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Faulty epigenetic reprogramming of somatic nuclei is likely to be a major cause of low success observed in all mammals produced through somatic cell nuclear transfer (SCNT). It has been demonstrated that the developmental competence of SCNT embryos in several species were significantly enhanced via treatment of histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) to increase histone acetylation. Here we report that 50nM TSA for 10h after activation increased the developmental competence of porcine SCNT embryos constructed from Landrace fetal fibroblast cells (FFCs) in vitro and in vivo, but not at higher concentrations. Therefore, we optimized the application of another novel HDACi, Scriptaid, for development of porcine SCNT embryos. We found that treatment with 500nM Scriptaid significantly enhanced the development SCNT embryos to the blastocyst stage when outbred Landrace FFCs and ear fibroblast cells (EFCs) were used as donors compared to the untreated group. Scriptaid increased the overall cloning efficiency from 0.4% (untreated group) to 1.6% for Landrace FFCs and 0 to 3.7% for Landrace EFCs. Moreover, treatment of SCNT embryos with Scriptaid improved the histone acetylation on Histone H4 at lysine 8 (AcH4K8) in a pattern similar to that of the in vitro fertilized (IVF) embryos.
Somatic cell nuclear transfer (SCNT) to derive cloned embryos is a promising technology with potential applications in both agriculture and regenerative medicine (Campbell et al., 2007; Prather, 2007; Yang et al., 2007). It clearly indicates that epigenetic modifications accumulated in the somatic nuclei can be fully reprogrammed into the totipotent state of early preimplantation embryos, although the efficiency is still low (Tamada and Kikyo, 2004), and the mechanism by which this remodeling occurs is not known. This causes the overall cloning efficiency to be very low. In most mammalian species studied thus far, the survival rate to birth for cloned blastocysts is only about 1–5%, compared to a 30–60% birth rate for in vitro fertilized (IVF)-produced blastocysts (Wilmut et al., 2002). Accumulating evidence suggests that pigenetic reprogramming of DNA and histone in the SCNT embryo is defective, and may result in abnormal epigenetic modifications (Dean et al., 2001; Kang et al., 2001; Ohgane et al., 2004; Santos et al., 2003), and abnormal gene expression profiles also have been found in placenta and live cloned animals (Humpherys et al., 2002; Inoue et al., 2002; Jiang et al., 2008; Suemizu et al., 2003). These abnormal epigenetic modifications and gene expression patterns are likely associated with the overall low success rate of cloning (Kishigami et al., 2006).
It is thought that during SCNT, an adult somatic pattern of epigenetic modification that is normally very stable must be reversed within a short period of time after the nuclei are fused with or injected into the recipient cytoplasm but before zygotic genome activation (Zuccotti et al., 2000). Considering the reprogramming of nuclei following nuclear transfer only happens in a limited time, the relaxation of chromatin structure by histone acetylation, which corresponds to a transcriptionally permissive state, might contribute to successful cloning. It has been shown recently the HDAC inhibitor Trichostatin A (TSA) can significantly improve the mouse reproductive cloning efficiency (Kishigami et al., 2006; Rybouchkin et al., 2006) and the adult male and female outbred mice, ICR, were successfully cloned only when TSA was applied (Kishigami et al., 2007). Although TSA application resulted in great improvement in mouse somatic cloning, the effect of TSA treatment on cloning efficiency remains controversial. Beside mouse, some studies showed TSA treatment resulted in higher preimplantation embryonic development in pigs (Li et al., 2008a; Zhang et al., 2007), cattle (Ding et al., 2008; Iager et al., 2008), and rabbits (Shi et al., 2008b); however, others obtained the opposite results or thought that TSA treatment has detrimental effects on the in vitro development of SCNT embryos. Meng et al. (2009) found the offspring from TSA-treated embryos died within an hour to 19 days while four rabbit pups of the TSA-untreated group have grown into adulthood, and three of them produced offspring. Wu et al. (2008) also reported that cells treated with 50ng/mL TSA resulted in significantly lower blastocyst development (9.9 vs. 20%) in bovine. In addition, TSA is known to be teratogenic (Svensson et al., 1998) and results in a significant reduction of embryo development (Van Thuan et al., 2009) as well as the success rates of cloning (Svensson et al., 1998; Tsuji et al., 2009) as well as more severe placentomegaly (Kishigami et al., 2006) when the concentration is high or exposure is long (more than 14h). Therefore, it is necessary to investigate if the HDACi treatment has beneficial effects on somatic cloning in species other than the mouse, and especially the effect on the full-term development. In this study we investigated the effect of Scriptaid, an HDACi with low toxicity that enhances transcriptional activity and protein expression (Su et al., 2000) during SCNT. Scriptaid treatment of highly inbred NIH miniature pig (Zhao et al., 2009) and inbred mice (C57BL/6, C3H/He, DBA/2 et al) (Van Thuan et al., 2009) after SCNT improves the clonability of these embryos. The objective of this study was to investigate, optimize, and compare the application of TSA and Scriptaid on the reprogramming of somatic nuclei following SCNT using Truline® Landrace donor cells and to verify the action of Scriptaid.
All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified. All of the solutions and media were filtered by a 0.22-μm filter.
Landrace fetal fibroblast cells (FFCs) and ear fibroblast cells (EFCs) were established as previously described (Lai and Prather, 2003).
Briefly, 35-day-old fetuses or ear notch from 1-week-old piglets were recovered and rinsed three times with DPBS. After removal of the head, intestine, liver, limbs, and heart, the remaining tissues of day 35 fetus were finely minced into pieces (1mm3) using scissors in DPBS. The ear notch was cleaned, the hair removed, and minced into pieces. Minced tissue were then digested with Collagenase (200U/mL) and Dnase I (25K U/mL) in Dulbecco's Modified Eagle Medium (DMEM) plus 15% fetal bovine serum (FBS; Hyclone, Logan, UT) for 4–5h at 38.5°C and 5% CO2 in air. After digestion and rinsed, the cell pellet was seeded in a 75-cm2 culture flask and left to culture until confluent. After confluence, cells were frozen in FBS containing 10% DMSO. One day or 2 days earlier before nuclear transfer, cells were thawed, cultured, and subsequently used between passages 1–4. Single cell suspension was prepared by trypsinization of the cultured cells and then resuspension in oocyte manipulation medium (25mM HEPES-buffered TCM199 with 3mg/mL BSA) prior to SCNT.
The oocytes used in this study were from two different resources. For in vitro development experiments, the oocytes were collected and matured as previously described (Hao et al., 2004). Briefly, ovaries were collected from prepubertal gilts at slaughterhouse and transported to the lab at 37°C. The cumulus–oocyte complexes (COCs) were aspirated from the follicles in the size between 3–6mm with an 18-gauge needle attached to a 10-cc syringe. Only the COCs with multiple layers of intact cumulus cells and uniform cytoplasm were selected and rinsed three times in TL–HEPES containing 0.1% (w/v) polyvinyl alcohol (PVA) for maturation. A group of 70–80 COCs were placed into 500μL of maturation medium (TCM 199; Gibco BRL Grand Island, NY) supplemented with 0.1% polyvinylalcohol (PVA) (w/v), 3.05mM D-glucose, 0.91mM sodium pyruvate, 1μg/mL Gentamicin, 0.57mM cysteine, 0.5μg/mL luteinizing hormone, 0.5μg/mL follicle stimulating hormone and 10ng/mL epidermal growth factor) in the four-well plate (Nunc, Roskilde, Denmark) cover with mineral oil. COCs were matured for 44h (if used for IVF) or 40h (if used for SCNT) at 38.5°C and 5% CO2 in air, 100% humidity.
To determine the in vivo development of SCNT embryos, maturing oocytes from sows were purchased from ART Inc. (Madison, WI) and shipped to the lab overnight in their commercial maturation medium #1. Twenty-four hours after being placed in the maturation medium #1 (provided by ART), the oocytes were transferred to fresh medium #2 and cultured for a total of 40h of maturation.
After maturation, COCs were vortexed in HEPES-buffered Tyrode's medium containing 0.1% hyaluronidase and 0.01% PVA for 4min to remove the cumulus cells. Only the oocytes having an extruded first polar body (PB) with uniform cytoplasm were used for the recipient of SCNT. MII oocytes were placed in the manipulation medium supplemented with 7.5μg/mL cytochalasin B and enucleated by aspirating the PB and MII chromosomes and a small amount of surrounding cytoplasm using a 17–20-μm beveled glass pipette. A single donor cell was introduced into the perivitelline space and placed adjacent to the recipient cytoplasm. The resulting karyoplast–cytoplast complexes (KCCs) were placed in fusion medium (0.3M mannitol, 1.0mM CaCl2, 0.1mM MgCl2, and 0.5mM HEPES, pH adjusted to 7.0–7.4) and subjected to 2 DC pulses (1-sec interval) of 1.2kV/cm for 30μsec provided by a BTX Electro-cell Manipulator 200 (BTX, San Diego, CA) for the fusion/activation. Couplets were then washed and incubated for 20min in Porcine Zygote Medium-3 (PZM3, pH 7.3) (Lai and Prather, 2003) supplemented with 3mg/mL BSA medium and evaluated for fusion under a stereomicroscope. Reconstructed embryos were cultured in four-well cell culture plates containing 500μL of PZM3 medium at 38.5°C and 5% CO2 in humidified air.
IVF was carried out as previously described (Hao et al., 2006). Briefly, MII oocytes were washed three times in the fertilization medium [modified Tris-buffered medium (mTBM) containing 2mg/mL BSA and 2mM caffeine]. Approximately 30–35 oocytes were transferred into 50μL droplets of fertilization medium covered with mineral oil that had been equilibrated for 4h at 38.5°C in 5% CO2 in air. A 0.1-mL frozen semen pellet was thawed at 38.5°C in 10mL sperm washing medium [Dulbecco phosphate-buffered saline (DPBS, Gibco BRL Grand Island, NY)] supplemented with 1mg/mL BSA (pH 7.3). After washing twice by centrifugation (1900×g, 4min), spermatozoa were resuspended with fertilization medium to a concentration of 1×106cells/mL. Fifty microliters of the sperm suspension solution was added to the fertilization droplets, giving a final sperm concentration of 0.5×106cells/mL. The incubation of oocytes and sperm was for 4–6h. After fertilization, oocytes were washed three times and cultured in 500μL PZM3 medium in four-well Nunclon dishes at 38.5°C in 5% CO2 in air.
Stock solutions of TSA and Scriptaid were dissolved in dimethyl sulfoxide (DMSO) at 100nM and 1mM, respectively, and stored at −20°C. Following electrical activation or fertilization the SCNT or IVF embryos were treated with various concentrations of TSA for 10h Scriptaid for 14–16h in PZM3. After treatment, embryos were washed three times before transferring into a four-well cell culture plate containing 500μL PZM3 medium, and then cultured at 38.5°C in 5% CO2 in humidified air for either over night or 6 days. Cleavage and blastocyst formation were evaluated on days 2 and 6, respectively, with the day of SCNT or IVF designated day 0.
Day 1 SCNT zygotes (more than 100) were transferred to the oviducts of surrogates on the day of, or 1 day after, the onset of estrus. Pregnancy was diagnosed on Day 25 (day 0 was the day of SCNT), and then was checked regularly at 2-week intervals by ultrasound examination. All of the cloned piglets were delivered by cesarean section on day 117 of gestation and hand raised. All animals were treated according to preapproved institutional animal care and use protocol.
For day 6 embryos derived from IVF, zonae pellucidae were removed by Pronase treatment to eliminate the attached sperm. FFCs used for indirect immunofluorescence were cultured on an eight-well glass surface chamber (Nunc, Rochester, NY) and treated with 500nM Scriptaid for 14–16h. Embryos and cells were washed in PBS, fixed for 15min in 4% paraformaldehyde in PBS, and permeabilized with 0.2% Triton X-100 in PBS for 30min at room temperature (RT). Samples were blocked overnight at 4°C in 1% BSA in PBS (blocking solution). The samples were stained with a rabbit polyclonal antibody to AcH4K8 (Millipore, Bedford, MA) diluted 1:250 overnight at 4°C. After extensive washing, samples were treated with a secondary antibody of Alexa Fluor 594 goat anti rabbit IgG (Molecular Probe, Eugene, OR) diluted in 1:800 for 2h at RT. After washing three times with PBS with 0.1% polyvinylpyrrolidone, embryos and cells were mounted on slides in mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratory Inc., Burlingame, CA). Day 6 blastocysts with good morphology were selected for cell number comparison. After being fixed in 4% paraformaldehyde in PBS for 15min at RT, embryos were mounted on slides in mounting medium containing DAPI. Groups of embryos stained without primary antibody or secondary antibody or both antibodies were used as negative controls to examine the specificity of the reaction. At least 10 oocytes/embryos were processed for each treatment group and the experiments were replicated three times. Slides were analyzed under an epifluorescent microscope (Nikon, Tokyo, Japan) equipped with a digital camera. Images were captured and quantified using Nikon NIS element software. To make relative comparisons, we kept settings for exposure and image capture constant and all images were assembled without any adjustment of contrast or brightness to the images.
SCNT embryos using Landrace FFCs as the donor cells were treated with various concentrations of TSA (0, 5, 50, 100, or 200nM) for 10h after activation. The MII oocytes from the same batch without treatment were in vitro fertilized and used as an additional control group. Day 2 cleavage and day 6 blastocyst rates were used to evaluate in vitro development and day 1 SCNT zygotes were transferred to the oviducts of surrogates on the day of, or 1 day after, the onset of estrus for testing subsequent developmental potential.
SCNT embryos using Landrace FFCs as the donor cells were treated with Scriptaid at various concentrations (0, 250, 500, or 1000nM) for 14–16h after activation. Developmental rates to the two-cell and blastocyst stage were determined on day 2 and 6. Full-term development was also tested by transferring day 1 SCNT zygotes to surrogates.
SCNT embryos and Landrace FFCs treated without (Control) or with 500nM Scriptaid for 14–16h were collected at the end of treatment for detecting the acetylation level on AcH4K8.
Experiments were repeated at least three times, and data expressed as proportions (percentages) were analyzed with chi-squared tests and data on the number of cells as well as signals of AcH4K8 intensity were analyzed by analysis of variance (ANOVA) using SAS 6.12.
Landrace FFCs (female, passage 1–4) were used as donors, and enucleated oocytes at the Metaphase II (MII) stage were used as recipients, for making cloned embryos. After activation, SCNT embryos were treated with TSA at 0, 5, 50, 100, or 200nM in embryo culture medium for 10h. Preimplantation development of SCNT and fertilized embryos was examined from the two-cell to the blastocyst stage (Table 1). Treatment of 50nM TSA increased the percentage blastocyst (p<0.05) compared to the untreated group. However, TSA had no effect on percentage cleavage at 24 and 48h and blastocyst quality judged by total cell number.
Embryo transfer results demonstrated a doubling of the cloning efficiency in the TSA treatment group (0.8 vs. 0.4%, Table 2), reflecting the increased blastocyst development during culture in vitro. Three of four piglets from the TSA treatment group died of heart failure (a hole was found on the heart by necropsy) 2–3 weeks after birth, and one of the three was also found to have a fever, septic infection, and was lethargic; the other one is healthy and is currently 5 months old. The piglet from the untreated group couldn't walk and was euthanized at 2 weeks of age.
The same Landrace FFCs as for Experiment 1 were used for the reconstruction of SCNT embryos to optimize the concentration of Scriptaid. After activation, SCNT embryos were treated with various concentrations of Scriptaid at 0 (control), 250, 500, or 1000nM for 14–16h. The development of SCNT embryos treated with 500nM Scriptaid was higher than the untreated group (p<0.05) (Table 3). However; similar to TSA, Scriptaid treatment had no effect on 48h cleavage or the quality of the blastocysts as determined by total cell number relative to the IVF, untreated SCNT, and Scriptaid treated SCNT embryos. Because SCNT embryos treated with 500nM Scriptaid resulted in the highest developmental potential to blastocyst in vitro, 500nM Scriptaid for 14–16h after activation was used for all subsequent experiments. Scriptaid treated day 1 SCNT zygotes, which reconstructed with three landrace cell lines from three different individuals (one male FFCs, one male EFCs, and one female EFCs), were transferred to surrogates to test the full-term development. Five of five surrogates became pregnant in the Scriptaid treatment group when male landrace FFCs were used as donors and 13 piglets were born from five litters with cloning efficiency of 1.6% (Table 2). Ten of 13 piglets are healthy (2 died 2 days after birth, whereas the other one could not walk and was euthanized soon after birth). When landrace EFCs were used as donors, two of two surrogates became pregnant and delivered in the Scriptaid treatment group but no pregnancy was established in the Scriptaid untreated group. Ten healthy piglets were born from these two litters with a cloning efficiency of 3.7%.
To provide a mechanism for the improved development of the SCNT embryos after Scriptaid treatment we measured the level of histone acetylation in one-cell stage embryos. No signals could be detected in the embryos or cells stained without primary or secondary antibodies suggesting specificity of staining of the primary antibody (data not shown). As shown in Figure 1, treatment of 500nM Scriptaid for 14–16h increased acetylation level as determined by acetylation of histone 4 at lysine residue 8 (AcH4K8) in the “pro”-nucleus of SCNT embryos and donor cells when compared to the untreated group. To confirm the difference, average optical intensity of AcH4K8 was measured using Nikon NIS element software. The results displayed significant difference in AcH4K8 level between Scriptaid treatment group and control in both cells and embryos (Fig. 2, p<0.01). Scriptaid treatment made the histone acetylation level in SCNT embryos more similar to the IVF cohort.
In the present study, we investigated the effect of TSA and Scriptaid on the in vitro and in vivo development potential of SCNT embryos using various landrace FFCs and EFCs. We found that treatment with a novel HDACi, Scriptaid, enhanced the developmental potential of reconstructed embryos in vitro and in vivo. This may be a result of improved reprogramming from increased histone acetylation in the somatic nuclei.
In Experiment 1, the development of SCNT embryos to the blastocyst stage after treatment with TSA after activation was twice than that of untreated groups. There was no difference of embryo quality as judged by total cell number at the blastocyst stage. From the conditions used in this study, we found treatment of 50nM for 10h achieved the best results in the SCNT embryos. Embryo transfer results showed that TSA treatment can produce viable cloned piglets and, although not significant, numerically increased the cloning efficiency from 0.4 to 0.8%.
In addition to TSA, Scriptaid is a novel HDAC inhibitor that belongs to an existing class of hydroxamic acid-containing HDAC inhibitors (Su et al., 2000). When 500nM Scriptaid was used on SCNT embryos using same donor cells as Experiment 1, the blastocyst rate was more than twice as much as those of untreated groups (25 vs. 11%). Embryo transfer results showed that the cloning efficiency was 1.6 or 3.7% in the Scriptaid treatment group when FFCs or EFCs used as donor cells, respectively (Table 2). However, the cloning efficiency in the untreated group when FFCs or EFCs were used as donor cells is only 0.4 or 0%, respectively (Table 2). In total, 23 piglets were obtained from seven litters of the Scriptaid treatment group. Thus, both Scriptaid and TSA teatment improve the developmental potential of SCNT embryos.
We observed some abnormalities during the cloning treated with HDACi in the current study, 3 of 4 cloned piglets died of heart failure in the TSA treatment group, and 3 of 23 from the Scriptaid treatment were abnormal. Despite some abnormal piglets from the TSA and Scriptaid treatment, it is difficult to make any conclusions about abnormalities caused by HDACi because a significant percentage of cloned offspring are normal and healthy and a piglet from the control TSA group was also abnormal. However, it still needs to note that there was a higher percentage (75%, three of four pigs) of abnormalities in the TSA treatment group.
Some cloned animals with abnormal phenotypes that reach sexual maturity can be naturally bred, and all of their offspring show normal phenotypes, suggesting that the abnormalities of the clones are due to epigenetic aberrancies rather than genetic mutations (Prather, 2006; Tamashiro et al., 2002). Besides DNA methylation, histone modification (acetylation, methylation, phosphorylation, and ubiquitination) is another important epigenetic modification to the chromatin structure. Acetylation, the introduction of an acetyl group, usually occurs on the lysine residues of core histones (Kouzarides, 2007; Surani et al., 2007; Wang et al., 2007). Changes in DNA methylation, histone methylation, and histone acetylation are tightly linked to the transcriptional state of genes in those modified regions (Armstrong et al., 2006; Li, 2002). Histone acetylation–deacetylation is a dynamic process during embryogenesis and differentiation, and is related to transcriptional regulation by the status of the chromosome structure. Histone acetylation emerges as a central switch that allows interconversion between permissive and repressive chromatin structures and domains. Increased histone acetylation levels on most amino acid residues leads to relaxed binding of the nucleosome to DNA and/or linker histones, relaxation of the chromatin structure, and formation of a transcriptionally permissive state (Hebbes et al., 1988; Hong et al., 1993; Lee et al., 1993; Zlatanova et al., 2000). Histone deacetylation, frequently followed by histone methylation, establishes a base for highly repressive chromatin structures, such as heterochromatin (Eberharter and Becker, 2002). These principles are not only the heart of transcriptional regulation but are also likely to govern other processes involving chromatin substrates, including replication, site-specific recombination, and DNA repair (Roth et al., 2001; Wolffe and Hayes, 1999). Thus, transcriptional activation within a permissive domain frequently correlates with additional targeted acetylation of histone at promoter nucleosomes (Brown et al., 2000; Forsberg and Bresnick, 2001).
Trichostatin A can induce the hyperacetylation of the core histones, resulting in structural changes in the chromatin that permit transcription. These changes include decreasing DNA methylation and thus activation of genes, which are key for development (Cervoni and Szyf, 2001), and we believe the Scriptaid may have a similar function. Scriptaid treatment could increase the AcH4k8 intensity in both the “pro”-nuclear area of SCNT embryos and also somatic donor cells (Figs. 1 and and2).2). However, the intensity AcH4K8 in the untreated SCNT embryos was lower when compared to IVF cohort embryos. After treatment with Scriptaid for 14–16h, the histone acetylation level is increased in the SCNT embryos where it more closely resembles the IVF embryos. Similar results also have been observed in several studies that treatment of SCNT embryos with HDACi altered the histone acetylation in a manner similar to that in normal embryos (Iager et al., 2008; Shi et al., 2008a; Wang et al., 2007). TSA treatment produced eight-cell stage bovine embryos with levels of acetylation on histone H4 at lysine 5 (AcH4K5) similar to fertilized counterparts and significantly greater than in control SCNT embryos (p<0.05) (Iager et al., 2008). Similar results after TSA treatment in histone acetylation of SCNT embryos were also observed in the mouse (Wang et al., 2007) and rabbit (Shi et al., 2008a). Thus, the histone acetylation of a somatic genome in the cloned embryos may not be sufficient, and HDACi treatment increased the histone acetylation level, which induces a more open chromatin configuration, allowing access to transcription factors (Li, 2002) and enhancing the DNA demethylation of the somatic nuclei after SCNT, which is a necessary step of nuclear reprogramming (Armstrong et al., 2006; Simonsson and Gurdon, 2004). One study showed that treatment of cloned embryos with TSA causes them to transcribe mRNA similar to in vivo derived embryos (Li et al., 2008b). Additionally, valproic acid, another HDAC inhibitor, improves reprogramming efficiency of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells by more than 100-fold over the untreated group (Huangfu et al., 2008a, 2008b).
In this study, we focused on the application and optimization of a novel HDACi, Scriptaid, on improving pig cloning efficiency and partially explained the mechanism. However, further studies are still needed to elucidate which cluster of genes are affected by HDACi treatment, thus improving the cloning efficiency. The effect of Scriptaid on the cloning efficiency in other species should also be investigated.
In conclusion, Scriptaid improves the in vitro and in vivo development of pig SCNT embryos, and could produce healthy cloned offspring. We also suggest that epigenetic reprogramming of the somatic nuclei after SCNT is deficient and hypoacetylation of the core histone might be a limiting factor for successful reprogramming.
The authors thank all the members in both Dr. Prather's lab and the National Swine Resource and Research Center (NSRRC). We appreciate funding from the National Institutes of Health National Center for Research Resources RR018877 and RR013438.
The authors declare that no conflicting financial interests exist.