were counterstained with Hoechst 33342 to visualize the chromosomes, and they then were examined under a fluorescence microscope (BZ-8000; Keyence, Osaka, Japan).

Injection of Cumulus Cell Nuclei into Porcine Oocytes

Injection of cumulus cell nuclei into 30-h cultured intact oocytes or 18-h cultured enucleated oocytes was performed using a Piezo-micromanipulating system (Prime Tech Ltd.). The oocytes were incubated in culture medium supplemented with 15 µg/ml cytochalasin B and 10 µg/ml Hoechst 33342 for 10 min at 37°C. The cumulus cells, which had been isolated from the COCs by a denuding process, were gently aspirated in and out of the injection pipette (inner diameter, 7–9 µm) until their nuclei were largely devoid of visible cytoplasmic material. Then, one cumulus cell nucleus was injected into each oocyte. After injection, the oocytes were cultured for 3 h until nuclear envelope breakdown (NEBD) was observed. Some injected oocytes were examined for their nuclear states after they had been mounted on glass slides, fixed with acetic acid-ethanol (1:3), and stained with 0.75% (w/v) acetoorcein solution.

Maturation-Promoting Factor and Mitogen-Activated Protein Kinase Activity Assay

Ten denuded oocytes were lysed in 2.5 µl assay buffer [27] and stored at –80°C until use. Maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity was evaluated in terms of histone H1 kinase and myelin basic protein (MBP) kinase activity, respectively, as described in previous reports [26, 28]. The lysates (2.5 µl) were added to 2.5 µl of 2.5 µM cAMP-dependent protein kinase inhibitor (Sigma), 5 µl of a 2-mg/ml concentration of histone H1 (Sigma), 2.5 µl of a 10-mg/ml concentration of MBP, and 5 µl of 0.1 mM [{gamma}-32P]ATP (0.4 mCi/ml; Amersham Pharmacia Biotech); the samples were incubated at 37°C for 30 min. After the reaction, 5 µl of 5x Laemmli buffer was added to each lysate, which was then denatured at 100°C for 5 min and subjected to SDS-PAGE. The bands of phosphorylated histone H1 and MBP were visualized after autoradiography.

Statistical Analysis

Student t-test was used to evaluate the results. A probability of P < 0.05 was considered to be statistically significant.

RESULTS

GV-Stage Oocytes Exhibit High HDAC Activity in the Nucleus and Cytoplasm

To determine whether HDACs are activated after GVBD in porcine oocytes, we performed an HDAC activity assay during the in vitro maturation of porcine oocytes. In our in vitro maturation system, porcine oocytes undergo GVBD at approximately 24 h of culture, and they reach the M1 and M2 stages at 30 h and 48 h of culture, respectively. Thus, we collected porcine oocytes at the indicated times and subjected them to HDAC activity assays. As shown in Figure 1A, high levels of HDAC activity were detected in GV-stage oocytes at 0 h of culture. The level of total HDAC activity at 0 h of culture was similar to that at 24 h of culture, when almost all of the oocytes had undergone GVBD. High levels of HDAC activity were maintained until 48 h of culture, when most oocytes reached M2. Thus, we found that total HDAC activity in porcine oocytes does not change significantly throughout maturation, indicating that HDAC activity in GV-stage oocytes is sufficient to induce global histone deacetylation observed after GVBD.


Figure 01
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  		FIG. 1. Histone deacetylase activity in porcine oocytes. A) Porcine oocytes were cultured for the indicated periods. The HDAC activity of each 100 oocytes was assayed as fluorescent intensity, and activities were expressed as relative values compared with those of crude HDACs extracted from about 104 MCF4 cells. The activity of MCF4 cells (104 cells) treated with 0.5 µM trichostatin A (TSA+) is also shown as a negative control. The values indicate the mean ± SD of three independent experiments. There was no significant difference among culture periods. B) Histone deacetylase activity in the nucleus and cytoplasm of GV-stage oocytes. Noncultured oocytes were enucleated as described in Materials and Methods, and the HDAC activity of 100 karyoplasts (nucleus) or 100 cytoplasts (cytoplasm) was assayed. The values indicate the mean ± SD of three independent experiments. There were no significant differences in levels of HDAC activity in the nucleus and the cytoplasm.

We next determined whether the HDAC activity observed in GV-stage oocytes is present in the nucleus. Using a micromanipulator, 0-h cultured oocytes were enucleated, and the enucleated oocytes and isolated karyoplasts were used for HDAC activity assays. As shown in Figure 1B, we found that the nucleus exhibited high levels of HDAC activity, i.e., approximately half of the total HDAC activity observed in 0-h cultured oocytes. Previously, we reported that porcine HDAC1 is located primarily in the nucleus [20]. Therefore, it is likely that HDAC activity in the nucleus represents active HDAC1. Interestingly, high HDAC activity was also detected in the cytoplasm, and this activity did not differ significantly from that in the nucleus of 0-h cultured oocytes. This result suggests that cytoplasmic HDACs might be involved in global histone deacetylation.

High Levels of Class I HDACs Are Present in the Nucleus of GV-Stage Oocytes

To assess class I HDAC activity in porcine oocytes, we next carried out an HDAC activity assay in the presence of VPA, a class I HDAC-specific inhibitor. Treatment with VPA significantly inhibits the total HDAC activity of 0-h cultured oocytes in a dose-dependent manner (Fig. 2A). Thus, active class I HDACs appear to be predominant in porcine oocytes. The HDAC activity of oocytes treated with 1.0 or 4.0 mM VPA did not significantly differ from that of oocytes treated with 0.5 µM TSA, a nonspecific HDAC inhibitor, which completely inhibited global histone deacetylation of lysine 14 of H3 (H3K14) and lysine 8 of H4 (H4K8) in our previous report [9]. We therefore consider 1.0 mM VPA sufficient for the inhibition of class I HDAC activity in porcine oocytes, and 1.0 mM VPA was used for the following studies.


Figure 02
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  		FIG. 2. Effects of VPA on HDAC activity in porcine oocytes. A) The HDAC activity of each 100 noncultured oocytes was assayed in the presence of various concentrations of VPA; TSA (0.5 µM) was used for comparison. The values indicate the mean ± SD of three independent experiments. Values designated by different letters indicate significant differences (P < 0.05). B) The HDAC activity of 100 karyoplasts or 100 cytoplasts of noncultured oocytes was assayed in the presence of VPA (1.0 mM). The values shown indicate the mean ± SD of three independent experiments. Values associated with asterisks are significantly different from those of control groups (P < 0.05).

To assess class I HDAC activity in the nucleus and in the cytoplasm, isolated karyoplasts and enucleated oocytes were treated with 1.0 mM VPA and were used for the HDAC activity assay. As shown in Figure 2B, treatment with 1.0 mM VPA almost completely inhibited HDAC activity in the nucleus. In contrast, 1.0 mM VPA has no significant effect on HDAC activity in the cytoplasm (P < 0.05). These results suggest that class I HDACs, including HDAC1, are primarily present in the nucleus but are not located in the cytoplasm.

Inhibition of Class I HDACs Does Not Prevent Global Histone Deacetylation

To determine whether class I HDACs are involved in global histone acetylation, we next added VPA to the culture medium, and the states of histone acetylation in M1 and M2 oocytes were examined by immunostaining. In intact oocytes, high levels of histone acetylation were observed at the GV stage in instances of both H4K8 and H3K14, and levels were remarkably decreased at M1 and M2 stages (Fig. 3). In contrast, 0.5 µM TSA maintained unaltered levels of histone acetylation from the GV stage to M2. These results confirmed the findings of our previous report [9]. However, we found that 1.0 mM VPA had no effect on deacetylation in M1 or M2 oocytes, even though the degree of HDAC activity inhibited by 1.0 mM VPA was almost the same as that inhibited by 0.5 µM TSA in 0-h cultured oocytes (Fig. 2A). These results suggest that class I HDACs, which are primarily present in the nucleus, are not required for global histone deacetylation observed after GVBD, and HDACs other than class I HDACs are required for global histone deacetylation.


Figure 03
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  		FIG. 3. Effects of VPA on global histone deacetylation in porcine oocytes. Porcine oocytes were cultured for 0, 30, and 48 h without inhibitors (–) or with 1.0 mM VPA or 0.5 µM TSA, and they were immunostained with antibodies specific for lysine 8-acetylated histone H4 (acH4K8) or lysine 14-acetylated histone H3 (acH3K14). GV, Noncultured oocytes at the GV stage; M1, 30-h cultured oocytes at first metaphase; M2, 48-h cultured oocytes at second metaphase. Arrowheads indicate the second metaphase chromosome. Each sample was counterstained with Hoechst 33342 to visualize the DNA (lower panels). Experiments were repeated at least three times, and more than 30 oocytes were examined in each experimental group. At least 90% of the examined oocytes in each experimental group showed the same signal strength. Bar = 20 µm.

Global Histone Deacetylation Occurs in Cumulus Cell Nuclei Injected into M1 Oocytes

To confirm that the histone acetylation pattern in somatic cells is maintained during mitosis, we performed an immunostaining analysis of cumulus cells during mitosis. In the cumulus cells, acetylation levels of both H4K8 and H3K14 at metaphase were almost identical to those at interphase (Fig. 4). These results demonstrate that histone acetylation patterns in cumulus cell nuclei are maintained during mitosis, which is consistent with the findings of a previous study of NIH 3T3 cells [8].


Figure 04
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  		FIG. 4. Global histone deacetylation in porcine cumulus cells. Isolated porcine cumulus cells were cultured and immunostained (top panels) with antibodies specific for lysine 8-acetylated histone H4 (acH4K8) or lysine 14-acetylated histone H3 (acH3K14), and were counterstained with Hoechst 33342 to visualize the DNA (middle panels). Bright-field images of the some cells are also shown (bottom panels). Arrowheads and arrows indicate the cells at M phase and interphase, respectively. Bar = 10 µm.

It was previously reported that histone acetylation levels decreased markedly in somatic nuclei that had been transferred into M2 oocytes [8, 12]. To determine whether or not the cytoplasm of maturing oocytes can also cause deacetylation in somatic cell nuclei, we injected cumulus cell nuclei at interphase into M1 oocytes, and the states of histone acetylation in the cumulus cell nuclei 3 h after injection were examined by immunostaining. In the cumulus cell nuclei immediately (0 h) after injection, histone acetylation of both lysines was higher than that of the M1 chromosomes (Fig. 5). When these injected oocytes were cultured for 3 h, the injected nuclei at interphase underwent chromosome condensation, and histone acetylation in both lysines decreased remarkably in the injected nuclei. The acetylation levels in the injected nuclei were comparable to those of the M1 chromosomes from oocytes. The decrease in acetylation levels in the cumulus cell nuclei was due to deacetylation by HDAC, as TSA treatment inhibited the deacetylation of the cumulus cell nuclei and M1 chromosomes. This result suggests that maturing oocytes can also induce histone deacetylation in somatic cell nuclei. In addition, we propose that the injection of cumulus cell nuclei into maturing oocytes is a useful method for the assessment of the histone deacetylation potential of maturing oocytes.


Figure 05
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  		FIG. 5. Global histone deacetylation in porcine cumulus cell nuclei injected into M1 oocytes. The cumulus cell nuclei were injected into 30-h cultured M1 oocytes. After injection, the oocytes were cultured for 3 h in medium with or without 0.5 µM TSA, and were then subjected to immunostaining with antibodies specific for lysine 8-acetylated histone H4 (acH4K8) or lysine 14-acetylated histone H3 (acH3K14). Each sample was counterstained with Hoechst 33342 to visualize the DNA (lower panels). Arrowheads indicate the injected cumulus cell nuclei. Experiments were repeated at least two times, and more than 20 oocytes were examined in each experimental group. At least 90% of the examined oocytes in each experimental group showed the same signal strength. Bar = 20 µm.

GV Contents, Including Class I HDACs, Are Not Required for Global Histone Deacetylation

As described above, although class I HDAC activity is primarily present in the nucleus (Fig. 2B), global histone deacetylation occurred in M1 and M2 oocytes treated with VPA (Fig. 3). These results suggest that nuclear HDAC itself is not required for the global histone deacetylation observed after GVBD. To examine this possibility, we removed the nuclei from noncultured oocytes and injected the cumulus cell nuclei at interphase into the enucleated oocytes, and histone acetylation status of the injected nuclei was then examined by immunostaining after NEBD. The experimental design is shown in Figure 6A.


Figure 06
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  		FIG. 6. Effect of enucleation on meiosis and the global histone deacetylation of porcine oocytes. A) Scheme of cumulus cell nuclei injection into enucleated oocytes. Noncultured GV-stage oocytes were enucleated and cultured for 18 h until MPF activity had reached the maximum level, as shown in B, in order to induce NEBD in injected somatic cell nuclei. The enucleated oocytes were injected with porcine cumulus cell nuclei at 18 h of culture. Three hours after injection, NEBD and chromosome condensation were observed in the cumulus cell nuclei, as shown in D. B) Intact and enucleated oocytes were cultured for up to 30 h and were then subjected to MPF and MAPK assays. The phosphorylated bands of histone H1 (upper panel) and MBP (lower panel) are shown as MPF and MAPK activity, respectively. C) MPF and MAPK activities of enucleated oocytes immediately after injection with porcine cumulus cells nuclei (18 h) and 3 h after injection (21 h). D) Typical status of the injected cumulus cell nuclei observed 0 h and 3 h after injection (upper panel and lower panel, respectively). Bar = 10 µm. E) The enucleated and 18-h cultured oocytes were injected with cumulus cell nuclei and then were cultured further for an additional 3 h in medium with 1.0 mM VPA or 0.5 µM TSA, or without any inhibitor. The oocytes were immunostained with antibodies specific for lysine 8-acetylated histone H4 (acH4K8) or lysine 14-acetylated histone H3 (acH3K14). Each sample was counterstained with Hoechst 33342 to visualize the DNA (lower panels). Experiments were repeated at least two times, and more than 20 oocytes were examined in each experimental group. At least 90% of examined oocytes in each experimental group showed the same signal strength. Bar =10 µm.

As it has been previously reported that MPF activation is more rapid in enucleated oocytes than in intact oocytes [29], we confirmed the level of MPF activity during in vitro culture of enucleated oocytes (Fig. 6B). Low levels of MPF activity were observed at the initiation of the culture period, and MPF activity in enucleated oocytes just after enucleation was identical to that of control oocytes. Then, MPF activity in enucleated oocytes rose to a maximum level at 18 to 24 h, and decreased until 27 h of culture, although the MPF activity in the control oocytes rose to a maximum level at 30 h of culture. In contrast, MAPK activity in the enucleated oocytes remained high at 30 h of culture, indicating that the decrease in MPF activity at 30 h could not be attributed to degenerative changes in the enucleated oocytes. These results were consistent with those of a previous report [29]. Therefore, cumulus cell nuclei were injected into enucleated oocytes at 18 h of culture to induce NEBD, and then the injected oocytes were cultured for an additional 3 h. We confirmed that the MPF activity in the injected oocytes remained high at 3 h after culture (Fig. 6C), indicating that no deterioration of activity had taken place due to the manipulation of the injected oocytes. Moreover, the cumulus cell nuclei underwent chromosome condensation following NEBD (Fig. 6D), suggesting that cytoplasmic factors in the enucleated oocytes were accessible to the cumulus cell chromosomes.

Finally, the histone acetylation status of the injected cumulus cell nuclei was examined by immunostaining (Fig. 6E). Immediately (0 h) after injection, the histone acetylation levels in the nuclei were high in both H4K8 and H3K14. Strikingly, the histone acetylation levels of both lysines dramatically decreased 3 h after injection, and the degree of histone acetylation was very similar to that in those injected into M1 oocytes (Fig. 5). This decrease in acetylation levels in the nuclei was due to global histone deacetylation by HDACs, as TSA maintained histone acetylation at levels unchanged from those observed just after injection. In contrast, VPA had no effect on histone deacetylation in the injected cumulus cell nuclei, as described above. Taken together, these results revealed that global histone deacetylation does not require nuclear contents, including class I HDACs, and instead requires cytoplasmic factors, including HDACs other than class I HDACs.

DISCUSSION

In the present study, we examined whether class I HDACs present in the nucleus are involved in the global histone deacetylation observed after GVBD in porcine oocytes. Previously, we and others reported that HDAC1, a class I HDAC, is present in the nuclei of porcine oocytes, where H3 and H4 are highly acetylated [20, 21]. Thus, it is conceivable that HDACs in the nuclei are inactive and then activated by cytoplasmic factors after GVBD. Therefore, we first examined the fluctuations in total HDAC activity during porcine oocyte maturation. Interestingly, we observed high levels of HDAC activity at the GV stage, and these levels of activity were maintained throughout the maturation period. This is the first report to demonstrate that total HDAC activity remains unaltered during the meiotic maturation of mammalian oocytes. In addition, we detected high levels of HDAC activity in isolated nuclei; this activity was comparable to that in the cytoplasm. These results strongly suggest that HDACs in the nucleus are already active during the GV stage, without activation by cytoplasmic factors after GVBD. Our findings refute the notion that the cytoplasmic factors required for global histone deacetylation are activators of inactive HDACs in the nucleus.

We investigated whether or not the high levels of HDAC activity detected in the nucleus could be attributed to class I HDACs, such as HDAC1 and HDAC2, as reported by immunocytochemical analysis [13, 20]. A class I-specific HDAC inhibitor, VPA [30], which also induces HDAC2 degradation [31], almost completely inhibited the HDAC activity in isolated nuclei. This result indicates that the active HDACs present in the nuclei primarily belong to class I. In contrast, VPA was not found to inhibit HDAC activity in the cytoplasm, suggesting that HDACs other than those of class I are present in the cytoplasm. It has been reported in Xenopus oocytes that HDAC activity at the GV stage depends on the amount of nucleic HDACm, a homolog of HDAC1, and that HDAC activity in the cytoplasm is much lower than that in the nucleus as a result of the translocation of active HDACm into the nucleus soon after synthesis [22]. The results of the present study suggest for the first time that mammalian GV-stage oocytes exhibit high cytoplasmic HDAC activity that is comparable to that of nucleic HDACs, due to the accumulation of HDACs other than those of class I, unlike in Xenopus oocytes.

In order to investigate the requirement of class I HDACs, which are primarily found in the nucleus, for global histone deacetylation, maturing porcine oocytes were treated with VPA, and their acetylation status was examined. The results showed that VPA was unable to prevent histone deacetylation in both M1 and M2 oocytes. In mouse oocytes, the involvement of class I HDACs in global histone deacetylation has been suggested by immunocytochemical experiments in which the HDAC1 signal was observed on meiotic chromosomes after GVBD [8]. We have also obtained the same result in porcine oocytes [20]. On the other hand, no localization of the HDAC1 signal on chromosomes after GVBD has been reported recently in studies of mouse and porcine oocytes [13, 21]. This discrepancy is thought to be the result of differences in antibody specificity and reactive conditions used for immunostaining. The present results using VPA suggest more directly than those obtained by immunostaining that class I HDACs, including HDAC1, are not involved in global histone deacetylation.

As HDAC activity in the nucleus is primarily due to class I HDACs, the present results indicate that HDACs in the nucleus are not required for global histone deacetylation. To confirm this possibility, we removed nuclei from immature porcine oocytes just after collection, and their global histone deacetylation activity was examined by the injection of cumulus cell nuclei, which were highly acetylated, even in the M phase. This experiment showed a dramatic decrease of histone acetylation levels in the injected cumulus cell nuclei after NEBD, thus confirming that all HDAC activity in the nucleus is dispensable for global histone deacetylation during porcine oocyte maturation. In contrast, TSA completely prevented histone deacetylation in injected cumulus cell nuclei, suggesting that cytoplasmic HDACs other than those belonging to class I are involved in global histone deacetylation.

In this study, although high class I HDAC activity in the nucleus was detected at GV stage, nuclear HDACs itself were not required for global histone deacetylation. It has been reported in mouse GV-stage oocytes that inhibition of HDAC activity by TSA induced the striking change in chromatin structure, which underwent a dramatic decondensation and acquired a diffuse conformation over the entire GV [32]. Therefore, class I HDACs in the nucleus might be involved in the maintenance of chromatin structure during GV stage. More detailed studies are required to elucidate the role of class I HDACs in mammalian oocytes.

The present results indicated that not only class I HDACs present in the nucleus, but also all other materials in the nucleus are dispensable for global histone deacetylation. It has been reported in somatic cells that HDACs alone cannot exert direct effects on chromatin, but instead required several cofactors and formed HDAC complexes in order to associate with the chromatin [33–35]. Retinoblastoma-associated protein p48 (RbAp48/46), a cofactor of the HDAC complex, promotes the binding of HDAC1 and HDAC2 to the chromatin [36, 37]. The presence of various cofactors, including RbAp48/46, has also been reported, primarily in the nuclei of somatic cells and Xenopus oocytes [22, 38, 39]. Thus, it is noteworthy that all cofactors involved in global histone deacetylation might be present in the cytoplasm of GV-stage oocytes.

In summary, as the nuclear envelope sequesters chromatin from cytoplasmic HDACs and cofactors, H3 and H4 are highly acetylated during the GV stage of mammalian oocytes. Germinal vesicle breakdown enables the association of these cytoplasmic factors to the chromatin and induces global histone deacetylation. In this study, we also showed that cytoplasmic HDACs other than class I are involved in global histone deacetylation. In Xenopus oocytes, although high HDAC activity was reported at GV stage due to the presence of HDACm in the nucleus [22], global histone deacetylation during meiotic maturation has not been detected, unlike in mammalian oocytes. Therefore, our findings generate the notion that mammalian oocytes acquired the global histone deacetyalation in an evolutionary process by accumulating cytoplasmic HDACs other than class I. In somatic cells, global histone deacetylation is not observed even in M phase, suggesting the lack of HDACs and/or cofactors required for the global histone deacetylation. This indicates that the global histone deacetylation is mediated by the meiotic oocyte-specific HDACs and/or cofactors that are not present in the somatic cells. Further experiments are necessary to identify those HDACs and/or the cofactors involved in the oocyte-specific global histone deacetylation during meiosis.

FOOTNOTES

1Supported by Grant-in-Aid for Scientific Research 19380155 to K.N., and by a Research Fellowship for Young Scientists grant 19-2573 from the Japan Society for the Promotion of Science to T.E. Back

Correspondence: 2Kunihiko Naito, Department of Animal Resource Sciences, Graduate School of Agricultural Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. FAX: 81 3 5841 8191; e-mail: aknaito{at}mail.ecc.u-tokyo.ac.jp

Received: 20 December 2007.

First decision: 26 January 2008.

Accepted: 21 February 2008.

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