[Cell Cycle 5:7, 766-774, 1 April 2006]; ©2006 Landes Bioscience
Histone Deacetylation is Required for Orderly Meiosis
Report
ABSTRACT
Histone acetylation is associated with a diversity of chromatin-related processes in
mitosis. However, its roles in mammalian oocyte meiosis are largely unknown. In the
present study, we first investigated in detail the acetylation changes during porcine oocyte
maturation using a panel of antibodies specific for the critical acetylated forms of histone
H3 and H4, and showed meiosis stage-dependent and lysine residue-specific patterns of
histone acetylation. By using trichostatin A (TSA), a general inhibitor of histone deacetylases
(HDACs), we further determined that selective inhibition of histone deacetylation (thereby
maintaining hyperacetylation) delayed the onset of germinal vesicle breakdown and
produced a high frequency of lagging chromosomes or chromatin bridges at anaphase
and telophase I (AT-I), suggesting that histone deacetylation is required for orderly meiotic
resumption and accurate chromosome segregation in porcine oocytes. In addition, we
examined the localization and expression of HDAC1 by performing immunofluorescence
and immunoblotting analysis. The results showed that subcellular translocation, expression
level and phosphorylated modification of HDAC1 were temporally regulated and likely
to coparticipate in the establishment of histone acetylation profiles in oocyte meiosis.
School; Chinese Academy of Sciences; Beijing, China
3Department of Veterinary Pathobiology; University of Missouri-Columbia;
Columbia, Missouri USA
ABBREVIATIONS
Previously published as a Cell Cycle E-publication:
http://www.landesbioscience.com/journals/cc/abstract.php?id=2627
INTRODUCTION
.D
IEN
ES
ACKNOWLEDGEMENTS
BIO
histone acetylation, deacetylation, porcine oocyte,
meiosis, HDAC1, TSA
20
06
LA
ND
We thank Chun-Ming Wang, Zhi-Sheng Zhong
and Li-Jun Huo, Shi-Wen Li, for their technical
assistance; Ling Gu, Jing-He Liu, Jun-Cheng
Huang for helpful discussions and critical reading
of the manuscript. This work was supported by the
National Natural Science Foundation of China
(No. 30430530, 30225010, 30570944) and the
Special Funds for Major State Basic Research
Project of China.
©
NOTE
Supplemental material can be found at:
http://www.landesbioscience.com/journals/cc/
supplement/wangCC5-7-sup.pdf.
766
In eukaryotes, the fundamental building component of chromatin is the nucleosome,
which is composed of 147 base pairs of DNA and an octamer containing two copies of
each of the core histone proteins H2A, H2B, H3 and H4. The histone amino termini
exposed on the nucleosome surface can be modified post-translationally by reversible
acetylation,1 which is determined by the combined activities of two enzyme families, the
Histone Acetyltransferases (HATs) and Deacetylase (HDACs).2 HATs and HDACs are
classified into many subfamilies that are mostly conserved from yeast to human.3 It has
been widely accepted that histone acetylation is associated with diverse chromatin-related
processes, such as nucleosome assembly,4 DNA replication, repair5,6 and transcription.7,8
All core histones are acetylated in vivo; modifications of histone H3 and H4 are,
however, much more extensively characterized than those of H2A and H2B.9,10 The critical
sites for acetylation include at least four highly conserved lysines (K) in histone H4 (K5,
K8, K12, K16) and two in histone H3 (K9, K14).11 Accumulated evidence suggests that
acetylation of individual histones, and even of particular lysine residues on the same histone,
may exert specific functional effects by directly initiating defined changes in chromatin
conformation or by regulating protein-histone interactions.4,12,13 Recent results have
shown that global levels of histone acetylation are dramatically changed during progression
through mitosis in various cell lines.14-16 However, entirely different opinions have been
presented by several research groups about the effects of hyperacetylated histones on
chromosome conformation, and cell cycle progression during mitosis.14,16,17,18 Therefore,
the roles of histone acetylation in the cell cycle still need further analysis.
Like in mitosis, the dramatic changes in chromatin structure and function also take
place in meiosis. On the other hand, there are many events that specifically occur during
meiotic progression, including successive M phases without an intervening DNA replication
phase, pairing of homologous chromosomes, and asymmetric cell division, which raises
questions about particular histone acetylation patterns that may be different during oocyte
SC
KEY WORDS
HDAC, histone deacetylase; HAT, histone acetyltransferase; TSA, trichostatin A;
GVBD, germinal vesicle breakdown
CE
Original manuscript submitted: 11/12/05
Revised manuscript submitted: 02/10/06
Manuscript accepted: 02/24/06
ON
*Correspondence to: Qing-Yuan Sun; State Key Laboratory of Reproductive
Biology; Institute of Zoology; Chinese Academy of Sciences; #25 Beisihuanxi Rd;
Haidian, Beijing 100080, China; Tel.: 8610.8262.7593; Fax: 8610.6256.5689;
Email: [email protected]
OT
D
1State Key Laboratory of Reproductive Biology; Institute of Zoology and 2Graduate
IST
RIB
UT
E
.
Qiang Wang1,2
Shen Yin1,2
Jun-Shu Ai1,2
Cheng-Guang Liang1,2
Yi Hou1
Da-Yuan Chen1
Heide Schatten3
Qing-Yuan Sun1,*
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2006; Vol. 5 Issue 7
Histone Acetylation in Meiotic Oocyte
maturation and may play meiosis-specific roles. To date, dynamic
histone modifications in oocyte meiosis and embryonic development
have only been fragmentally reported,19,20,21 and their results showed
that histone acetylation is the dynamic and reversible epigenetic
marker during development of the oocyte and preimplantation
embryo; meanwhile, other researchers revealed that the acetylation
state of H4/K12 and global histone H4 in mouse oocyte fluctuated
temporally during meiotic maturation.22,23 Detailed changes of
histone acetylation in meiosis have not been thoroughly examined,
and to our knowledge, its roles during oocyte maturation are nearly
unknown.
Here, we first investigated the histone acetylation patterns of
various lysine residues during porcine oocyte maturation, presenting
meiosis stage-dependent and lysine residue-specific profiles. Next,
we explored whether preventing histone deacetylation with trichostatin A (TSA), a specific inhibitor of HDACs,24 thereby maintaining
hyperacetylated histones, would disrupt the orderly progression of
meiotic maturation. Our results suggest that histone deacetylation is
required for orderly meiotic resumption and accurate chromosome
segregation in porcine oocytes. In addition, we examined the localization and expression of HDAC1 in various specific oocyte stages to
gain insights into the mechanism underlying histone deacetylation.
MATERIALS AND METHODS
All chemicals used in this study were purchased from Sigma Chemical
Company (St. Louis, MO) except for those specifically mentioned.
Antibodies. Rabbit polyclonal anti-acetyl lysine 5 in histone H4
(AcH4/K5) antibody, anti-acetyl lysine 8 in histone H4 (AcH4/K8) antibody,
anti-acetyl lysine 12 in histone H4 (AcH4/K12) antibody, anti-acetyl lysine
14 in histone H3 (AcH3/K14) antibody and anti-HDAC1 antibody were
purchased from Upstate Biotechnology (Lake Placid, NY); goat polyclonal
anti-acetyl lysine 16 in histone H4 (AcH4/K16) antibody and anti-actin
antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA);
rabbit polyclonal anti-acetyl lysine 9 in histone H3 (AcH3/K9) antibody
was purchased from Cell Signaling Technology (Beverly, MA); human
ANA-Centromere Auto-antibody (CREST) was purchased from Cortex
Biochem (Sanleandro, CA); anti-α-tubulin antibody was obtained from
Sigma (St Louis, MO).
Oocyte collection and culture. Ovaries were collected from prepubertal
gilts at a local slaughterhouse and transported to the laboratory within
1–2 hour. Oocytes were aspirated from antral follicles (3–8 mm in diameter)
with an 18-gauge needle fixed to a 20 ml disposable syringe. After washing
three times with maturation medium (see below), oocytes with a compact
cumulus and evenly granulated ooplasm were selected for maturation
culture. The medium used for maturation culture was improved TCM-199
(Gibco, Grand Island, NY) supplemented with 75 µg/ml potassium
penicillin G, 50 µg/ml streptomycin sulphate, 0.57 mM cysteine, 0.5 µg/ml
FSH, 0.5 µg/ml LH, and 10 ng/ml EGF (epidermal growth factor). A group
of 30 oocytes was cultured in a 100 µl drop of maturation medium for up
to 44 hours at 38.8˚C in an atmosphere of 5% CO2 and saturated humidity.
Trichostatin A (TSA) treatment. TSA solution prepared in dimethyl
sulfoxide (DMSO) was diluted in maturation medium to yield a final
concentration of 10 ng/ml, 100 ng/ml and 1000 ng/ml, respectively.
To explore the role of histone deacetylation in meiotic resumption,
fully-grown oocytes were cultured for 24 hours or 30 hours in maturation
medium containing different doses of TSA, respectively.
To study the effects of histone deacetylation on meiotic progression
through prometaphase I to metaphase II, fully grown oocytes were cultured
for 24 hours and subsequently treated with 100 ng/ml TSA for 12 hours
(24 hours + TSA 12 hours). Correspondingly, 0.1% DMSO was included
as a control.
Finally, oocytes were freed of cumulus cells by treatment with 300 IU/ml
hyaluronidase and repeated pipetting. Cumulus-free oocytes were used in
the following experiments.
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Figure 1. Nuclear maturation of porcine oocytes during in vitro culture.
Experiments were repeated three times for each time point examined. (GV,
germinal vesicle; Pro-MI, prometaphase I; MI, metaphase I; AT-I, anaphase I
and telophase I; MII, metaphase II).
Immunofluorescence microscopy. After removing the zona pellucida in
acidic Tyrodes medium (pH 2.5), oocytes were fixed with 4% paraformaldehyde in PBS (PH 7.4) for at least 30 minutes at room temperature. Cells
were permeabilized with 1% Triton X-100 overnight at 37˚C, followed by
blocking in 1% BSA-supplemented PBS (blocking solution) for 1 hour and
incubation overnight at 4˚C with primary antibodies diluted 1:300. After
three washes in PBS containing 0.1% Tween 20 and 0.01% Triton X-100
(washing solution) for 5 minutes each, the oocytes were labeled with secondary
FITC (fluorescein isothiocyanate) conjugated antibody diluted 1:100 for 1
hour at room temperature. Nuclear status of oocytes was evaluated by staining
with 10 µg/ml PI (propidium iodide) for 5 minutes. Following extensive
washing, samples were mounted between a coverslip and glass slide supported
by four columns of mixture of petroleum jelly and paraffin (9:1). Cells were
observed under a Confocal Laser-Scanning Microscope (Zeiss LSM 510
META, Germany) as soon as possible. Nonspecific staining was determined
by substituting primary antibodies with normal rabbit IgG. Each experiment
was repeated three times, and at least 40 oocytes were examined each time.
The same instrument settings were used for each replicate.
Western blot analysis. For detection of AcH4/K5, proteins from 300
oocytes at each time point were collected in SDS sample buffer and heated
to 100˚C for 5 minutes. After cooling on ice and centrifugation at 10,000
g for 3 minutes, samples were frozen at -20˚C until use. The total proteins
were loaded on a 15% SDS-PAGE gel, resolved and electroblotted onto a
nitrocellulose membrane. Membranes were blocked in Tris-buffered saline
containing 0.1% Tween 20 and 5% low fat dry milk for 1 hour and then
incubated with anti-AcH4/K5 antibody overnight at 4˚C. After multiple
washes in Tris-buffered saline containing 0.1% Tween 20 and incubation
with anti-rabbit horseradish peroxidase linked antibody, the labeled proteins
were detected using enhanced chemiluminescence.
For detection of HDAC1, proteins from 200 oocytes were extracted and
loaded on a 12% SDS-PAGE gel. All other steps were the same as those
described for the AcH4/K5 detection. Equal protein loading was confirmed
by detection of β-actin.
Evaluation of nuclear status. Orcein staining was employed according to
the procedures described by Sun et al.25 with minor modifications. Denuded
oocytes were mounted on slides, fixed in acetic acid: ethanol (1:3 v/v) for at
least 48 hours, stained with 1% orcein, and examined with a phase-contrast
microscope.
Statistical analysis. The data were pooled from at least three replicates
and analyzed by Chi-square test. Differences at p < 0.05 were considered to
be significant.
RESULTS
Nuclear maturation of porcine oocytes during in vitro culture. As shown
in Figure 1 at 0 hour of culture most oocytes were at the germinal vesicle
(GV) stage (94.3%); by 24 hours of culture, 90.9% of oocytes underwent
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767
Histone Acetylation in Meiotic Oocyte
Figure 2. The acetylation patterns of histone H4 lysine 5 (H4/
K5) during porcine oocyte meiosis. Oocytes at distinct stages
of meiosis are immunolabeled with antibody against AcH4/
K5: GV, noncultured oocytes at germinal vesicle stage (A, A',
A''), the nucleolus is indicated by “N”; L-GV, oocytes at late
germinal vesicle stage (B, B', B''); E-GVBD, oocytes at early
germinal vesicle breakdown stage (C, C', C''); Pro-MI,
oocytes at first prometaphase (D1, D1', D1''; D2, D2',D2'');
MI, oocytes at first metaphase (E, E', E''); AT-I, oocytes at first
anaphase and telophase (F, F', F''); MII, oocytes at second
metaphase (G, G', G''). Arrows indicate the first polar bodies.
Each sample is counterstained with PI to visualize DNA. Inserts
show magnified image of AcH4/K5 and DNA. Bars, 30 µm.
germinal vesicle breakdown (GVBD) and proceeded to the
prometaphase I (Pro-MI) stage. The proportion of oocytes at
the metaphase I (MI) stage increased to 44.2% when the
oocytes were examined at 30 hours, and 50.6% of oocytes
were at anaphase and telophase I (AT-I) stage by 36 hours of
culture. The proportion of oocytes at the MII stage was
75.1% after 44 hours of culture. These data obtained by
orcein staining are a crucial prerequisite for the following
experimental design.
Meiosis stage-dependent and lysine residue-specific
changes in histone acetylation during porcine oocyte meiosis.
Fully-grown porcine oocytes are arrested at diplotene stage of
the first meiotic prophase. The period between the diplotene
stage to the onset of germinal vesicle breakdown (GVBD) can
be divided into four well-defined stages, based on chromatin
changes, nucleolus and nuclear membrane disappearance.26,27
In GV-I, nuclear membrane and nucleolus are clearly visible
and chromatin forms a ring or horseshoe around the nucleolus;
In GV-IV, the last stage, the nuclear membrane is less distinct
and the nucleolus disappears completely, and chromatin is
seen as an irregular network. These two stages are termed GV
and late GV stage (L-GV), respectively, in this paper. Just
after GVBD, the nuclear membrane entirely vanished and
network-like chromatin is visible, which we call the early
GVBD stage (E-GVBD). With further condensation of
chromatin, the oocytes proceed to prometaphase I (Pro-MI),
and subsequently progress to anaphase and telophase I (AT-I),
followed by oocytes entering meiosis II and becoming arrested
at metaphase II (MII).28,29
To investigate the detailed dynamic changes in histone
acetylation, porcine oocytes at the different developmental
stage were immunolabeled with a panel of antibodies specific
for the acetylated forms of histone H3 and H4 including
AcH4/K5, AcH4/K8, AcH4/K12, AcH4/K16, AcH3/K9 and
AcH3/K14. The results showed that, in GV stage, intense fluorescence signals for all acetylated lysines colocalized with
DNA around the nucleolus (Figs. 2A', 3A', 4A'; S1A', 2A',
3A'). Importantly, the acetylation levels of six lysine residues
were uniformly decreased in the L-GV stage, showing negative
fluorescence on the condensed chromatin and weak acetylation
signals on the dispersed GV chromatin (Figs. 2B', 3B', 4B';
S1B', 2B', 3B'). With the resumption of meiosis, the complete
deacetylation of histones took place in the E-GVBD stage
oocytes (Figs. 2C', 3C', 4C'; S1C', 2C', 3C'). These results
indicate that histone deacetylation may be required for the
orderly progression through GV to L-GV or even E-GVBD
stage. As the oocytes progressed into Pro-MI, discerning
acetylation patterns have been observed among various
lysines of histone H3 and H4. In Pro-MI stage oocytes,
AcH4/K5 staining became evident in some oocytes (Fig. 2D1')
but was absent in other oocytes (Fig. 2D2'), while the signal
of AcH4/K16 was barely detectable (Fig. 4D'). Based on
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2006; Vol. 5 Issue 7
Histone Acetylation in Meiotic Oocyte
Table 1
The acetylation profiles of various lysine
residues in histone H3 and H4 during porcine
oocyte meiotic maturation
Histone/Lysine
GV
L-GV
E-GVBD
Pro-MI
MI
ATI
MII
AcH3/K9
+
±
-
+
+
+
+
AcH3/K14
+
±
-
+
+
+
+
AcH4/K5
+
±
-
+-
-
+
-
AcH4/K8
+
±
-
+
+
+
+
AcH4/K12
+
±
-
+
+
+
+
AcH4/K16
+
±
-
-
-
?
-
Intense, decreasing and no fluorescence signals are denoted with + , ±, and - , respectively. ?, not
completely determined.
these results, we concluded that temporary acetylation of H4K5 may appear
between E-GVBD and MI stage. Interestingly, H4/K5 and K16 were
completely deacetylated in MI oocytes (Figs. 2E'; 4E'), and after transient
reacetylation in AT-I (Figs. 2F'; 4F'), they were dramatically deacetylated again
at MII chromosomes (Figs. 2G'; 4G'). The intense signals of AcH4/K5 and
K16 were still present in the first polar body (Figs. 2G', 4G'; arrows), which
strongly indicates that the histone acetylation of oocytes is regulated by a
meiosis stage-dependent mechanism, and the first polar bodies, which have
escaped from the normal cell cycle, are beyond the control of this mechanism.
Here, we show an interesting phenomenon that exclusively appeared on
H4K16. We find about 64% (n = 75) of the chromosomes segregating
toward opposite poles in AT-I oocytes have a distinct acetylation level on
H4/K16 (Fig. 4F'); the chromosomes with slightly acetylated H4/K16
(double arrows) perhaps would be sequestered in the ooplasm. However, we
are puzzled about the lost or weak signals of H4K16 in the remaining 36%
of AT-I oocytes. Is this difference the result of the specificity of the antibody
or the degree of exposure to the AcH4/K16 site? We are in the process of
clarifying this contradictory result. In contrast to the dynamic acetylation of
H4/K5 and K16, H4/K8, 12 and H3/K9, 14 maintain a constant acetylation
state during cell progression through Pro-MI to MII (Figs. 3; S1, 2, 3). The
acetylation states of various lysine residues in porcine oocytes are summarized
in Table 1. These results clearly show that histone acetylation is a meiosis stagedependent and lysine residue-specific process during meiotic maturation.
Prevention of histone deacetylation delays the onset of GVBD in
porcine oocytes. Since the acetylation states of all lysine residues examined
in this study were uniformly and dramatically reduced from GV stage to
L-GV and E-GVBD stage, we wished to determine whether meiotic resumption of porcine oocytes was disrupted by preventing histone deacetylation
during this course. Thus, GV oocytes were cultured for 24 or 30 hours in
maturation medium containing different doses of TSA, and the GVBD rates
were evaluated by orcein staining as illustrated in Figure 5A.
At 24 hours, the GVBD rate was significantly decreased in the 100 ng/ml
TSA group (30.4%, n = 148) and 1000 ng/ml TSA group (29.1%, n = 127)
compared to the control group (85.6%, n = 118) and 10 ng/ml TSA group
(86.2%, n = 109). These data indirectly tell us that a high proportion of
oocytes were arrested at the GV stage by TSA treatment. To gain better
insights into this process, we used confocal microscopy to examine the
arrested oocytes, and, unexpectedly, found that most of them (75.7%, n = 74)
had remained at the GV-III stage (data not shown) characterized by condensed
chromatin clumps or strands around the nucleolus,26,27 indicating that the
sharp deacetylation may occur through GV-III to GV-IV stage. With longer
(30 hours) incubation in TSA, the GVBD rates of the 100 ng/ml group
(60.7%, n = 122) and 1000 ng/ml TSA group (61.0%, n = 118) were significantly increased compared to their counterparts at 24 hours of treatment.
However, they were still statistically lower than in controls (92.8%, n = 113)
and the 10 ng/ml group (90.8%, n = 109). This result indicates that prevention of histone deacetylation does not block porcine oocytes at the GV stage.
Moreover, similar to the cumulus-oocyte complex, denuded oocytes were
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Figure 3. The acetylation patterns of histone H3 lysine 9 (H3/K9) during
porcine oocyte meiosis. Oocytes at distinct stages of meiosis are immunolabeled with antibody against AcH3/K9: GV, noncultured oocytes at germinal
vesicle stage (A, A', A''), the nucleolus is indicated by “N”; L-GV, oocytes at
late germinal vesicle stage (B, B', B''); E-GVBD, oocytes at early germinal
vesicle breakdown stage (C, C', C''); Pro-MI, oocytes at first prometaphase
(D, D', D''); MI, oocytes at first metaphase (E, E', E''); AT-I, oocytes at first
anaphase and telophase (F, F', F''); MII, oocytes at second metaphase (G,
G', G''). Arrows indicate the first polar bodies. Each sample is counterstained
with PI to visualize DNA. Inserts show magnified image of AcH3/K9 and
DNA. Bars, 30 µm.
also arrested at the GV stage by TSA treatment (data not shown), suggesting
that TSA affects meiotic resumption by direct action on the oocytes but not
through the pathway mediated by cumulus cells.
Simultaneously, to determine whether the histone deacetylation was
efficiently prevented in the above experiments, analysis of acetylation level
was performed by detecting AcH4/K5 which is a reliable marker of hyperacetylated histone H4.14 The resulting Western blots showed that (Fig. 5B)
100 and 1000 ng/ml TSA apparently prevented the deacetylation of H4/K5
through GV to GVBD. Taken together, these results and other results
reported previously,30,31 lead to the conclusion that 100 ng/ml TSA (≈330 nM)
effectively inhibited all HDACs activity under our experimental conditions.
Accordingly, this concentration was also applied in the following studies.
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Histone Acetylation in Meiotic Oocyte
Figure 4. The acetylation patterns of histone H4 lysine 16 (H4/K16) during
porcine oocyte meiosis. Oocytes at distinct stages of meiosis are immunolabeled with antibody against AcH4/K16: GV, noncultured oocytes at germinal
vesicle stage (A, A', A''), the nucleolus is indicated by “N”; L-GV, oocytes
at late germinal vesicle stage (B, B', B''); E-GVBD, oocytes at early germinal
vesicle breakdown stage (C, C', C''); Pro-MI, oocytes at first prometaphase
(D, D', D''); MI, oocytes at first metaphase (E, E', E''); AT-I, oocytes at first
anaphase and telophase (F, F’, F’’); MII, oocytes at second metaphase (G,
G', G''). Arrows indicate the first polar bodies; double arrows indicate
weak signal of AcH4/K16 in segregating chromosomes. Each sample is
counterstained with PI to visualize DNA. Inserts show magnified image of
AcH4/K16 and DNA. Bars, 30 µm.
Histone hyperacetylation induced by TSA treatment produces chromosome segregation defects in AT-I oocytes. Previous findings on the
complete deacetylation of H4/K5 and K16 at MI (Figs. 2E'; 4E') prompted
us to ask what would happen in meiosis if the MI–stage oocytes were highly
770
acetylated and allowed to enter AT-I? To address this issue, we applied TSA
to inhibit histone deacetylation and thereby induced hyperacetylation after
24 hours of in vitro maturation, a time point where a high proportion of
oocytes are at Pro-MI stage (Fig. 1), to avoid the known effects of TSA on
GVBD (Fig. 5A).
As expected, deacetylation was efficiently inhibited in MI oocytes treated
with TSA (Fig. 6A; +TSA) when compared to the control group (Fig. 6A;
-TSA). Next, these oocytes maintaining histone hyperacetylation were
continuously exposed to TSA until 36 hours, at which time a large number
of them is expected to progress into AT-I. By performing confocal scanning
and statistical analysis, we did not observe any marked effects of hyperacetylation on the conformation of MI chromosomes or progression through
Pro-MI to AT-I (data not shown). However, we surprisingly observed a high
frequency of aberrant AT-I segregation in TSA-treated oocytes by immunostaining of spindles and chromosomes (Fig. 6B). To rule out any possible
artifacts caused by the PI dye, a more precise assay was conducted by
immunostaining of oocytes with CREST, an antibody of constitutive kinetochore proteins32,33 detecting the exact position of chromosomes. Similar
results were obtained (Fig. 6C) and two basic phenotypes were determined
that display defective separation including chromatin bridges (Fig. 6Bb, Ce;
arrows) and lagging chromosomes (Fig. 6Bc, Cf; arrows). The typical segregation in AT-I oocytes was also detected (Fig. 6Ba; Cd), showing chromosomes
or kinetochores moving evenly away from the equator toward opposite
poles. Meanwhile, our quantitative analysis (Fig. 6D) showed that 38.3% of
the total examined AT-I oocytes (n = 94) in the TSA treatment group exhibited abnormal segregation, which is significantly higher than in the control
group (5.3%, n = 75).
Taken together, these results indicate that histone deacetylation is essential
for the accurate chromosome segregation in porcine oocytes.
Subcellular localization and expression of HDAC1 during porcine
oocyte maturation. It has been widely documented that HDAC activity is
regulated in multiple ways including protein-protein interaction, post-translational modification, abundance, as well as by subcellular distribution.34,35
However, little is known about the pathway by which HDACs regulate the
acetylation status of histones during mammalian oocyte maturation. Hence,
we additionally examined the localization and expression of HDAC1, loss of
which resulted in a substantial reduction of cellular HDAC activity and
hyperacetylation of histone H3 and H4.36,37 The oocytes were immunolabeled
with the anti-HDAC1 antibody and costained with PI to confirm the stage
of meiotic maturation. In GV-stage oocytes, HDAC1 was detected in the
GV but most of it accumulated in chromatin-depleted domains (Fig. 7Aa';
double arrows). Importantly, just after GVBD, HDAC1 was concentrated
to the periphery of condensed chromatin (Fig. 7Ab'). However, no apparent
HDAC1 signal was detected in the metaphase chromosomes (Fig. 7Ac').
These results indicate that the localization of HDAC1 is temporally regulated,
and may be involved in the establishment of histone acetylation profiles
during oocyte meiosis.
Recently published reports have identified that, as a phosphoprotein,
HDAC1’s enzymatic activity and interaction with other corepressors are
closely related with transformation between its phosphorylated and nonphosphorylated isoforms which could be separated by SDS-PAGE.35,38-41
In this study, the expression of HDAC1 in oocytes cultured for 0, 24, 36,
44 hours was investigated by performing Western blotting (Fig. 7B). Two
distinct bands were clearly distinguished in three samples (lane 1–3), so it is
reasonable to assume that they respectively correspond to the phosphorylated
and nonphosphorylated HDAC1. Strikingly, the expression of HDAC1
underwent gradual changes during porcine oocyte maturation. In the oocytes
collected at 0 and 36 hours, two isforms of HDAC1 were simultaneously
detected, and especially, the nonphosphorylated (nonactivated) HDAC1
was predominant in GV-staged oocytes, which provides a possible explanation
for the high levels of histone acetylation in GV. On the other hand, the
phosphorylated isoform was preponderant in the oocytes collected at 24 and
44 hours which might account for the reduced acetylation in GVBD or MII
oocytes. Very low expression of HDAC1 (Fig. 7B, lane 4) detected by
Western blotting may contribute to its negative signal in metaphase oocytes
examined by immunofluorescene (Fig. 7Ac'). Taken together, our results
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2006; Vol. 5 Issue 7
Histone Acetylation in Meiotic Oocyte
Figure 5. Prevention of histone deacetylation delays the onset of
GVBD. (A) Fully grown oocytes were cultured for 24 hours (grey
bars) or 30 hours (white bars) with 0.1% DMSO (control), 10 ng/ml,
100 ng/ml or 1000 ng/ml TSA and the GVBD rate was evaluated
by orcein staining. The graph shows the mean + SME of the results
obtained in three independent experiments. Different superscripts
indicate statistical differences (p < 0.05); (B) AcH4/K5 immunoblotting
of cellular extracts from oocytes cultured for 0 hour, 24 hours with
0.1% DMSO (control), 10 ng/ml, 100 ng/ml, or 1000 ng/ml TSA.
A control blot shows equivalent levels of protein loading between
the various samples.
provide strong indications that subcellular localization, expression
level and phosphorylated modification of HDAC1 are likely to
coparticipate in the regulation of histone acetylation during
porcine oocyte maturation.
DISCUSSION
Genetic and biochemical assays have established that
(de)acetylation of lysine residues in the N-termini of histones
plays fundamental roles in diverse chromatin-associated
processes.4 Confocal scanning in the present study revealed
that distinct lysines in histones exhibit a uniform deacetylation
tendency with gradual chromatin condensation through
GV to L-GV (Fig. 2A', B'; 3A', B’; 4A', B';
S1, 2 and 3). Such chromatin condensation
could be disrupted at GV-III stage by TSA
treatment, indicating that histone deacetylation is required for meiotic resumption
in porcine oocytes perhaps by affecting the
state of chromatin condensation through
GV to L-GV stage. The mechanisms by
which histone (de)acetylation affect chromatin conformation are thought to
include two major pathways. First, histone
acetylation may modulate interactions
between histone tails and the DNA or alter
the internucleosomal association.42 Second,
the histone tail acetylation also provides
specific binding surfaces for some chromosomal proteins that subsequently induce a
more open and transcriptionally competent
conformation.43,31 Based on our observations, we cannot easily accept the conclusion
that high levels of histone acetylation always
persists in GV stage and deacetylation initiates from GVBD in oocyte meiosis.19,21 As
the oocytes progress into the MI stage and
through the MII stage, the signals of
AcH4/K5 and K16 dramatically disappear
in metaphase chromosomes (Figs. 2E';
4E'). Interestingly, we also observed a
reestablishment of highly acetylated Figure 6. Histone hyperacetylation induced by TSA treatment produces chromosome segregation defects
chromatin at AT-I stage when the chromatin in AT-I oocytes. (A) Immunofluorescence detection of AcH4/K5 in MI oocytes treated with 0.1% DMSO
(-TSA) or 100 ng/ml TSA (+TSA). Bars,10 µm.; (B) Immunostaining of spindles with α-tubulin (green) and
is still highly condensed but decondensation chromosomes with PI (red) in normal (a) and defective (b,c) AT-I oocytes; (C) Immunostaining of kinetohas begun (Figs. 2F'; 4F'). Similar observa- chores with CREST (green) and chromosomes with PI (red) in normal (d) and defective (e,f) AT-I oocytes.
tions have been reported for mitosis.14 Bars, 5 µm.; (D) Quantitative analysis of the induction of chromosome segregation defects after treatment
However, previous studies19 and our recent in the absence or presence of 100 ng/ml TSA. The graph shows the mean + SME of the results obtained
results (unpublished data) showed that the in three independent experiments. Different superscripts indicate statistical differences (P < 0.05).
acetylation of H4/K5 was not detectable at
AT-I stage in mouse oocyte meiosis, which
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general rule that histone acetylation is correlated with gene activity and deacetylation
events create repressive chromatin. Therefore,
we hypothesize that the acetylation of particular lysines in metaphase chromosomes may
be involved in specific events in oocyte
meiosis, which awaits further experimentation.
Indeed, studies utilizing yeast cells revealed
that acetylation of H4/K8 and K12 is not
correlated with transcriptional activity;45
and AcH4/K8 mediates the recruitment of
SWI/SNF complex, which is a chromatinremodeling ATPase;46 in addition, pericentric
heterochromatin in flies47 and silent loci in
yeast48 are marked by acetylation of H4/K12.
It has been extensively reported that
incubation of mammalian cell lines with
inhibitors of HDACs induces an arrest in
G1 or G2/M phase.31,49,50 On the other hand,
simultaneous substitution of four conserved
lysine residues in histone H4 by glutamine
activates the G2/M checkpoint and introduction of a single acetylatable lysine in the
mutant histone tail suppresses the G2/M
cell-cycle defects in yeast.51,52 All these data
suggest that histone (de)acetylation is linked
to cell cycle control. In support of this, we
found that preventing histone deacetylation
evidently arrested porcine oocytes at the G2
phase in the first meiotic cycle (Fig. 5A).
The above results raise the possibility that
improper chromosome condensation can
affect kinetochore-microtubule interaction,
and induce checkpoint activation and cell
cycle arrest in the G2 phase. In contrast,
effects of TSA treatment on the GVBD were
not observed in mouse oocytes,19 and we
cannot discuss the differing results because
the acetylation profiles in detailed stages of
Figure 7. Localization and expression of HDAC1 during porcine oocyte maturation. (A) The GV (a), mouse oocyte maturation were not reported
GVBD (b) and M (c) oocytes were immunostained with anti-HDAC1 antibody and costained with PI. and also the concentration of TSA employed
Inserts show magnified image of HDAC1 and DNA localization. Bars, 30 µm.; (B) Oocytes cultured for in this paper was unclear. In addition, no
0, 24, 36 or 44 hours were analyzed by immunoblotting. Bars on the right indicate the positions of apparent effects of histone hyperacetylation
two possible isoforms, and the faster migrating band corresponds to nonphosphorylated HDAC1. A
induced by TSA on the metaphase chromocontrol blot shows equivalent levels of protein loading between the various samples.
some configuration and progression through
Pro-MI to MII were observed during porcine
is greatly different from our conclusion mentioned above. These data oocyte meiosis, which was consistent with Kruhlak’s study in mitosis14
revealed that the patterns of histone acetylation were not identical but contradicts subsequently published results.16,18,53 All these data
among various cell types and the acetylation level was not always indicate that the effects of histone hyperacetylation on cell cycle
well correlated with chromatin conformation.
progression may correlate with cell types. More advanced methods
Kim et al.19 reported that the acetylation of all lysine residues and technologies are perhaps needed to clarify this controversy.
except H4/K8 was not observed in metaphase chromosomes during
Although the effects of hyperacetylation on chromosome segregamouse oocyte meiosis. Interestingly, we obtained totally different tion in mitosis have been previously reported,17,18,54 their influence
results, since we have detected the signals of acetyl-H4/K8, K12 and on the segregation of homologous chromosomes in oocyte meiosis
H3/K9, 14 at MI and MII stages in porcine oocyte (Figs. 3E', G'; was up to now largely unknown. Here, we revealed that inhibition
S1, 2, 3). Our results strongly indicate that the acetylation is not of histone deacetylation and thereby maintaining hyperacetylation
constantly lost in metaphase chromosomes. On the other hand, the can lead to a high frequency of lagging chromosomes and chromatin
transcriptional activity is essentially absent in fully grown oocytes bridges in AT-I oocytes (Fig. 6B and C). Accurate segregation of
and subsequent completion of meiosis is achieved by using stored homologous chromosomes or sister chromatids during anaphase is a
maternal transcripts.44 The results above offer the exceptions to the critical event in meiosis or mitosis. Any error in this process may
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Histone Acetylation in Meiotic Oocyte
result in aneuploid embryo formation, which causes early embryo
death, spontaneous abortion and genetic diseases.55 Chromosome
segregation depends on the establishment of physical and biochemical interactions between spindle microtubules and specialized
chromosomal regions localized at centromeric heterochromatin.56,57
Furthermore, considerable evidence suggests that underacetylated
state of histones in centromeric chromatin is essential for the recruitment of specific heterochromatin protein, such as Swi6 and
HP1.17,18,54,58,59 In addition, methylation of H3/K9 completes a
binding site for the chromodomain of HP1.60,61 Importantly, TSA
treatment not only induces histone hyperacetylation but also results
in reduction of H3/K9 methylation.62 Therefore, it is conceivable
that the inhibition of deacetylation may prevent the methylation of
H3, which in turn leads to a loss of association of HP1 or Swi6 with
the centromeric heterochromatin domain, and, as a result, weakened
kinetochores with impaired interactions with spindle microtubules
are maintained, causing persistent defects in chromosome segregation.17 In fact, the chromosome segregation is regulated by a
complicated and coordinated system, which is supported by recent
studies on cohesion, condensin and the phosphorylation of serine 10
in H3.63,64,65
It was reported that HDAC1 localized in the nonchromatin
domains in the GV and colocalized with chromosomes after GVBD
in mouse oocytes.19 Contrary to these results, our studies in porcine
oocytes showed that HDAC1 also localized in the chromatin-depleted
spaces at GV stage but then translocated to the periphery of condensed
chromosomes at GVBD stage (Fig. 7A), which supports the model
proposed by Kruhlak et al.14 Further investigation of HDAC1 distribution in other types of oocytes will be helpful to understand these
differences.
Clearly, such versatile acetylation patterns in histones during
oocyte maturation (Table 1) are possibly not operated by a single
HDAC1. Different HATs and HDACs, which display distinct specificities toward the histone acetylation sites,66 may coparticipate in
this process. The expression, function of various HDACs and the
mechanisms by which their activities are regulated needs to be further
elucidated.
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