In oocyte maturation in Xenopus laevis, nuclear material induces rapid maturation and is required for entry into meiosis II. Nuclear material contains a large number of RNAs and proteins, including histone deacetylase (HDAC); however, it is not known which materials induce accelerated maturation. The HDAC activity modifies transcription rate and is required for normal meiosis; however, its function in oocyte maturation is still unclear. We investigated the function of HDAC activity, which is localized in the nuclear material, in the regulation of the speed of oocyte maturation. Inhibition of HDAC activity with trichostatin A (TSA) induced hyperacetylation of histone H3 and prolonged oocyte maturation. In contrast, increase in HDAC activity with an injection of FLAG-tagged maternal histone deacetylase (HDACm-FLAG) mRNA induced deacetylation of histone H3 and reduced the duration of oocyte maturation. Cdc2 kinase, Cdc25C or mitogen-activated protein kinase (MAPK), which are key regulators of the meiosis, were activated coincidently with maturation progression. In oocytes, the mRNA level of Cdc25C, an activator of Cdc2, was increased by HDACm-FLAG mRNA-injection; in contrast, the mRNA level of Cdc2 inhibitor Wee1 was increased by TSA treatment. These results suggest that HDAC activity is involved in the control of maturation speed through the regulation of mRNA levels of cell cycle regulators. Thus, HDACm is a candidate for the nuclear material component that induces rapid maturation in Xenopus oocytes.
Meiotic oocyte maturation is the final step of oogenesis and is a prerequisite process for the immature oocyte to become fertilizable (Masui & Clarke 1979). In almost all species, immature oocytes are arrested in the first meiotic prophase (prophase I) or the late G2 phase. They resume meiosis usually in response to hormonal stimulation for example, progesterone stimulation (Sagata 1996). Initiation of maturation induces the activation of maturation promoting factor (MPF), a key G2/M phase regulator in eukaryotic cells that consists of Cdc2 kinase and Cyclin B (Nurse 1990). MPF is activated in meiosis I, then transiently inactivated and reactivated to enable entry into meiosis II (Gerhart et al. 1984; Furuno et al. 1994). In vertebrates, oocyte maturation is arrested again at the second meiotic metaphase (metaphase II) before fertilization by a cytoplasmic factor called cytostatic factor (Masui & Markert 1971) whose essential component is Mos kinase (Sagata et al. 1989b). Mos kinase induces the activation of MPF and maintains its activity at a high level in meiosis II.
Maturation promoting factor activity is regulated by the phosphorylation status of Cdc2 kinase. Activation requires the dephosphorylation of the Thr-14 and Tyr-15 sites of Cdc2 kinase, and the Wee1/Myt1 kinases inactivate MPF by phosphorylating these sites of Cdc2 (Parker & Piwnica-Worms 1992; Heald et al. 1993; McGowan & Russell 1993; Booher et al. 1997; Fattaey & Booher 1997; Liu et al. 1997). Cdc25 phosphatase dephosphorylates both of these sites and activates Cdc2 (Millar et al. 1991; Strausfeld et al. 1991; Trunnell et al. 2011). Thus, the Wee1/Myt1 family is an inactivator and Cdc25 is an activator of Cdc2/MPF in oocytes.
The activation of MPF in meiosis I leads to nuclear envelope disassembly and the nuclear material (or nucleoplasm) then mixes with the cytoplasm of the maturing oocyte (Miller et al. 1989). The large nucleus of the immature oocyte is called the germinal vesicle (GV), and disassembly of its nuclear envelope in meiosis I is referred to as germinal vesicle breakdown (GVBD). This is followed by chromosome condensation and spindle formation in meiosis I (Masui & Clarke 1979). Distributed nuclear material is required for sperm chromatin decondensation and cleavage in fertilized eggs (Katagiri & Moriya 1976; Balakier & Tarkowski 1980). The nuclear material contains a number of restored mRNAs and proteins, such as histones and DNA polymerases that are used immediately after fertilization (Davidson 1986). In starfish oocytes, nuclear material is required for the activation of MPF upon maturation (Kishimoto et al. 1981; Picard & Doree 1984). In vertebrate species, however, nuclear material has long been thought to be unnecessary for MPF activation and oocyte maturation. In a previous study, we re-evaluated the role of nuclear material in the meiotic cell cycle of the amphibian Xenopus laevis oocytes and showed that the germinal vesicle contains a factor or factors that play important roles in the meiotic cell cycle, particularly MPF reactivation and entry into meiosis II (Iwashita et al. 1998). Moreover, we found that nuclear material induces rapid maturation in oocytes. The oocytes injected with nuclear material undergo accelerated maturation, whereas enucleated oocytes undergo prolonged maturation. These results suggest that nuclear material contains cell cycle accelerator, but the mechanism, which regulates and accelerates the progression of maturation, remains unclear.
Nuclear material contains a number of restored proteins including histone deacetylase (HDAC) (Ryan et al. 1999). The transcription rate from chromatin is closely regulated by the acetylation status of core histones, which is determined by the equilibrium between the activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) (Wolffe & Hayes 1999; Sterner & Berger 2000). Reversible acetylation of core histones plays an important regulatory role in the transcription of specific genes and the replication of chromatin in eukaryotic cells. HDAC activity reduces the acetylation of histones in nuclei and regulates the rate of transcription in cells. Maternal histone deacetylase (HDACm), a homologue of human HDAC I, accumulates in the nucleus of Xenopus oocytes and is organized in a multiprotein complex (Ryan et al. 1999). HDACm is an active subunit of the HDAC complex and its content represents histone deacetylase activity during early developmental stages in Xenopus (Ryan et al. 1999). In oocyte maturation, HDAC activity is thought not to be essential for the progression of meiosis because inhibition of HDAC activity by HDAC inhibitors did not stop GVBD and entry into meiosis II; however, HDAC activity is required for chromosome condensation during oocyte maturation (Magnaghi-Jaulin & Jaulin 2006). Thus, the regulation of HDAC activity is essential for normal oocyte maturation, but its detailed function in maturation is still unclear.
In the present study we re-evaluated the role of HDAC activity distributed in the nuclear material during the meiotic cycle of oocytes of the amphibian Xenopus. We found that HDACm-FLAG mRNA-injection induced accelerated cell cycle progression in progesterone-stimulated oocytes in contrast to the prolongation of maturation induced by HDAC inhibition. Our results suggest that HDAC activity in the germinal vesicle plays an important role in regulating the speed of maturation and induces rapid maturation in Xenopus oocytes.
Materials and methods
Preparation and culture of Xenopus oocytes
Stage VI Xenopus oocytes were defolliculated by collagenase treatment, microinjected, treated with progesterone (to induce maturation), and cultured in modified Barth's solution at 20–22°C, as described (Furuno et al. 1994; Sagata 1996).
Oocytes were enucleated in half-strength modified Barth's solution with 18G syringe needle (TERUMO, Japan) exactly as described (Iwashita et al. 1998). In brief, germinal vesicle alone was removed from immature oocytes as follows. A small puncture was first made on the animal pole, and then germinal vesicle comes out from oocytes. Then the germinal vesicles and cytoplasms were collected. The collected oocytes and cytoplasm were homogenized in an extraction buffer (EB; 0.01 mL per oocyte) on ice and briefly centrifuged (the EB buffer contained: 80 mmol/L β-glycerophosphate, 20 mmol/L ethylene glycol tetraacetic acid (EGTA), 15 mmol/L MgCl2, 0.1 mmol/L dithiothreitol [DTT], 20 mmol/L leupeptin, 10 mmol/L pepstatin, 2 mmol/L phenylmethylsulfonyl fluoride [PMSF], 10 mg/mL aprotinin, 1 mmol/L Na3VO4, 1 mmol/L NaF, pH 7.5). The supernatant obtained was mixed with equal amounts of 2× Laemmli's sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromphenol blue, 0.125 M Tris HCl, pH 7.5). The collected germinal vesicles were directly mixed with 1× Laemmli's sample buffer.
Oocytes were injected with glass needles prepared with a glass needle puller PC-10 (Narishige, Japan). The defolliculated oocytes were incubated in modified Barth's solution with 3% Ficoll PM 400 (GE Healthcare, Sweden), and were injected with 60 ng of mRNA (30 nL water) into each oocyte with Nanoject II microinjector (Drummond Scientific Company, USA). The mRNA was diluted in di-ethyl-pyro-carbonate- treated water at a concentration of 2.0 μg/μL. The injected oocytes were incubated in modified Barth's solution for 12 h at 20°C before progesterone treatment. For progesterone treatment, oocytes were incubated in the presence of 1 μg/mL of progesterone at 23°C.
0.1 mmol/L of trichostatin A (TSA; Sigma, Japan), dissolved in dimethyl sulfoxide (DMSO; Sigma, Japan) was prepared as a stock solution. TSA was added in modified Barth's solution at a final concentration of 100 nmol/L. Stage VI oocytes were pre-incubated in the presence or absence of 100 nmol/L of TSA for 12 h. As a control, oocytes were treated with DMSO instead of TSA at a final concentration of 0.1%.
Mos monoclonal antibody was obtained from abcam (ab5480, Japan). Cdc25C monoclonal antibody and phospho-MAP kinase (Thr202) polyclonal antibody was obtained from Santa Cruz Biotechnology (sc-73370 and sc-101760MA, USA). Anti-acetyl-Histone H3 polyclonal antibody was obtained from Millipore (06-599, Japan). Wee1 polyclonal antibody was obtained from invitrogen (51-1700, USA). Anti-Cdc2 p34 (PSTAIRE) polyclonal antibody was purchased from Santa Cruz Biotechnology (sc-53, USA). Anti glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polyclonal antibody was used as a loading control and was purchased from abcam (ab9485, Japan). Anti FLAG monoclonal antibody was purchased from Sigma (F3165, Japan). Anti-proliferating cell nuclear antigen (PCNA) polyclonal antibody was purchased from Santa Cruz Biotechnology (sc-7907, USA).
Western blot analysis
Treated oocytes were collected, centrifuged, and dissolved in Laemmli's sample buffer and heated at 98°C for 3 min. Aliquots were then subjected to 7.5% and 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) for detection of Cdc25C, Wee1 and MAP kinase, Mos, FLAG, GAPDH, respectively. 12.5% SDS–PAGE was used for detection of Cdc2. After electrophoresis, proteins on the gel were transferred to 12.5 cm × 8.5 cm of nitrocellulose membrane (Hybond ECL: Amersham Pharmacia Biotech, USA) by electrophoresis at 100 mA for 35 min using a transfer apparatus (Cima Biotech, USA) with a transfer buffer composed of 20 mmol/L Tris, 150 mmol/L glycine and 20% methanol. Following transfer, the membrane was incubated in a blocking buffer of TBS-T (20 mmol/L Tris–HCl, 150 mmol/L NaCl, 0.1% Tween-20, pH 7.5) containing 4% skimmed milk (Gibco Oriental, Japan) at room temperature for 1 h. The membrane was incubated with primary antibody (1:2000) in Can Get Signal Immunoreaction Enhancer Solution (TOYOBO, Japan) for 1 h at room temperature. After washing with TBS-T, the membrane was then incubated for 1 h with goat antirabbit IgG (H + L) antibody conjugated with horseradish peroxidase (1:2000; Promega, USA) for phospho-MAP kinase, acetyl-Histone H3, Wee1, GAPDH, PCNA and Cdc2 detection, or goat antimouse IgG antibody conjugated with horseradish peroxidase (1:2000; Promega, USA) for Mos, FLAG and Cdc25C phosphatase detection. After membrane washing with TBS-T, the enzyme reactions were detected by an enhanced chemiluminescence assay using a Luminata Forte western HRP substrate (millipore, USA) and a LAS-4000 image analyzer (Fujifilm, Japan).
Isolation of ribonucleic acid
RNA was isolated with a TRIzol Reagent kit (Invitrogen, Japan). The collected 10 oocytes were mixed with 0.8 mL of the TRIzol Reagent, homogenized by vigorous pipetting and left at room temperature for 5 min. After addition of 0.06 mL of chloroform, oocytes were incubated at room temperature for 3 min. Following centrifugation at 12 000 g for 10 min, the aqueous phase was collected, mixed with 0.4 mL of isopropyl alcohol, and left at room temperature for 10 min. After centrifugation, the precipitated RNA was collected, washed with 70% ethanol, and dissolved in RNase-free water. The RNA was treated with DNase I (Nippon Gene, Japan) for 30 min and extracted by standard methods. The DNA-free RNA was used for the detection of mRNAs by real-time reverse transcription–polymerase chain reaction (RT–PCR).
Reverse transcription–polymerase chain reaction
Cdc25C and Wee1 mRNA level in oocytes was assayed by RT–PCR using a real-time PCR LightCycler System V3 (Roche Molecular Biochemicals, Germany) and a QuantiTect SYBR Green RT-PCR Kit (Qiagen, Japan). Total RNA (500 ng), forward primer and reverse primer (10 pmol each) were mixed with the kit solution containing reverse transcriptase, deoxyribonucleoside triphosphates (dNTPs), Taq DNA polymerase and fluorescence dyes in a 20 μL solution. After one cycle of reverse transcription at 50°C for 20 min and activation of Taq polymerase, PCR was performed for 40 cycles. The PCR was a cycle of 94°C for 15 s, 64°C for 15 s and 72°C for 20 s. All reactions were carried out in triplicate from three separate samples. Each RNA samples were obtained from 10 treated oocytes. The amounts of each mRNAs were obtained from a standard curve. The expression level of Cdc25C mRNA and Wee1 mRNA were normalized by the amount of 18S rRNA. The total RNAs were electrophoresed on a 1.5% agarose gel-containing ethidium bromide. The amount of 18S rRNAs were assayed by a LAS-4000 image analyzer (Fujifilm, Japan). To visualize the relative amount of PCR products, PCR was stopped at the log-linear phase after 32 cycles for Cdc25C and 38 cycles for Wee1. The PCR products were detected by electrophoresis with a 100 bp DNA ladder (TAKARA, Japan) on a 1.5% agarose gel-containing ethidium bromide. The PCR primers used are as follows:
Cdc25C forward: 5′- CGA TAC ATC ACT GGA GAG AC-3′
Cdc25C reverse: 5′-CTT GGT GGT GCA TTG GGC AG-3′
Wee1 forward: 5′- GGG GAC CTT GGT CAT GTG AC-3′
Wee1 reverse: 5′-CAA CTC CCT CTC AAG CAT GG-3′.
Sequence analysis of PCR products
The identity of the PCR products and cloned HDACm-FLAG was confirmed by nucleotide sequencing that was performed in the Biotechnology Center of Akita Prefectural University.
Cloning of Xenopus HDACm cDNA and synthesis of mRNA
We cloned a 1440 bp cDNA sequences of Xenopus HDACm (GenBank accession no. X78454.1) using primers, forward primer (5′-AATGGCGCTGACTCTAGGAA-3′) and reverse primer (5′-CCATAGTTGAAGGATCAGAC-3′). Total RNA was isolated by using a TRIZOL (Invitrogen, USA) with chloroform extraction. Total RNA was reverse transcribed and amplified to obtain HDACm cDNA using Quantitect SYBR Green RT–PCR kit (Quiagen, Germany). After one cycle of reverse transcription at 50°C for 20 min and activation of Taq polymerase, PCR was performed for 40 cycles. The PCR was a cycle of 94°C for 15 s, 64°C for 15 s and 72°C for 90 s. Then, to produce FLAG-tagged HDACm, we used forward primer (5′-ACCATGGCGCTGACTCT-3′) and reverse primer (5′-TCACTTGTCATCGTCGTCCTTGTAGTCGACTGATTTG-3′) in PCR with Taq polymerase (M300F; Promega, USA) for 40 cycles. The PCR was a cycle of 94°C for 15 s, 50°C for 15 s and 72°C for 90 s. The PCR product was subcloned into pGEM-T Easy vector (Promega, USA) and sequenced. To create a construct of ΔN HDACm, the construct of HDACm was digested by Hind III (TAKARA, Japan), and was ligated by T4 DNA Ligase (Promega, Japan). Then, to add Xho I site and Xba I site in constructs, we used forward primer (5′-GATCTCGAGCCACCATGGCGCTGACTCTA-3′) and reverse primer (5′-GTTTCTAGATTACTTGTCATCGTCGTC-3′) in PCR for HDACm-FLAG construction. The other forward primer (5′-GATCTCGAGAGCTTCACATCAGCCCA-3′) was used to create ΔN HDACm-FLAG construction. All PCR reactions to create HDACm-FLAG and ΔN HDACm-FLAG construction were performed with Taq polymerase (M300F; Promega, USA) for 40 cycles. The PCR was a cycle of 94°C for 15 s, 50°C for 15 s and 72°C for 90 s. After amplification with PCR, the amplified products were digested with Xho I (TOYOBO, Japan) and Xba I (TAKARA, Japan) and were subcloned into pT7G(UK+) (Nakajo et al. 1999). mRNA was synthesized from a T7 promoter using the Megascript T7 kit (Ambion, USA) as instructed and is capped with m7G(5′)ppp(5″) (Ambion, USA). The mRNA was resuspended in di-ethyl-pyro-carbonate- treated water at a concentration of 2.0 μg/μL.
Inhibition of histone deacetylase activity induced prolongation of oocyte maturation
To study the function of HDAC activity in the regulation of oocyte maturation, we treated oocytes with an HDAC inhibitor trichostatin A (TSA). TSA is a hydroxamic acid-derived compound and is a potent class I and class II HDAC inhibitor (Khan et al. 2008). Inhibition of HDAC activity induces the hyperacetylation of histone H3 proteins (Fig. 1a). Maternal HDAC activity is localized in the nuclear material in Xenopus oocytes (Ryan et al. 1999). We manually isolated germinal vesicles from oocytes and investigated the acetylation level of histone H3 in germinal vesicles and the cytoplasm. Histone H3 in germinal vesicles was hyperacetylated by treatment with TSA (Fig. 1b). We used GAPDH as a loading control of cytoplasm. Proliferating cell nuclear antigen (PCNA), which is localized in the nuclei, was used as a nuclear loading control (Leibovici et al. 1990). Next, we evaluated the effect of hyperacetylation of histone H3 during oocyte maturation. Oocytes treated with or without TSA were induced to mature by treatment with progesterone and the progression of oocyte maturation was observed. Oocytes show a white spot at the animal pole coincident with GVBD at meiosis I, and we therefore observed the time course of the appearance of the white spot. We found that inhibition of HDAC activity by TSA treatment induced a delay in the time course of appearance of the white spot (Fig. 2). The activities of Cdc2 and MAPK, which are key regulators of cell division, were measured by detection of their phosphorylated form with western blot analysis in TSA treated oocytes. Cdc2 kinase is inactivated before meiosis by phosphorylation at its inhibitory site of Tyr-15. The resumption of meiosis induces the activation of Cdc25 phosphatase and it dephosphorylates Tyr-15 of Cdc2, which allows activation of Cdc2 kinase. Compared with control oocytes, TSA-treated oocytes showed a delay in the disappearance of Tyr-15 phosphorylated and inactivated form of Cdc2 (Fig. 3a). The phosphorylation and activation of MAPK was accompanied by Cdc2 activation and progression of oocyte maturation. The time course of the appearance of the phosphorylated/activated form of MAPK was measured by western blot analysis. Compared with control oocytes, TSA-treated oocytes showed a delay in the appearance of phosphorylated and activated form of MAPK (Fig. 3a). The activation of MAPK was delayed in synchrony with the appearance of the white spot (Figs 2, 3a). The phosphorylation and activation of Cdc25C phosphatase, an activator of Cdc2, was also showed a delay in TSA treated oocytes compared with the controls (Fig. 3a). In TSA treated oocytes, the amount of Mos, which phosphorylates and activates MAPK (Nebreda & Hunt 1993), was at a high level compared with control oocytes at 550 min after progesterone treatment (Fig. 3b). There was no significant difference in the amount of Wee1 kinase, which inhibits Cdc2 activity, between TSA treated and control oocytes at 450 and 550 min after progesterone treatment (Fig. 3b).
These results suggest that the inhibition of HDAC activity by TSA treatment does not affect entry into meiosis I, but delays the time course of oocyte maturation with slower activation of Cdc2, MAPK, or Cdc25C.
Increase in histone deacetylase activity induced accelerated oocyte maturation
We next investigated the effect of upregulation of HDAC activity on oocyte maturation. Most HDAC activity can be accounted for by the maternal HDAC (HDACm) accumulates in the nuclear material in Xenopus oocytes and early embryos. We amplified HDACm cDNA with FLAG-tag (HDACm-FLAG) by RT–PCR and inserted it into the appropriate vector. We synthesized HDACm-FLAG mRNA and injected it into oocytes. The HDACm-FLAG protein has a conserved region, which is an enzyme core and is needed for HDAC activity in N-terminal half (Leipe & Landsman 1997). Therefore, we created an N-terminus-deleted mutant (ΔN HDACm-FLAG) as a null mutant that is an inactive form of HDACm-FLAG and injected ΔN HDACm-FLAG mRNA into oocytes as a control (Fig. 4a). HDAC activity should induce a decrease in the acetylation status of histones in chromatin. The injected and translated HDACm-FLAG and ΔN HDACm-FLAG were detected by western blot analysis using anti-FLAG antibody (Fig. 4b). Injected HDACm is reported to be translocated to the nucleus where it is free to interact with the endogenous chromatin (Smillie et al. 2004). Acetylation of histone H3 was decreased in the HDACm-FLAG mRNA-injected oocytes (Fig. 4c). Oocytes injected with HDACm-FLAG mRNA underwent rapid maturation after progesterone stimulation compared with control oocytes injected with ΔN HDACm-FLAG mRNA or water (Fig. 5). Injection of HDACm-FLAG mRNA alone did not induce oocyte maturation without progesterone stimulation (data not shown).
We observed the activation time of proteins that regulate the speed of maturation after progesterone stimulation. Phosphorylation at Thr-161 of Cdc2 resulted in the active form of Cdc2 to migrate slightly faster than the nonphosphoryated/inactive form.
In HDACm mRNA injected oocytes, a faster disappearance of Tyr-15 phosphorylated and the inactivated form of Cdc2 than the control (ΔN HDACm-FLAG mRNA-injected) oocytes was shown (Fig. 6a). MAPK and Cdc25C phosphatase were also rapidly activated in the HDACm-FLAG mRNA-injected oocytes compared with the controls (Fig. 6a). There was no significant difference in the amount of Mos and Wee1 kinase between HDACm-FLAG mRNA injected and control oocytes at 350 and 550 min after progesterone treatment (Fig. 6b).
Acetylation status of histone affects accumulation of specific mRNAs of cell cycle regulators
Acetylation status strongly affects the structure of the chromosome and modulates the expression of multiple genes. We investigated which mRNAs of cell cycle regulators were affected in their transcription rate by the injection of HDACm-FLAG mRNA or by TSA treatment.
We measured the effects of HDACm-FLAG mRNA injection or TSA treatment on the accumulation of mRNAs of Wee1 and Cdc25C, which are representative inhibitors and promoter of cell cycle progression. In TSA-treated oocytes, Wee1 mRNAs were increased compared with control and HDACm-FLAG mRNA-injected oocytes (Fig. 7). In HDACm-FLAG mRNA injected oocytes, Cdc25C mRNA-increased compared with control and TSA-treated oocytes (Fig. 7). These results are consistent with the finding that HDACm-FLAG mRNA-injected oocytes showed rapid maturation and TSA-treated oocytes showed prolonged maturation after progesterone stimulation, and suggest that HDAC activity is involved in the control of maturation speed through the regulation of mRNA levels of cell cycle regulators such as Cdc25C.
In developing Xenopus oocytes, many mRNAs and proteins are synthesized and restored before meiosis. The fully grown (stage VI) oocytes are not metabolically dormant, but synthesize a steady level of total RNAs (LaMarca et al. 1973). The chromatin of oocyte nuclei is organized as lampbrush chromosomes to ensure a high rate of transcription. Acetylation and deacetylation of histones play important roles in chromatin remodeling and consequently change the expression of specific genes in eukaryotic cells. The balance of HATs and HDACs determines the acetylation status of chromatin in eukaryotic cells such as Xenopus oocytes.
The Xenopus maternal HDAC, HDACm, shows sequence homology to other putative histone deacetylases and undergoes deacetylation of histone proteins in oocyte chromatin (Ladomery et al. 1997). It accumulates in the germinal vesicle and forms a multiprotein complex with retinoblastoma-associated protein p48 (RbAp48/46). HDACm is synthesized during oogenesis and decreases in amount after the mid-blastula stage when the cell cycle slows to a normal rate and cells begin to differentiate (Ryan et al. 1999). This suggests that HDAC activity is closely related to the regulation of the cell cycle in the early development of Xenopus. HDAC plays an important role in gene regulation induced by T3 during metamorphosis in Xenopus. TSA, an inhibitor of HDAC, blocks normal metamorphosis at the tadpole stage (Sachs et al. 2001). TSA also blocks tail regeneration (Tseng et al. 2011); therefore, normal HDAC activity is required during Xenopus regeneration.
In meiosis, HDACm activity is necessary for correct chromosome condensation, but is not required for the progression of and exit from meiosis or for entry into mitosis (Magnaghi-Jaulin & Jaulin 2006). However, the possibility that HDAC activity is necessary for the regulation of maturation speed has not been investigated. The cell cycle comprises an ordered series of events that are required for the faithful duplication of one eukaryotic cell into two genetically identical daughter cells. If the events of cell cycle progression occur out of order, abnormal cell division occurs leading to cell death or apoptosis. The precise regulation of cell cycle speed is therefore essential for the precise progression of mitosis and meiosis (Kastan & Bartek 2004). Our present results suggest that HDAC activity in the germinal vesicle mediates the speed of maturation in Xenopus oocytes. Inhibition of HDAC activity with TSA-induced hyperacetylation of nuclear histone H3 (Fig. 1) and prolonged the period of maturation required to reach meiosis I (Fig. 2). In contrast, injection of HDACm-FLAG mRNA, which induced repression of histone acetylation (Fig. 4), shortened this period of maturation (Fig. 5). These results are consistent with our past report that nuclear material, which contains HDACm protein, induces rapid oocyte maturation. The result that injection of HDACm-FLAG mRNA alone did not induce oocyte maturation (data not shown) is also consistent with our past report that injection of nuclear material alone does not induce oocyte maturation.
The finding that TSA treatment prolongs meiosis was confirmed by the time course of activation of Cdc25C, Wee1, MAPK, and Cdc2 kinase. Activation of Cdc2 kinase and MAPK was delayed in TSA-treated oocytes and rapidly induced in HDACm-FLAG mRNA-injected oocytes (Figs 3a, 6a). Cdc25C phosphatase is activated at the G2/M transition by phosphorylation at serine and threonine residues by MPF. Activation of Cdc25C was delayed in TSA-treated oocytes, but was induced rapidly in HDACm-FLAG mRNA-injected oocytes (Figs 3a, 6a). These results suggest that HDAC activity in the germinal vesicle accelerates the course of maturation through activation of cell cycle regulators. In TSA-treated oocytes, Mos accumulated at a high level compared with control oocytes in 100% GVBD oocytes (550 min after progesterone treatment, Fig. 3b). This result suggests that hyper-acetylation of histones induce the transcriptional activation of Mos kinase (Sagata et al. 1989a).
Wee1 and Myt1 kinase are representative inhibitors of the progression of cell division in the regulation of the cell cycle. Wee1 is a nuclear kinase, whereas Myt1 associates with the cell membrane and represses the activation of MPF. Wee1 protein is not detectable in fully grown oocytes and becomes detectable after completion of GVBD. Wee1 protein is translated and accumulated in the course of oocyte maturation. In contrast, Myt1 is detectable in fully grown oocytes, but its role in oocyte maturation is still unclear (Gaffre et al. 2011). In our past report, the absence of Wee1 during meiosis I ensures the normal meiotic cell cycle. In contrast, ectopic expression of Myt1 has little effect on the meiotic cell cycle (Nakajo et al. 2000). Thus, the activity of Wee1 is important for the regulation of maturation speed during meiosis I in Xenopus oocytes. Our quantitative RT–PCR results showed that the accumulation of Wee1 mRNA in TSA-treated oocytes was greater than in control oocytes (Fig. 7). This result suggests that the upregulation of Wee1, caused by TSA treatment, induces a delay in maturation; however, Wee1 protein was not detectable before progesterone treatment and was not accumulated at a high level after progesterone treatment (Fig. 6). This contradiction suggests that Wee1 mRNA was inhibited to be translated in oocytes before progesterone stimulation even if the accumulation of Wee1 mRNA increased and another cell cycle regulator induced a delay in maturation. Xenopus Wee1 was reported to cause a dose dependent delay in a cell division (Mueller et al. 1995). In contrast, mRNA for Cdc25C, an activator of Cdc2, accumulated in oocytes injected with HDACm-FLAG mRNA. These results, that a high level of HDAC activity induced Cdc25C mRNA accumulation and that a low level of HDAC activity induced Wee1 mRNA accumulation, suggest that these changes induced by histone acetylation status alter the speed of oocyte maturation. Oocyte maturation is a process that depends largely on post-transcriptional events. The status of histone acetylation changes mainly the transcription rate of cell cycle regulators. We speculate that the changes of accumulation of mRNA of cell cycle regulators like Cdc25C induced by the changes of histone acetylation status might induce the rapid response to progesterone stimulation and produces a rapid cell cycle progression. There is another possibility, which is that histone deacetylase inhibitor increased mRNA stability of cell cycle regulators (Hirsch & Bonham 2004). We will investigate the stability of mRNAs of cell cycle regulators like Wee1 or Cdc25C in TSA-treated or HDACm-FLAG mRNA injected oocytes.
Many studies have indicated that histone acetylation is associated with transcriptional activation and that histone deacetylation leads to gene repression (Grunstein 1997; Wade et al. 1997; Peterson 2002). However, a recent study concluded that histone acetylation may lead to not only gene activation but also to gene repression (Davie 2003; Glaser et al. 2003). Our results suggest that acetylation activity in oocytes has different effects on the transcription of Cdc25C and Wee1 mRNA and ensures the normal progression of oocyte maturation.
In eukaryotes, HDAC does not work in isolation, but works with transcription factors or corepressors such as Sin3 with which it forms multiprotein complexes. Sin3, a corepressor of HDAC, forms a multiprotein complex (Sin3 complex) in Xenopus oocytes (Wade et al. 1998). The Sin3 complex interacts with its target chromatin location sequence-specifically. Investigation of the role of Sin3 in oocyte maturation may provide clearer evidence of the integrity of HDAC activity in meiosis progression.
The present study showed that HDACm is a candidate for the component of nuclear material that induces rapid maturation in Xenopus oocytes. In addition, since nuclear material contains many proteins or mRNAs, it is possible that multiple factors that accumulate in the germinal vesicle can induce rapid maturation. Speedy (Spy1) is able to induce rapid maturation of Xenopus oocytes resulting in the induction of GVBD and activation of MPF. Spy1 could therefore be a candidate for an inducer of rapid maturation in nuclear material (Lenormand et al. 1999). Further investigation is needed on the relationship between factors that can induce rapid maturation, such as Spy1 and the regulation of histone acetylation status in oocyte maturation.
This work was supported in part by a Grant-in-Aid for Young Scientists (B) from JSPS (No. 17770197) and Akita Prefectural University President's Research Project. We thank the staff of the Biotechnology Center in Akita Prefectural University for their help in DNA sequence analysis.