Scriptaid Improves In Vitro Development and Nuclear Reprogramming of Somatic Cell Nuclear Transfer Bovine Embryos

Abstract

The present study evaluated the effect of Scriptaid, a novel histone deacetylase inhibitor (HDACi), on the in vitro development of somatic cell nuclear transfer (SCNT) bovine embryos. Average fluorescence intensity of two epigenetic markers (H3K9ac and H3K9m2) at two-cell, eight-cell, and blastocyst stages, and the expression levels of two developmental important genes (Oct4 and IFN-t) at the blastocyst stage were also examined to assess the influence of Scriptaid on the nuclear reprogramming of bovine SCNT embryos. The results showed that treatment with 500 nM Scriptaid for 14 h after activation significantly increased the cleavage rate, blastocyst formation rate, and blastocyst hatching rate of SCNT embryos compared with those of nontreated counterparts, but the total number of blastomeres per blastocyst did not differ. Scriptaid treatment also significantly increased the immunofluorescent signal for H3K9ac in SCNT embryos at two-cell, eight-cell, and blastocyst stages, and the fluorescent signal for H3K9m2 was decreased at two-cell, eight-cell, and blastocyst stages. The expression levels of Oct4 and IFN-t were significantly higher in Scriptaid-treated SCNT blastocysts than in Scriptaid nontreated SCNT blastocysts. The results indicated that Scriptaid treatment improved the in vitro developmental capacity and the nuclear reprogramming of bovine SCNT embryos.

Introduction

During somatic cell nuclear transfer(SCNT), differentiated somatic cells such as mammary epithelial cells (Wilmut et al., 1997), cumulus cells (Wakayama et al., 1998), oviduct epithelial cells (Kato et al., 1998), and fetal fibroblast cells (Baguisi et al., 1999) were used as the donor cells to produce cloned animals. Somatic cell is in a highly differentiated state that has differentiation-specific epigenetic markers and no differentiation potential compared with stem cell (Hochedlinger and Jaenisch, 2006). After the injection of the donor nuclei into the oocyte cytoplasm, donor nucleus undergoes dramatic changes that result in an erasure of tissue-specific epigenetic markers and reestablishment of a totipotency state within a short period of time that is referred to as nuclear reprogramming (Campbell et al., 1993; Santos and Dean, 2004). Maternal factors in oocyte cytoplasm, such as MPF and MAP kinase were demonstrated to be important in nuclear reprogramming (Campbell et al., 1993; Lee and Campbell, 2006). The incomplete removal of old labels or the incomplete establishment of new labels might result in abnormal epigenetic modifications and abnormal gene expression patterns. Further, developmental defects including abortion might occure (Cezar, 2003).

Epigenetic modifications of the genome involves genomic methylation, histone modifications, and chromatin remodeling (Bird, 2002; Li, 2002). Extremely higher DNA methylation and lower histone acetylation levels have been commonly observed in the cloned embryos (Beaujean et al., 2004; Dean et al., 2001). Accordingly, artificially repairing the abnormal epigenetic modifications was proposed as a possible solution to improve cloning efficiency. HDACi could induce histone hyperacetylation on preimplantation embryos (Das et al., 2010; Iager et al., 2008; Oliveira et al., 2010; Wu et al., 2008), Therefore, several kinds of HDACis have been used to enhance the nuclear reprogramming of SCNT embryos, such as TSA (Ding et al., 2008; Enright et al., 2003; Kishigami et al., 2006; Li et al., 2008b; Wang et al., 2011b), NaBu (Das et al., 2010; Shi et al., 2003; Yang et al., 2007), Scriptaid (Van Thuan et al., 2009; Zhao et al., 2009, 2010), and VPA (Miyoshi et al., 2010). TSA is a widely tested HDAC inhibitor that has been shown to enhance epigenetic reprogramming of SCNT embryos. But the effect of TSA is inconsistent in different species. In mice, TSA was demonstrated to improve both the in vitro and in vivo development of SCNT embryos (Kishigami et al., 2006; Rybouchkin et al., 2006). However, in bovine, some studies showed that TSA treatment could significantly improve the preimplantation development of SCNT embryos (Ding et al., 2008; Enright et al., 2003), whereas others indicated that TSA had detrimental effect on the preimplantation development of fertilized embryos (Oliveira et al., 2010) and SCNT embryos (Wu et al., 2008). Thus, the efficacy of HDACi on the developmental capacity of embryos seems to vary in different species, and it is necessary to find out an agent that would be more effective in supporting the development of bovine SCNT embryos.

Scriptaid is a novel HDAC inhibitor, which has relatively higher histone acetylation activity and lower cellular toxicity compared with TSA (Su et al., 2000). It has been reported that Scriptaid treatment could significantly increase the in vitro and in vivo development of cloned inbred mice embryos (Van Thuan et al., 2009). Similar results were also obtained in pigs. Previous studies showed that Scriptaid could significantly improve the cloning efficiency of inbred miniature pigs and normal pigs (Zhao et al., 2009, 2010). The authors suggested that Scriptaid has two main characteristics: the induction of lower cellular toxicity compared to other HDACis, and the improvement of cloning efficiency in mice and pigs. Thus, we hypothesized that Scriptaid would have beneficial effects on in vitro production of bovine SCNT embryos. Growing evidence suggests that interactions exist among different modifications on the histone tails (Wang et al., 2001). We supposed that Scriptaid would affect not only histone acetylation but also histone methylation.

It was demonstrated that histone acetylation could increase gene expression (Struhl, 1998). The possible mechanisms is that acetylation on the amino-terminal tails of histones neutralizes the positive charge of the histone tails and decreases their affinity for DNA, thereby altering nucleosomal conformation and increasing the accessibility of transcriptional regulatory proteins to chromation templates (Struhl, 1998). Taken together, histone acetylation could result in increased transcriptional activity.

The objective of this study was to assess the effects of Scriptaid treatment on the fluorescent intensity of two epigenetic markers (H3K9ac and H3K9me2) in preimplantation embryos produced by SCNT, examine the effects of Scriptaid on the expression of two developmental important genes (Oct4 and IFN-t), and finally determine the effects of Scriptaid treatment on the in vitro development and nuclear reprogramming of bovine SCNT embryos.

Materials and Methods

All chemicals used in this study were purchased from the Sigma Chemical Company (St. Louis, MO, USA) unless otherwise noted. Disposable, sterile plasticware was purchased from Nunclon (Roskilde, Denmark).

All procedures in this experiment were approved by the Animal Care and Use Committee of Northwest A & F University and performed in accordance with animal welfare and ethics.

Oocytes collection and in vitro maturation (IVM)

Bovine ovaries were obtained from local slaughter houses and transported to the laboratory within 4 h after being slaughtered in sterile 0.9% NaCl saline at 15–20°C (Wang et al., 2011c) in a thermos bottle. Cumulus oocyte complexes (COCs) were aspirated from 2 to 8 mm antral follicles using a 12-gauge disposable syringe. The COCs with evenly granulated cytoplasm and enclosed by more than three layers of compact cumulus cells were selected and washed three times in TCM-199 (Gibco, Grand Island, NY, USA) and then were transferred into maturation medium [TCM-199 supplemented with 10% (v/v) fetal bovine serum (FBS), 1 μg/mL 17β-estradiol, and 0.075 IU/mL human menopausal gonadotropin], and then incubated at 38.5°C in a humidified incubator of 5% CO₂ in air for approximately 20 h.

Donor cells preparation

Donor somatic cells were derived from a 1-week-old Holstein heifer. Ear skin was collected and minced with sterile scissors in a 35-mm Petri dish. Explants (approximately 1 mm in diameter) were cultured in Dulbecco's Modified Eagle's medium (DMEM, Gibco) containing 10% FBS, 1 mM sodium pyruvate, 100 IU/mL penicillin, and 100 mg/mL streptomycin under 5% CO₂ in air at 37.5°C. Once cells reached 90% confluence, they were trypsinized and reconstituted at a concentration of 1×10⁶ cells/mL. The second to fifth passage of cell line were assayed by conventional chromosome analysis. Diploid cells with normal morphology were utilized as nuclei donors.

Somatic cell nuclear transfer (SCNT)

Nuclear transfer was performed essentially as we described earlier (Wang et al., 2011a). Briefly, matured oocytes were denuded of cumulus cells by treatment with 0.1% bovine testicular hyaluronidase in phosphate-buffered saline (PBS); only oocytes with a first polar body were selected and used for SCNT. The selected oocytes were transferred into droplets of PBS supplemented with 7.5 μg/mL cytochalasin B (CB) and 10% FBS. Enucleation was performed with a 20-μm (internal diameter) glass pipette by aspirating the first polar body and a small amount of surrounding cytoplasm. Enucleation was confirmed by staining the oocytes with 10 μg/mL Hoechst 33342 for 15 min. Enucleated oocytes were subsequently reconstructed by injecting a donor cell into the perivitelline space. The oocyte–cell couplets were vertically sandwiched by a pair of platinum microelectrodes connected to the micromanipulator in a droplet of fusion medium (0.27 M mannitol, 0.1 mM MgSO₄, 0.5 mM HEPES, and 1 mg/mL BSA). Then a double electrical pulse of 35 V for 10 μsec was applied for oocyte–cell fusion (Liu et al., 2007). Successfully reconstructed embryos were kept in mSOF (Takahashi and First, 1992), containing 5 μg/mL cytochalasin B for 2 h until activation. All fused embryos were further activated in 5 μM Ionomycin for 5 min followed by 4 h exposure to 1.9 mM 6-dimethynopyridine in mSOF.

In vitro fertilization (IVF)

After maturation, the COCs were transferred into several microdrops of Fert-Talp medium supplemented with 30 mg/mL heparin, 1.65 mg/mL hypotaurine, 0.27 mg/mL epinephrine, and 4.5 mg/mL penicillamine, and the frozen–thawed spermatozoa from a Holstein bull were used to inseminate the oocytes. Motile spermatozoa were selected by a swim-up technique (Parrish et al., 1986) and inseminated in a concentration of 1×10⁶ mL⁻¹. Oocytes and spermatozoa were incubated together for 24 h. Presumptive zygotes were denuded by treatment with 0.1% bovine testicular hyaluronidase in PBS.

In vitro embryo culture

Embryos were washed three times in the culture medium. Then they were treated with various concentrations of Scriptaid in mSOF for different durations as experimental design. After treatment, embryos are washed three times in culture medium and then cultured in mSOF medium (supplemented with 8 mg/mL fatty acid-free of bovine serum albumin (BSA), 1% MEM nonessential amino acid solution and 2% MEM essential amino acid solution) under mineral oil for 6–8 days. All incubations were done at 38.5°C in a humidified incubator of 5% CO₂ in air. Cleavage, blastocyst formation and hatching rates were recorded at 24, 144, and 192 h after IVF and SCNT.

Immunodetection of H3K9ac and H3K9m2

Embryos at different developmental stages (two-cell, eight-cell, and blastocyst) were immunostained with antibodies against acetyl-histone H3 lysine 9 and dimethyl-histone H3 lysine 9 (Abcam, Cambridge, UK). Embryos were washed in 0.2% PVA–PBS, fixed for 20 min in 4% paraformaldehyde in PBS, and permeabilized with 0.2% Triton X-100 in PBS for 30 min at room temperature (RT), and then blocked in 2% BSA in PBS overnight at 4°C. After blocking, embryos were transferred to the primary antibodies (rabbit polyclonal to histone H3 acetyl K9 or mouse monoclonal to histone H3 dimetyl K9) diluted (1:500) in blocking solution (2% BSA in PBS) overnight at 4°C. Then embryos were washed three times for 5 min each in 0.2% PVA–PBS and incubated for 2 h at RT in the presence of 1:500 diluted FITC-labeled secondary antibodies (goat anti-rabbit IgG or goat anti-mouse IgG, Beyotime, NanTong, China). After extensive washing for three times (10 min each), embryos were mounted on microscope slides using a drop of mounting medium containing 10 μg/mL of Hoechst 33342 for chromatin visualization. Experiments were repeated at least three times with 10–15 embryos in each replication. Slides were examined by epifluorescence using a Nikon eclipse Ti-S microscope equipped with a Nikon DS-Ri1 digital camera (Nikon, Tokyo, Japan). Images were individually captured and transported to computer with Nikon NIS element software. All images were captured using the same settings and without any adjustment of constant or brightness. Fluorescence was measured by analysis of the images with Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). Images were first converted to grayscale and inverted. After correcting optical density (average cytoplasmic intensity were measured for normalization to background), all individual nuclei of embryos at the two-cell and eight-cell stages, and 30 nuclei per blastocyst were outlined, integrated optical density (IOD), and area of those nuclei was measured, the average normalized fluorescence intensity for a single embryo was represented by “sum IOD/sum area.”

Number counting of nuclei in the blastocysts

Day 7 blastocysts with good morphology were selected for cell number counting. After being fixed in 4% paraformaldehyde in PBS for 15 min at RT, groups of 5 to 10 blastocysts were mounted on slides in mounting medium containing 10 μg/mL of Hoechst 33342. Slides were examined by epifluorescence using a Nikon eclipse Ti-S microscope equipped with a Nikon DS-Ri1 digital camera (Nikon, Tokyo, Japan). Images were individually captured and transported to computer with Nikon NIS element software. Total cell number of blastocysts were counted using Nikon NIS element D 8.0 software.

Quantitative real-time PCR

For each group, three to four blastocysts from the same batch were randomly chosed and pooled for Total RNA extraction. There were three replications; a total of 10 blastocysts for each group were used. Total RNA was isolated using the Cells-to-Signal^™ Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The quality and quantity of the extracted RNA were determined using an Epoch Multi-Volume Spectrophotometer System (BioTek, VT, USA), then the total RNA samples were treated with RNA-free Dnase I (Invitrogen) to digest the genomic DNA in the RNA samples, and then stored at −80°C. cDNA was produced and amplified using the cDNA synthesis kit (Takara, DaLian, China) according to the manufacturer's instructions. Primers for all genes were designed cross-intron by primer 5.0 software (Premier Biosoft International, PaloAlto, CA, USA), and were based on bovine RNA sequences found in the Genbank (Table 1). The specificity of all primers were tested using a BLAST analysis against the genomic NCBI database, PCR amplicons were characterized using Mfold (Zuker, 2003) and sequenced for verification.

Table 1.

Primer Sequences and PCR Conditions Used for Real-Time PCR

Gene	Primer sequences	Annealing temperature (°C)	Product size (bp)	Sequence accession
Oct4	F-5′-ACTCTTCGGTCCCATTCCC-3′ R-5′-CCTTCCTCTGCCCCCTATT-3′	56	121	NM_174580
IFNt	F-5′-AGGTTAAGAAAGATGGGTGGAGA-3′ R-5′-ACTGCTGACAAAGTATCGGCTAA-3′	56	167	NM_001015511
H2a	F-5′- CTCGTCACTTGCAACTTGCTATTC-3′ R-5′-CCAGGCATCCTTTAGACAGTCTTC-3′	56	148	NM_178409

All PCR reactions were performed in triplicate in a 20 μL reaction volume on the quantitative real-time PCR StepOne plus system (ABI, Carlsbad, CA, USA) using the SYBR Premix Ex Taq^™ II (Takara, DaLian, China), contain 10 μL 2×SYBR Green premix, 0.8 μL of forward and reverse primers (20 pmol/mL), respectively, 2 μL embryonic cDNA and RNase-, DNase-free water. The PCR reaction was initiated for 30 sec at 95°C to activate hot-start Taq polymerase followed by 40 cycles of denaturation at 95°C for 5 sec, then 56°C for 20 sec, extension at 72°C for 20 sec, and during which fluorescence was measured and was followed by a melt curve analysis to confirm a single gene-specific peak and to detect primer/dimer formation. A negative template control of DEPC water was used to ensure that there was no nucleotide contamination. The relative amount of each mRNA was determined using the 2^−▵▵Ct method (Rutledge and Cote, 2003) and calculated by dividing the intensity of the corresponding reference gene H2a.

Experimental design and statistical analysis

Experiment 1

SCNT embryos were treated with various concentrations of Scriptaid (0, 200, 500, or 1000 nM) for 14 h after activation. Cleavage rates (24 h), blastocyst formation rates (144 h), hatching rates (192 h), and total cell number of blastocysts (168 h) were recorded to assess the in vitro developmental capacity of each group.

Experiment 2

SCNT embryos were treated with 500 nM Scriptaid for different durations (0, 7, 14 or 21 h) after activation. Cleavage rates (24 h), blastocyst formation rates (144 h), hatching rates (192 h), and total cell number of blastocysts (168 h) were recorded.

Experiment 3

SCNT embryos treated with or without 500 nM Scriptaid for 14 h and IVF embryos (control) were collected at two-cell (24 h), eight-cell (72 h), and blastocyst (168 h) for detecting the immunofluorescent signal for histone acetylation (H3K9ac) and methylation (H3K9m2).

Experiment 4

SCNT blastocysts treated with or without 500 nM Scriptaid for 14 h and IVF blastocysts were collected for qRT-PCR. Expression levels of developmental important genes Oct4 and IFN-t were examined.

Experiments were repeated at least three times; each replicate of the experiments was carried out using oocytes matured on the same day to remove any batch effect of oocytes. All embryos were allocated randomly to each treatment group. Data expressed as proportions (percentages) were analyzed with chi-squared test using SPSS 13.0 software and data on the number of blastocyst cells, H3K9ac and H3K9m2 intensity values and expression levels of genes were analyzed by one-way ANOVA and Tukey's LSD test using SPSS 13.0 software. Differences were considered significant at p<0.05.

Results

Effect of Scriptaid on the in vitro development of SCNT embryos

As shown in Table 2, treatment of 500 nM Scriptaid signigicantly increased the cleavage rates (24 h), blastocyst formation rates (144 h), and blastocyst hatching rates (192 h) compared to the nontreated group. However, no significant differences in the cleavage rates, blastocyst formation rates, and blastocyst hatching rates were found among 200 nM, 1000 nM Scriptaid treatment groups, and nontreated group. The average number of cells in blastocysts treated with different concentrations of Scriptaid were not significantly different from that in nontreated blastocysts.

Table 2.

Effects of Concentration of Scriptaid on the In Vitro Development of Bovine SCNT Embryos

Concentration of Scriptaid (nM)	No. of Embryos cultured (replicates)	No. (%) of two-cell at 24 h	No. (%) of Blastocysts at 144 h	No. (%) of Hatched blastocysts at 192 h	Mean no.±SD of cells in blastocysts
0	165 (4)	116 (70)^b	54 (33)^b	22 (41)^b	101.2±3.2^a
200	173 (4)	124 (72)^ab	58 (34)^ab	27 (47)^ab	98.5±3.8^a
500	169 (4)	131 (78)^a	68 (40)^a	37 (54)^a	99.8±4.2^a
1000	174 (4)	124 (71)^b	60 (34)^ab	27 (45)^ab	98.4±5.6^a

Values with different superscripts (a, b) within a column are significantly different (p<0.05).

Cleavage percentage: No. of embryos cleaved/No. of embryos cultured.

Blastocyst percentage: No. of blastocysts/No. of embryos cultured.

Hatched blastocyst percentage: No. of hatched blastocysts/No. of blastocysts.

Table 3 showed that the cleavage rates, blastocyst formation rates, and blastocyst hatching rates of SCNT embryos treated with 500 nM Scriptaid for 7, 14, or 21 h were numerically higher than those from nontreated embryos, especially when treatment was performed for 14 h, which significantly increased the cleavage rate, blastocyst formation rate, and blastocyst hatching rate of embryos compared to those from nontreated embryos. Cleavage rate was also significantly increased at 21 h treatment group compared to nontreated group. However, treatment with Scriptaid for different durations had no effect on the average number of cells in blastocysts.

Table 3.

Effects of Duration of Treatment with 500 nM Scriptaid on the In Vitro Development of Bovine SCNT Embryos

Duration of treatment (h)	No. of Embryos cultured (replicates)	No. (%) of two-cell at 24 h	No. (%) of Blastocysts at 144 h	No. (%) of Hatched blastocysts at 192 h	Mean no.±SD of cells in blastocysts
0	178 (4)	127 (71)^b	56 (31)^b	23 (41)^b	101.6±3.4^a
7	164 (4)	120 (73)^ab	56 (34)^ab	25 (45)^b	100.5±4.2^a
14	163 (4)	128 (79)^a	66 (40)^a	35 (53)^a	102.2±5.8^a
21	163 (4)	126 (77)^a	59 (36) ^ab	28 (47) ^ab	99.6±2.6^a

Values with different superscripts (a, b) within a column are significantly different (p<0.05).

Cleavage percentage: No. of embryos cleaved/No. of embryos cultured.

Blastocyst percentage: No. of blastocysts/No. of embryos cultured.

Hatched blastocyst percentage: No. of hatched blastocysts/No. of blastocysts.

Effect of Scriptaid on the expression of epigenetic markers

After treatment with 500 nM Scriptaid for 14 h, the intensity of two epigenetic markers, H3K9ac and H3K9m2, at two-cell, eight-cell, and blastocyst stages of Scriptaid-treated SCNT embryos, nontreated SCNT embryos, and IVF embryos were measured. As shown in Figure 1, 500 nM Scriptaid treatment for 14 h resulted in an increased fluorescence signal for H3K9ac, but a decreased fluorescence signal for H3K9m2 in SCNT embryos at two-cell, eight-cell, and blastocyst stages compared with nontreated SCNT embryos at those three stages. The H3K9ac signal in SCNT embryos were significantly lower than in IVF embryos at two-cell, eight-cell, and blastocyst stages, after Scriptaid treatment, the signal of SCNT embryos increased to comparable levels to that of IVF counterparts. For H3K9m2, the flourescence signal in SCNT embryos were significantly higher than in their IVF counterparts at two-cell, eight-cell, and blastocyst stages, and after Scriptaid treatment, a downward trend in SCNT embryos was observed, but the reduction of fluorescence signal for H3K9m2 in Scriptaid treated SCNT embryos was not statistically significant compared with nontreated SCNT embryos at those three stages. The flourescence intensity of H3K9m2 in Scriptaid treated SCNT embryos were still significantly higher than in IVF embryos at those three stages.

FIG. 1.

Effect of Scriptaid on the expression of H3K9ac and H3K9m2 (A) and relative fluorescence intensity of H3K9ac and H3K9m2 (B). In Part A (A–I), acetylation of H3K9 (H3K9ac) in two-cell, eight-cell, and blastocyst stages SCNT embryos (treated with or without Scriptaid) and IVF embryos (control); (J–R) dimethylation of histone H3K9 (H3K9m2) in two-cell, eight-cell, and blastocyst stages SCNT embryos (treated with or without Scriptaid) and IVF embryos (control); (A′–I′, J′–R′) DNA staining of the same samples. The scale bar represents 200 μm. In Part B (black bars), Scriptaid-treated SCNT embryos; (gray bars) Scriptaid nontreated SCNT embryos; (open bars) IVF embryos. Average optical intensity was measured using Image-Pro Plus 6.0 software. The values are mean±SD. Values with different superscripts (a, b) within groups are significantly different (p<0.05).

Expression levels of developmental important genes in blastocyst

The expression levels of Oct4 and IFN-t in blastocysts were compared among Scriptaid-treated SCNT embryos, Scriptaid nontreated SCNT embryos, and IVF embryos. As shown in Figure 2, the expression levels of Oct4 and IFN-t in SCNT blastocysts were significantly lower than in IVF counterparts; after Scriptaid treatment, the expression levels of Oct4 and IFN-t were significantly improved and exhibited similar levels to their IVF counterparts.

FIG. 2.

Relative abundance of transcripts in blastocysts produced by IVF (open bars) and SCNT based on Q-PCR. Gray and black bars represent Scriptaid nontreated and treated SCNT embryos, respectively. Values with different superscripts (a, b) within groups are significantly different (P<0.05).

Discussion

Generally, the developmental defects of cloned embryos and animals were attributed to incomplete epigenetic reprogramming of donor nucleus. So HDAC inhibitors are used to improve the epigenetic modifications to increase the in vitro and even the full-term developmental capacity of SCNT embryos. TSA is the most commonly used HDACi, which was demonstrated to improve the cloning efficiency in the mouse (Kishigami et al., 2006; Rybouchkin et al., 2006), but in the other species, the results were inconsistent (Enright et al., 2003; Li et al., 2008a; Oliveira et al., 2010; Shi et al., 2008; Wang et al., 2011b; Wu et al., 2008). In the present study, we investigated the effect of Scriptaid, a novel HDACi, on the development capacity and epigenetic reprogramming of bovine SCNT embryos.

It was found that treatment with 500 nM Scriptaid for 14 h could signigicantly improve the cleavage rate, blastocyst formation rate, and hatching rate compared with the nontreated group. The results were consistent with that of previous studies performed in the pig and mice (Van Thuan et al., 2009; Zhao et al., 2009, 2010). Meanwhile, there was not any influence on the quality of blastocyst according to the total blastomere numbers, which might be attributed to the antiproliferative effects of HDACi on cells (Sambucetti et al., 1999).

It was also found that when treatment with Sciptaid for a longer time (21 h), the cleavage rate, blastocyst rate, and hatching rate were numerically higher than the nontreated group, and when using a higher concentration (1000 nM) of Scriptaid, the blastocyst rate and hatching rate were numerically higher and cleavage rate was significantly higher compared with the nontreated group, indicating that in the concentrations used, no toxic effect was observed. This result was consistent with the previous studies in mice and pigs (Van Thuan et al., 2009; Zhao et al., 2010). Conversely, treatment with TSA in a high concentration (>50 nM) or for a long time (more than 14 h) would significantly reduce the developmental capacity of preimplantation cloned bovine embryos (Wu et al., 2008), and some detrimental effects on full-term development were also found (Meng et al., 2009). This suggested that Scriptaid was more suitable in supporting the preimplantation development of bovine SCNT embryos than TSA.

H3K9 is an important site both for histone acetylation and methylation. In this study, we found that the average intensity of acetylation on H3K9 of SCNT embryos was significantly increased at two-cell, eight-cell, and blastocyst stages after Scriptaid treatment, indicating that histone acetylation of SCNT embryos could be increased to a normal degree by Scriptaid treatment. Similar results were obtained on different lysine residues including H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16 of SCNT embryos after different HDACi treatment in the previous studies (Das et al., 2010; Iager et al., 2008; Li et al., 2008a; Martinez-Diaz et al., 2010; Shi et al., 2008; Zhao et al., 2010). Meanwhile, we evaluated the intensity of H3K9m2 in the SCNT embryos. Although not significant, we observed a downward trend in average H3K9m2 intensity in Scriptaid-treated embryos compared to nontreated SCNT embryos. However, in a previous report (Martinez-Diaz et al., 2010), after treatment with TSA, a similar pattern of H3K9m2 in both treated and nontreated embryos was observed, and it was concluded that HDACi might not affect histone methylation in SCNT embryos. The discrepancy might be attributed to different species, different drugs, and the different stages of embryos evaluated. Given the evidences that dynamic interaction exists between H3K9 methylation and histone acetylation (Gilbert et al., 2007; Mutskov and Felsenfeld, 2004). Our results suggested that Scriptaid treatment could numerically reduce the intensity of H3K9m2 through increasing H3K9ac intensity.

It was known that dynamic interactions exist between histone acetylation, histone methylation, and gene transcriptions (Geiman and Robertson, 2002; Saccani and NatoLi, 2002; Struhl, 1998). Histone acetylases and deacetylases modulate transcriptional activity of specific promoters by locally perturbing chromatin structure, and increased histone acetylation could increase the transcriptional activity of genes (Struhl, 1998). Methylation of histone H3K9 is causally linked to formation of heterochromatin and to long-term transcriptional repression (Saccani and NatoLi, 2002), Therefore, decreased H3K9 methylation might also improve the transcriptional activity of genes. In order to confirm the effect of Scriptaid on gene expression of SCNT embryos, we also examined the relative mRNA abundance of two developmental important genes, Oct4 and IFN-t.

We found that Scriptaid treatment significantly improved the expression levels of Oct4 and IFN-t in SCNT blastocysts, indicating that Scriptaid could improve the transcriptional acitivity of genes in SCNT embryos. Oct4 is a key regulator of pluripotent cells across mammalian species and is supposed to be a critical factor controlling development of mammalian preimplantation embryos (Kirchhof et al., 2000; Kurosaka et al., 2004; Pesce and Scholer, 2001). It has been demonstrated that expression level of Oct4 in SCNT blastocyst was significantly lower than that in their IVF counterparts, which might lead to their compromised developmental potential (Aston et al., 2010; Kumar et al., 2007; Li et al., 2005). In the present study, Scriptaid treatment significantly increased the expression level of Oct4 in SCNT embryos, substantiating that Scriptaid improved the developmental capacity and enhanced the nuclear reprogramming of SCNT embryos. This result was consisted with the previous result that Scriptaid treatment enhanced the nuclear reprogramming of porcine SCNT embryos (Zhao et al., 2010).

IFN-t has a central role in the maternal recognition of pregnancy, and the failure to produce sufficient IFN-t leads to pregnancy loss (Demmers et al., 2001). In the present study, the expression level of IFN-t in SCNT blastocyst was increased to an equivalent expression level to that of their IVF counterparts after Scriptaid treatment, which might be linked with the higher blastocyst hatching rate.

Due to the low amount of RNA content of a single blastocyst, pooled blastocysts were used. However, pooled blastocysts for qPCR analysis might compromise intersample bias. If a single blastocyst extraction and entire cDNA amplification were evaluated instead, the results might be more precise.

In summary, this study showed that Scriptaid treatment improved the cleavage rate, blastocyst formation rate, and hatching rate of bovine SCNT embryos, increased the average intensity of H3K9 acetylation and transcriptions of Oct4 and IFN-t, and decreased the average intensity of H3K9 methylation, suggesting that Scriptaid was beneficial for the preimplantation development and nuclear reprogramming of bovine SCNT embryos. The effect of Scriptaid treatment on the full-term development of bovine SCNT embryos following embryo transfer is going on.

Footnotes

Acknowledgments

The authors are thankful to Younan Wang for providing the Holstein cow ovaries used in this study. This work was supported by grants from the National Key Project for Production of Transgenic Livestock, China (No.2008ZX-08007-004).

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

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