Effect of Cell Cycle Interactions and Inhibition of Histone Deacetylases on Development of Porcine Embryos Produced by Nuclear Transfer

Abstract

The aim of this study was to evaluate if the positive effects of inhibiting histone deacetylase enzymes on cell reprogramming and development of somatic cell nuclear transfer (SCNT) embryos is affected by the cell cycle stage of nuclear donor cells and host oocytes at the time of embryo reconstruction. SCNT embryos were produced with metaphase II (MII) or telophase II (TII) cytoplasts and nuclear donor cells that were either at the G_1–0 or G₂/M stages. Embryos reconstructed with the different cell cycle combinations were treated or not with the histone deacetylase inhibitor (HDACi) Scriptaid for 15 h and then cultured in vitro for 7 days. Embryos reconstructed with MII-G_1–0 and TII-G₂/M developed to the blastocyst stage with a higher frequency compared to the other groups, confirming the importance of cell cycle interactions on cell reprogramming and SCNT embryo development. Treatment with HDACi improved development of SCNT embryos produced with MII but not TII cytoplasts, independently of the cell cycle stage of nuclear donor cells. These findings provide evidence that the positive effect of HDACi treatment on development of SCNT embryos depends upon cell cycle interactions between the host cytoplast and the nuclear donor cells.

Introduction

Somatic cell nuclear transfer (SCNT) has been applied to study cellular reprogramming and also to produce cloned animals. Indeed, since the first cloned animal was generated from a somatic cell (Wilmut et al., 1997), many species have been cloned by SCNT, including mice (Wakayama et al., 1998), cattle (Cibelli et al., 1998), swine (Polejaeva et al., 2000), and other domestic and wild species (Meissner and Jaenisch, 2006). In swine, SCNT has been used mainly to produce genetically modified animal models for research given the physiological similarities of swine with humans (Wolf et al., 2014). However, SCNT efficiency remains very low, likely due to incomplete cellular reprogramming (Krishnakumar and Blelloch, 2013; Rodriguez-Osorio et al., 2012).

Cell reprogramming in SCNT embryos is regulated mainly by epigenetic events. Histone modifications and DNA methylation are the main factors involved in the reprogramming of a somatic cell into a pluripotent state (Morgan et al., 2005). Indeed, aberrant DNA and histone methylation (Kang et al., 2001; Santos and Dean 2004) and histone acetylation (Zhao et al., 2010b) patterns have been described in SCNT embryos when compared to control fertilized embryos. Epigenetic abnormalities can result in abnormal gene expression during early embryo development, which may compromise the health and viability of the cloned animals (Rideout et al., 2001).

Histone deacetylase inhibitors (HDACi) have been used to improve epigenetic reprogramming after SCNT in many species, including mice (Bui et al., 2010; Kishigami et al., 2006; Van Thuan et al., 2009), bovine (Wang et al., 2011), swine (Mao et al., 2015; Martinez-Diaz et al., 2010; Zhao et al., 2010a), and ovine (Wen et al., 2014) embryos. The acetylation status of SCNT embryos that were treated with the HDACi Scriptaid was shown to be similar to control fertilized embryos (Wen et al., 2014). Treatment of SCNT embryos with HDACi was shown to promote histone hyperacetylation and increase gene expression (Kretsovali et al., 2012), to alter DNA methylation (Xu et al., 2013), and to enhance DNA damage repair (Bohrer et al., 2014).

Besides epigenetic reprogramming, the cell cycle stage of nuclear donor cells and host oocytes is known to affect the development of SCNT embryos. Metaphase II (MII) stage oocytes are normally used as cytoplasts for SCNT. However, telophase II (TII) stage oocytes can also promote cell reprogramming after nuclear transfer (Baguisi et al., 1999; Bordignon and Smith, 1998, 2006; Chen et al., 2007). The coordination between the cell cycle stage of cytoplasts and nuclear donor cells is thought to be particularly important for maintaining normal ploidy in SCNT embryos (Campbell et al., 1996). It has been proposed that when MII oocytes are used as cytoplasts, the high activity of the maturation-promoting factor (MPF) induces nuclear envelope breakdown (NEBD) and causes a premature condensation of the chromosomes (PCC), which may result in a new cycle of DNA replication after the reformation of the nuclear envelope (Campbell et al., 1993). In this context, G₁/G₀ cells are better suited as nuclear donor cells; otherwise, the ploidy of the reconstructed embryo may be compromised.

On the other hand, when preactivated oocytes are used as cytoplasts, MPF levels are low and insufficient to promote NEBD and PCC. Hence, cells at any stages of the cell cycle could be transferred to preactivated oocytes without compromising the ploidy of the reconstructed embryos (Campbell and Alberio, 2003). Nevertheless, it has been proposed that the exposure of the donor chromatin to cytoplasmic factors after NEBD and PCC is essential for nuclear remodeling (Fulka et al., 1996). On the basis of this premise, MII cytoplasts would have a superior ability to promote nuclear reprogramming than preactivated cytoplasts. However, a recent study showed that the cytoplasm of interphase two-cell stage mouse embryos is able to reprogram somatic cells, but the proper coordination of the cell cycle between the host cytoplasm and the nuclear donor cell is crucial for successful development (Kang et al., 2014). This study indicates that nuclear reprogramming ability is not only present in interphase cytoplasts but also maintained after the first cell division.

In light of these previous findings, our objective in this study was to investigate if the effect of HDACi treatment on promoting cell reprogramming and development of SCNT embryos is affected by cell cycle stage interactions between the nuclear donor cells and the host cytoplasts.

Material and Methods

Chemicals

Unless otherwise indicated, chemicals and reagents were purchased from Sigma Chemical Company (Sigma-Aldrich, St. Louis, MO, USA).

Oocyte collection and in vitro maturation

Ovaries of prepubertal gilts were collected at a local slaughterhouse and transported to the laboratory in a saline solution (0.9% NaCl) at 30–35°C containing penicillin and streptomycin (100 UI/mL and 50 μg/mL, respectively). Cumulus–oocyte complexes (COCs) were aspirated from follicles ranging from 3 to 6 mm in diameter using a vacuum pump (vacuum rate of 15 mL water/min). COCs surrounded by a minimum of three cumulus cells layers and having a homogeneous granulated cytoplasm were selected for in vitro maturation (IVM). Groups of 35–40 COCs were cultured in 400 μL of maturation medium in four-well culture plates and in a humidified atmosphere of 5% CO₂ and 95% air at 38.5°C.

Maturation medium consisted of Tissue Culture Medium-199 (TCM-199; Life Technologies, Carlsbad, CA, USA), supplemented with 20% porcine follicular fluid, 1 mM of dibutyryl cyclic adenosine monophosphate (dbcAMP), 0.1 μg/mL cysteine, 10 ng/mL epidermal growth factor (EGF; Life Technologies), 0.91 mM sodium pyruvate, 3.05 mM d-glucose, 0.5 μg/mL luteinizing hormone (LH; Lutropin-V, Bioniche, Ontario, CA, USA), 0.5 μg/mL follicle-stimulating hormone (FSH; Folltropin-V, Bioniche, Ontario CA, USA), and 20 μg/mL gentamicin. After 22–24 h of maturation, oocytes were transferred to the same IVM medium, but without LH, FSH, and dbcAMP for an additional 20–22 h under the same conditions. After IVM, cumulus cells were removed by vortexing in TCM-199 HEPES-buffered medium (Life Technologies) supplemented with 0.1% hyaluronidase to prepare the oocytes for SCNT.

Nuclear donor cells culture and cell cycle synchronization

Porcine fibroblast cells were cultured in vitro in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 Ham (DMEM-F12), supplemented with 10% fetal bovine serum (FBS; Life Technologies) and 1% antibiotics (10,000 U/mL penicillin and 10,000 μg/mL streptomycin) at 37°C in 5% CO₂ and 95% air.

To obtain cells at the G_1–0 stage, fibroblasts were maintained in culture for at least 48 h after reaching confluence. To obtain cells at the G₂/M stage, fibroblasts were maintained in culture until reaching confluence and then used to prepare nonconfluent cultures by plating 50,000 cells per well on a six-well plate using 2 mL of culture medium. The cells were then trypsinized and fixed for cell cycle analysis at 0, 16, 20, 24, 28, 32, and 36 h after plating. The experiment was repeated three times.

Cell cycle analysis by flow cytometry

The cell culture medium was removed and cells were rinsed with phosphate-buffered saline (PBS). Cells were then trypsinized (0.25% trypsin-EDTA) and resuspended in ice-cold fixation solution (70% ethanol and 30% PBS) for 15 min. Fixed cells were then pelleted by centrifugation and resuspended in PBS until flow cytometry analysis. Before analysis, cells were resuspended in 1 mL of PBS containing 50 μg of propidium iodide and 100 μg of RNase at 37°C for 40 min. The DNA content of 10,000 cells was assessed by fluorescence-activated cell sorting (FACS) using a FACSVerse system (BD Biosciences, San Jose, CA, USA). The percentage of cells at G_1–0, S, or G₂/M was calculated using the FACSsuite Software (BD Biosciences).

Assessment of DNA and RNA synthesis in nuclear donor cells and reconstructed embryos

Detection of DNA and RNA synthesis was performed using the Click-iT^® EdU and Click-iT^® EU RNA Imaging Kits (Invitrogen, Life Technologies). Cells that were either confluent for more than 48 h or nonconfluent for 24–28 h after passage from confluent cultures were incubated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU; DNA synthesis) or 1 mM 5-ethynyluridine (EU; RNA synthesis) for 2 or 4 h. Cells were then fixed in 4% paraformaldehyde and stained according to the manufacturer's instructions. Samples were mounted on microscope slides, and DNA and RNA synthesis was evaluated using an epifluorescence microscope (DMI 4000B, Leica). To detect DNA and RNA synthesis in SCNT embryos, samples were incubated with either EdU or EU for 4 h starting at 4 or 8 h postfusion (hpf). DNA synthesis was quantified by assessing the grayscale signal of the nuclear area using the LAS AF software (Leica Microsystems). Nuclear diameter after staining with 10 μM Hoechst 33342 was also measured using the LAS AF software. The experiments were repeated three times.

Production of host cytoplasts, nuclear transfer, and embryo culture

Two different procedures were used to enucleate oocytes at MII (MII cytoplasts) and TII (TII cytoplasts). For MII cytoplasts, cumulus-free oocytes with a polar body were cultured in TCM-199 supplemented with 0.4 μg/mL demecolcine and 0.05 M sucrose for 60 min. This treatment resulted in a small protrusion in the ooplasmic membrane that contained the metaphase chromosomes. To prepare TII cytoplasts, cumulus-free oocytes were treated with 15 μM ionomycin for 5 min, and then cultured in Ca²⁺- free porcine zygote medium (PZM-3) supplemented with 10 μg/mL cycloheximide and 10 mM strontium chloride for 2 h. This treatment promoted oocyte activation and the extrusion of the second polar body. Either MII or TII oocytes were then transferred to TCM-199 HEPES-buffered medium supplemented with 2 mg/mL bovine serum albumin (BSA, fatty acid free), 20 μg/mL gentamicin, and 7.5 μg/mL cytochalasin B for 5–10 min and enucleated by removing the protruded chromatin and the first polar body (MII cytoplast) or the two polar bodies (TII cytoplasts).

A nuclear donor cell, synchronized either at the G_1–0 or G₂/M stage, was transferred into the perivitelline space of each enucleated oocyte and then fused electrically using a single DC pulse of 35 V for 50 μsec. Electrofusion was performed in a 0.28 M mannitol solution supplemented with 50 μM CaCl₂, 100 μM MgSO₄, and 0.1% BSA. Oocytes were then transferred to TCM-199 medium supplemented with 3 mg/mL BSA for 1 h to allow cell fusion.

Oocytes reconstructed with MII cytoplasts were exposed to ionomycin (15 μM) for 5 min and then transferred to Ca^2+- free PZM-3 supplemented with 10 mM strontium chloride, cytochalasin B (7.5 μg/mL), and cycloheximide (10 μg/mL) for 4 h. Oocytes reconstructed with TII cytoplasts were placed directly in PZM-3 after fusion. To assess the effect of HDACi treatment, a proportion of the oocytes was cultured in the presence of 500 nM Scriptaid for 15 h starting after ionomycin treatment (MII cytoplasts) or after fusion (TII cytoplasts). Both treated and control embryos were then washed in PZM-3 medium supplemented with 3 mg/mL BSA and cultured in a humidified atmosphere of 5% CO₂ and 95% air at 38.5°C. Culture medium was supplemented with 10% FBS on day 5. Cleavage rates were determined 48 h after nuclear transfer and blastocyst rates on day 7. Seven experimental replicates were performed using G_1–0 cells and six using G₂/M cells. All four groups tested were studied on the same replicate for each nuclear donor cell stage.

Embryo evaluation and cell counting

Embryos that developed to the blastocyst stage after 7 days in culture were separated, rinsed in PBS containing 0.1% polyvinyl alcohol (PBS-PVA), fixed for 15–20 min in 4% paraformaldehyde, and then stored in PBS containing 0.3% BSA and 0.1% Triton X-100 at 4°C. Nuclei were stained by exposing the embryos to 10 μM Hoechst 33342 for 10 min. Embryos were then mounted into slides using Mowiol, and nuclei were counted in each embryo using an epifluorescence microscope (DMI 4000B, Leica).

Statistical analysis

Data of cell cycle synchronization, nuclear diameter, DNA and RNA quantification signal, and number of cells at the blastocyst stage were analyzed by analysis of variance (ANOVA) followed by the LSMeans Student t-test using the JMP software (SAS Institute Inc., Cary, NC). Data were tested for normal distribution using the Shapiro–Wilk test and normalized when necessary. Results are presented as means ± standard error of the mean (SEM). Embryo cleavage and blastocyst rates were analyzed by a randomized complete block design (RCBD) to evaluate the effect of presence or absence of Scriptaid on embryo development. A single replication was considered a block with four treatment groups. To perform data analyses by parametric test, percentages were transformed by arcsine before being analyzed by ANOVA.

Results

Cell cycle synchronization and detection of DNA and RNA synthesis

To synchronize nuclear donor cells at the G_1–0 stages for use in the first experiment, cells were maintained in culture for at least 48 h after reaching confluency. The proportion of cells in G_1–0 (88.2 ± 4.3%), S (2.7 ± 1.9%), and G₂/M (9.1 ± 2.8%) confirmed that most of the cells were at the expected phase of the cell cycle (Fig. 1A, C, 0 h). To reduce the risk of using cells that were not at the G_1–0 stages, only the smaller cells (mean diameter = 13.9 μm), which represented the majority of cells in this experiment (Fig. 1B, arrowhead), were used for SCNT in the first experiment. Cell diameter was calculated using the LAS AF software, and smaller cells were chosen for nuclear transfer by visual comparison to the internal diameter of the injection pipette, which was ∼20 μm.

FIG. 1.

Synchronization of nuclear donor cells for SCNT. (A) Representative histogram showing the cell cycle distribution of cells that were maintained in culture for 48 h after reaching confluence. (B) Confluent cells after trypsinization. Only the smaller cells (arrowhead) were used for SCNT. (C) Proportion of cells at the different stages of the cell cycle from samples collected at different times after passage from confluent cultures. (D) Cells trypsinized at 24 h after plating from a confluent culture. Only the larger cells (black arrow) were used for SCNT.

To obtain G₂/M cells for the second experiment, confluent cells were trypsinized and plated in a nonconfluent density, which allows for a synchronized wave of cells resuming their cell cycle and progressing to S and then G₂ phase. As shown in Figure 1C, more than 30% of the cells have reached the G₂/M phases at 24–28 h after passage from confluent cultures. Only the larger cells (mean diameter = 25.9 μm) collected between 24 and 28 h after plating from confluent cultures (Fig. 1D, black arrow) were used for SCNT in the second experiment.

DNA and RNA synthesis was evaluated in cells synchronized at G_1–0 (confluent) and G₂/M (between 24 and 28 h after passage from confluent cultures). As expected, we observed fluorescent signal for DNA synthesis in most of G₂/M cells compared to only few positive cells in the G_1–0 group (Fig. 2A). On the other hand, positive fluorescent signal for RNA synthesis was detected in most cells from both groups, confirming transcriptional activity in all nuclear donor cells used in both experiments (Fig. 2B).

FIG. 2.

Detection of DNA (A) and RNA (B) synthesis in confluent (>48 h) and nonconfluent (24 h after passage from confluent culture) cell cultures.

Effects of cell cycle interactions and treatment with HDACi on development of SCNT embryos

In the first experiment, a total of 568 embryos were reconstructed using G_1–0 cells and MII or TII cytoplasts. Reconstructed embryos from each group were separated and cultured as controls (MII or TII groups) or treated with Scriptaid for 15 h (MII + S or TII + S groups). Cleavage rates were significantly higher (p < 0.05) in embryos reconstructed with MII (72.1%) than TII (45.7%) cytoplasts (Fig. 3A). Scriptaid treatment did not affect cleavage rates either in MII (MII 72.1% vs. MII + S 66.7%) or TII (TII 45.7% vs. TII + S 47.8%) cytoplasts. Development to the blastocyst stage was significantly higher in MII (19.8%) than TII (4.6%) cytoplasts (Fig. 3B). Blastocyst development was numerically higher after Scriptaid treatment of embryos produced with MII cytoplasts (MII 19.8% vs. MII + S 32.6%; p = 0.09), but not in embryos produced with TII cytoplasts (TII 4.6% vs. TII + S 6.2%; p = 0.73).

FIG. 3.

Development of embryos produced by SCNT. Cleavage and blastocyst rates of SCNT embryos reconstructed with G_1–0 cells (A and B) and G₂/M cells (C and D). Different letters indicate significant differences between groups (p < 0.05). (*) p = 0.09.

In the second experiment, a total of 448 embryos were reconstructed using G₂/M donor cells. Cleavage rates were not significantly different between MII (61%) and TII (51.9%) cytoplasts, as well as between control and Scriptaid-treated (MII 61% vs. MII + S 59.4%; TII 51.9% vs. TII + S 49.5%) cytoplasts (Fig. 3C). Interestingly, development to the blastocyst stage was significantly lower in MII (8.7%) compared to TII (22.2%) cytoplasts (Fig. 3D). Similar to the first experiment, Scriptaid treatment enhanced development in embryos reconstructed with MII (MII 8.7% vs. MII + S 16.6%) but not TII (TII 22.2% vs. TII + S 22.4%) cytoplasts (Fig. 3D).

Effect of cell cycle interactions on DNA and RNA synthesis and nuclear decondensation in SCNT embryos

DNA and RNA synthesis was evaluated in reconstructed embryos between 4–8 and 8–12 hpf. The mean nuclear diameter was similar at 8 and 12 hpf in either MII/G_1–0 or TII/G_1–0 embryos, and it was not affected by Scriptaid treatment (Fig. 4A). However, the average nuclear size was superior in MII/G_1–0 compared to TII/G_1–0 embryos either at 8 or 12 hpf (Fig. 4A). Similar results were observed in MII/G₂/M and TII/G₂/M embryos (Fig. 4B). This confirms that nuclear swelling in SCNT was affected mainly by the cytoplast cell cycle stage.

FIG. 4.

Nuclear diameter in SCNT embryos at 8 and 12 h after fusion. (A) Embryos produced from G_1–0 synchronized cells. (B) Embryos produced from G₂/M synchronized cells. MII, metaphase stage cytoplast; MII + S, metaphase stage cytoplast treated with Scriptaid; TII, telophase stage cytoplast; TII + S, telophase stage cytoplast treated with Scriptaid.

A DNA synthesis signal was detected in a higher proportion of embryos produced from MII cytoplasts compared to TII cytoplasts, independently of the nuclear donor stage G_1–0 versus G₂/M or Scriptaid treatment (Table 1 and Fig. 5). Indeed, most of the embryos reconstructed using MII cytoplasts have initiated DNA synthesis by 12 hpf, whereas the fluorescent signal for DNA synthesis was only detected in a small number of embryos derived from TII cytoplasts (Table 1 and Fig. 5). The intensity of the fluorescent signal for DNA synthesis was similar between Scriptaid-treated and control embryos either at 8 or 12 hpf (data not shown). RNA synthesis was not detected in any of the embryos evaluated at 8 and 12 hpf, independently of Scriptaid treatment.

FIG. 5.

Representative pictures showing nuclear diameter and DNA synthesis in SCNT produced from G_1–0 and G₂/M cells and fixed at 12 h after fusion. Bars, 75 μm.

Table 1.

Proportion of SCNT Embryos Presenting a Positive Fluorescent Signal for DNA Synthesis at 8 and 12 hpf

		G_1–0 cells, n (%)		G₂/M cells, n (%)
Cytoplast	Scriptaid	8 hpf	12 hpf	8 hpf	12 hpf
MII	–	3/10 (30%)	10/12 (83.3%)	4/6 (66.7%)	14/14 (100%)
	+	4/8 (50%)	9/11 (81.8%)	2/10 (20%)	6/6 (100%)
TII	–	2/14 (14.3%)	2/14 (14.3%)	0/11 (0%)	0/13 (0%)
	+	2/15 (13.3%)	1/14 (7.1%)	1/11 (9.1%)	0/12 (0%)

SCNT, somatic cell nuclear transfer; hpf, hours post fusion; MII, metaphase II; TII, telophase II.

Effect of cell cycle interactions and HDACi on embryo cell number

The total number of cells was counted in SCNT embryos that developed to the blastocyst stage (Table 2). Embryos reconstructed with G_1–0 cells and MII cytoplasts had a higher number of nuclei (31.5 ± 3.2) than those produced with G_1–0 cells and TII cytoplasts (15.3 ± 1.7). Scriptaid treatment did not affect embryo cell number either when using MII (MII 31.5 ± 3.2 vs. MII + S 36.7 ± 5.1) or TII (TII 15.33 ± 1.7 vs. TII + S 19 ± 2.3) cytoplasts (Table 2). In embryos reconstructed with G₂/M cells, there was no effect of cytoplast or Scriptaid treatment on the average number of cells per blastocyst.

Table 2.

Average Number of Nuclei in Day 7 SCNT Blastocysts Produced with G_1–0 or G₂/M Nuclear Donor Cells and MII or TII Cytoplasts

Cytoplast	Scriptaid	G_1–0 cells, n ± SEM	G₂/M cells, n ± SEM
MII	–	31.7 ± 3.2^a	17 ± 4.7
	+	36.7 ± 5.1^a	16 ± 0.5
TII	–	15.3 ± 1.7^b	23.3 ± 1.7
	+	19 ± 2.3^b	20.6 ± 1.4

a,b

Values with different superscripts differ significantly within the same column (p < 0.05).

SCNT, somatic cell nuclear transfer; MII, metaphase II; TII, telophase II; SEM, standard error of the mean.

Discussion

Somatic cell reprogramming has many applications in cell biology comprising the production of pluripotent cells for therapeutic uses and the creation of cloned animals. It has been shown that cell reprogramming after nuclear transfer is affected by a number of conditions such as epigenetic changes and cell cycle interactions between the nuclear donor cell and the host cytoplasm. Even though many studies have investigated the impact of these conditions on cell reprogramming and development of SCNT embryos (Bordignon et al., 2001; Campbell 1999; Dean et al., 2003; Niemann et al., 2008; Wells, 2013; Zhao et al., 2010b), the interplay between cell cycle stages at the time of embryo reconstruction and the epigenetic reprogramming has not been thoroughly investigated. In this study, our main goal was to evaluate if the positive effects of HDCAi treatment on cell reprogramming and development of SCNT embryos is affected by the cell cycle stage of nuclear donor cells and host oocytes at the time of embryo reconstruction. Our findings provide evidence that the response of SCNT embryos to HDACi treatment depends upon the stage of the host cytoplast but not the nuclear donor cell at the time of nuclear transfer.

Cell cycle coordination between the donor nucleus and the host cytoplast is thought to be important for preserving embryo ploidy and promoting nuclear reprogramming in SCNT embryos (Bordignon et al., 2001; Chia and Egli, 2013; Kang et al., 2014; Wells et al., 2003). In this context, it has been proposed that ploidy errors can be induced when G₂/M cells are used in combination with MII cytoplasts, whereas the use of preactivated cytoplasts would eliminate this problem (Campbell et al., 1996). Indeed, the high MPF activity present in MII cytoplasts may induce NEBD and the PCC of the transferred nucleus, which can result in DNA re-replication after nuclear envelope reformation. Thus, unless nuclear donor cells are used at the G₁ or G₀ stages, the ploidy of reconstructed embryos can be compromised (Campbell and Alberio, 2003). On the other hand, S/G₂ stage cells can be fully reprogrammed and generate healthy animals when transferred to preactivated cytoplasts (Bordignon and Smith, 2006). Therefore, it has been confirmed that nuclear donor cells at different cell cycle stages are amenable to reprogramming if transferred to a suitable cytoplasmic environment.

In the present study, we first confirmed that cell cycle mismatch between the nuclear donor cell and the host cytoplast resulted in low development of porcine SCNT embryos to the blastocyst stage. Similar to previous studies in other species, we found that SCNT embryos constructed with G_1–0 donor cells developed in higher frequency when using MII stage compared to TII stage cytoplasts. On the other hand, G₂/M donor cells resulted in higher development to the blastocyst stage when transferred to TII stage compared to MII stage cytoplasts. Interestingly, when G_1–0 donor cells were combined with TII cytoplasts and G₂/M donor cells with MII cytoplasts, development of SCNT embryos was 4.3- and 2.5-fold lower than the reverse cell cycle combinations, respectively. It is possible that the low development of embryos reconstructed with G₂/M donor cells and MII cytoplasts was due NEBD and PCC induced by the MII cytoplast, which may affect the ploidy of the developing embryo (Campbell and Alberio, 2003). On the other hand, the low development of embryos reconstructed with G_1–0 donor cells and TII cytoplasts was likely due to insufficient nuclear remodeling and chromatin reprogramming. In support of this, we observed that both nuclear swelling and DNA synthesis were markedly higher in SCNT embryos reconstructed with MII stage compared to TII stage cytoplasts.

Deficient cell reprogramming to a pluripotent state has been considered to be the main constraint affecting development of SCNT embryos in many animal species, including swine (Grupen, 2014; Liu et al., 2014). This is supported by the results of comparisons performed between IVF and SCNT embryos, which showed significant differences in gene expression (Wang et al., 2011), histone acetylation status (Wen et al., 2014), and genome integrity (Pereira et al., 2014). Further supporting the importance of cell reprogramming are other findings confirming that treatment of SCNT embryos with HDACi not only enhanced embryo development but also increased the efficiency of animal cloning from somatic cells (Bui et al., 2010; Chen et al., 2013; Kishigami et al., 2006; Martinez-Diaz et al., 2010; Zhang et al., 2014; Zhou et al., 2013). In addition to enhancing cell reprogramming, it has also been shown that HDACi treatment facilitates DNA damage repair in SCNT embryos (Bohrer et al., 2014).

To further investigate the effects of HDACi on SCNT, we produced embryos with different cell cycle interactions and then treated them with Scriptaid. Interestingly, we observed that HDACi treatment improved development of embryos reconstructed with MII cytoplasts but not with TII cytoplasts, independent of the nuclear donor cell cycle stage. This suggests that the positive effect of HDACi treatment depends on factors present in the host cytoplasts but not in the nuclear donor cell. Moreover, our findings suggest that the positive effect of HDACi on SCNT is quickly lost after oocyte activation because TII stage cytoplasts failed to respond to Scriptaid treatment.

Whether the observed benefit of HDACi treatment on development of SCNT embryos produced with MII cytoplasts was due to an enhancement in nuclear reprogramming or by facilitating the interaction between the transplanted interphase nucleus and the host MII cytoplast requires further investigation. It seems logical to speculate that both cytoplasts would have been affected equally by HDACi treatment if the effect was only due to the enhancement of nuclear reprogramming. On the other hand, it is possible that the HDACi effect depends on NEBD, which is known to occur when nuclei are transferred to MII but not preactivated cytoplasts. Previous studies revealed that HDACi treatment increased chromosome decondensation and nuclear volume, and enhanced DNA replication and RNA synthesis in mouse embryos produced by SCNT (Bui et al., 2010, 2011; Van Thuan et al., 2009). However, our data revealed that chromosome decondensation, nuclear swelling, and DNA synthesis were similar at 8 and 12 hpf in control and HDACi-treated embryos. This suggests that the positive effect on embryo development observed when SCNT embryos reconstructed using MII cytoplasts were treated with Scriptaid was not due to an earlier start of DNA synthesis and faster nuclear swelling. Moreover, we have also tested if HDACi would maintain RNA synthesis after SCNT, but were unable to detect RNA synthesis at 8 and 12 hpf in SCNT embryos.

In conclusion, findings of this study show that: (1) G_1–0 stage donor cells are better reprogrammed by MII cytoplast whereas G₂/M cells can better support SCNT embryo development if transferred to a preactivated TII stage cytoplast; (2) the positive effect of HDACi treatment on development of SCNT embryos depends on the cell cycle stage of the host cytoplast but not of the donor cell; and (3) the positive effect of HDACi treatment on SCNT embryos is likely dependent on factors present in MII but not preactivated oocytes.

Footnotes

Acknowledgments

This study was supported by the Brazilian council of Scientific and Technological Development (CNPq) and the Natural Sciences and Engineering Research Council (NSERC) of Canada.

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

References

Baguisi

, Behboodi

, Melican

D.T.

, Pollock

J.S.

, Destrempes

M.M.

, Cammuso

, Williams

J.L.

, Nims

S.D.

, Porter

C.A.

, Midura

, Palacios

M.J.

, Ayres

S.L.

, Denniston

R.S.

, Hayes

M.L.

, Ziomek

C.A.

, Meade

H.M.

, Godke

R.A.

, Gavin

W.G.

, Overström

E.W.

, and Echelard

(1999). Production of goats by somatic cell nuclear transfer. Nat. Biotechnol., 17, 456–461.

Bohrer

R.C.

, Duggavathi

, and Bordignon

(2014). Inhibition of histone deacetylases enhances DNA damage repair in SCNT embryos. Cell Cycle, 13, 2138–2148.

Bordignon

, and Smith

L.C.

(1998). Telophase enucleation: An improved method to prepare recipient cytoplasts for use in bovine nuclear transfer. Mol. Reprod. Devel., 49, 29–36.

Bordignon

, and Smith

L.C.

(2006). Telophase-stage host ooplasts support complete reprogramming of roscovitine-treated somatic cell nuclei in cattle. Cloning Stem Cells, 8, 305–317.

Bordignon

, Clarke

H.J.

, and Smith

L.C.

(2001). Factors controlling the loss of immunoreactive somatic histone H1 from blastomere nuclei in oocyte cytoplasm: A potential marker of nuclear reprogramming. Dev. Biol., 233, 192–203.

Bui

H.T.

, Wakayama

, Kishigami

, Park

K.K.

, Kim

J.H.

, Thuan

N.V.

, and Wakayama

(2010). Effect of trichostatin A on chromatin remodeling, histone modifications, DNA replication, and transcriptional activity in cloned mouse embryos. Biol. Reprod., 83, 454–463.

Bui

H.T.

, Seo

H.J.

, Park

M.R.

, Park

J.Y.

, Thuan

N.V.

, Wakayama

, and Kim

J.H.

(2011). Histone deacetylase inhibition improves activation of ribosomal RNA genes and embryonic nucleolar reprogramming in cloned mouse embryos. Biol. Reprod., 85, 1048–1056.

Campbell

K.H.

(1999). Nuclear equivalence, nuclear transfer, and the cell cycle. Cloning, 1, 3–15.

Campbell

K.H.

, and Alberio

(2003). Reprogramming the genome: Role of the cell cycle. Reprod. Suppl., 61, 477–494.

10.

Campbell

K.H.

, Ritchie

W.A.

, and Wilmut

(1993). Nuclear–cytoplasmic interactions during the first cell cycle of nuclear transfer reconstructed bovine embryos: Implications for deoxyribonucleic acid replication and development. Biol. Reprod., 49, 933–942.

11.

Campbell

K.H.

, Loi

, Otaegui

P.J.

, and Wilmut

(1996). Cell cycle co-ordination in embryo cloning by nuclear transfer. Rev. Reprod., 1, 40–46.

12.

Chen

C.H.

, Du

, Xu

, Chang

W.F.

, Liu

C.C.

, Su

H.Y.

, Lin

T.A.

, Ju

J.C.

, Cheng

W.T.

, Wu

S.C.

, Chen

Y.E.

, and Sung

L.Y.

(2013). Synergistic effect of trichostatin A and scriptaid on the development of cloned rabbit embryos. Theriogenology, 79, 1284–1293.

13.

Chen

D.Y.

, Jiang

M.X.

, Zhao

Z.J.

, Wang

H.L.

, Sun

Q.Y.

, Zhang

L.S.

, Li

R,C.

, Cao

H.H.

, Zhang

Q.J.

, and Ma

D.L.

(2007). Cloning of Asian yellow goat (C. hircus) by somatic cell nuclear transfer: Telophase enucleation combined with whole cell intracytoplasmic injection. Mol. Reprod. Dev., 74, 28–34.

14.

Chia

, and Egli

(2013). Connecting the cell cycle with cellular identity. Cell. Reprogram., 15, 356–366.

15.

Cibelli

J.B.

, Stice

S.L.

, Golueke

P.J.

, Kane

J.J.

, Jerry

, Blackwell

, Ponce de León

F.A.

, and Robl

J.M.

(1998). Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science, 280, 1256–1258.

16.

Dean

, Santos

, and Reik

(2003). Epigenetic reprogramming in early mammalian development and following somatic nuclear transfer. Semin. Cell Dev. Biol., 14, 93–100.

17.

Fulka

Jr. , First

N.L.

, and Moor

R.M.

(1996). Nuclear transplantation in mammals: Remodelling of transplanted nuclei under the influence of maturation promoting factor. BioEssays, 18, 835–840.

18.

Grupen

C.G.

(2014). The evolution of porcine embryo in vitro production. Theriogenology, 81, 24–37.

19.

Kang

, Wu

, Ma

, Li

, Tippner-Hedges

, Tachibana

, Sparman

, Wolf

D.P.

, Schöler

H.R.

, and Mitalipov

(2014). Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos. Nature, 509, 101–104.

20.

Kang

Y.K.

, Koo

D.B.

, Park

J.S.

, Choi

Y.H.

, Chung

A.S.

, Lee

K.K.

, and Han

Y.M.

(2001). Aberrant methylation of donor genome in cloned bovine embryos. Nat. Genet., 28, 173–177.

21.

Kishigami

, Mizutani

, Ohta

, Hikichi

, Thuan

N.V.

, Wakayama

, Bui

H.T.

, and Wakayama

(2006). Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer. Biochem. Biophys. Res. Commun., 340, 183–189.

22.

Kretsovali

, Hadjimichael

, and Charmpilas

(2012). Histone deacetylase inhibitors in cell pluripotency, differentiation, and reprogramming. Stem Cells Int., 2012, 184154.

23.

Krishnakumar

, and Blelloch

R.H.

(2013). Epigenetics of cellular reprogramming. Curr. Opin. Genet. Dev., 23, 548–555.

24.

Liu

, Li

, Lvendahl

, Schmidt

, Larsen

, and Callesen

(2014). In vitro manipulation techniques of porcine embryos: A meta-analysis related to transfers, pregnancies and piglets. Reprod. Fertil. Dev. Feb 24. doi: 10.1071/RD13329. [Epub ahead of print]

25.

Mao

, Zhao

M.T.

, Whitworth

K.M.

, Spate

L.D.

, Walters

E.M.

, O'Gorman

, Lee

, Samuel

M.S.

, Murphy

C.N.

, Wells

, Rivera

R.M.

, and Prather

R.S.

(2015). Oxamflatin treatment enhances cloned porcine embryo development and nuclear reprogramming. Cell. Reprogram., 17, 28–40.

26.

Martinez-Diaz

M.A.

, Che

, Albornoz

, Seneda

M.M.

, Collis

, Coutinho

A.R.

, El-Beirouthi

, Laurin

, Zhao

, and Bordignon

(2010). Pre- and postimplantation development of swine-cloned embryos derived from fibroblasts and bone marrow cells after inhibition of histone deacetylases. Cell. Reprogram., 12, 85–94.

27.

Meissner

, and Jaenisch

(2006). Mammalian nuclear transfer. Dev. Dynamics, 235, 2460–2469.

28.

Morgan

H.D.

, Santos

, Green

, Dean

, and Reik

(2005). Epigenetic reprogramming in mammals. Hum. Mol. Genet., 14 Spec. No. 1, R47–R58.

29.

Niemann

, Tian

X.C.

, King

W.A.

, and Lee

R.S.

(2008). Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning. Reproduction, 135, 151–163.

30.

Pereira

A.F.

, Melo

L.M.

, Freitas

V.J.

, and Salamone

D.F.

(2014). Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos. Zygote, 23, 485–493.

31.

Polejaeva

I.A.

, Chen

S.H.

, Vaught

T.D.

, Page

R.L.

, Mullins

, Ball

, Dai

, Boone

, Walker

, Ayares

D.L.

, Colman

, and Campbell

K.H.

(2000). Cloned pigs produced by nuclear transfer from adult somatic cells. Nature, 407, 86–90.

32.

Rideout

W.M.

3rd , Eggan

, and Jaenisch

(2001). Nuclear cloning and epigenetic reprogramming of the genome. Science, 293, 1093–1098.

33.

Rodriguez-Osorio

, Urrego

, Cibelli

J.B.

, Eilertsen

, and Memili

(2012). Reprogramming mammalian somatic cells. Theriogenology, 78, 1869–1886.

34.

Rogakou

E.P.

, Boon

, Redon

, and Bonner

W.M.

(1999). Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol., 146, 905–916.

35.

Santos

, and Dean

(2004). Epigenetic reprogramming during early development in mammals. Reproduction, 127, 643–651.

36.

Van Thuan

, Bui

H.T.

, Kim

J.H.

, Hikichi

, Wakayama

, Kishigami

, Mizutani

, and Wakayama

(2009). The histone deacetylase inhibitor scriptaid enhances nascent mRNA production and rescues full-term development in cloned inbred mice. Reproduction, 138, 309–317.

37.

Wakayama

, Perry

A.C.

, Zuccotti

, Johnson

K.R.

, and Yanagimachi

(1998). Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature, 394, 369–374.

38.

Wang

L.J.

, Zhang

, Wang

Y.S.

, Xu

W.B.

, Xiong

X.R.

, Li

Y.Y.

, Su

J.M.

, Hua

, and Zhang

(2011). Scriptaid improves in vitro development and nuclear reprogramming of somatic cell nuclear transfer bovine embryos. Cell. Reprogram., 13, 431–439.

39.

Wells

D.N.

(2013). Keith's MAGIC: Cloning and the cell cycle. Cell. Reprogram., 15, 348–355.

40.

Wells

D.N.

, Laible

, Tucker

F.C.

, Miller

A.L.

, Oliver

J.E.

, Xiang

, Forsyth

J.T.

, Berg

M.C.

, Cockrem

, L'Huillier

P.J.

, Tervit

H.R.

, and Oback

(2003). Coordination between donor cell type and cell cycle stage improves nuclear cloning efficiency in cattle. Theriogenology, 59, 45–59.

41.

Wen

B.Q.

, Li

J.J.

, Tian

S.J.

, Sun

S.C.

, Qi

, Cai

W.T.

, and Chang

Q.L.

(2014). The histone deacetylase inhibitor Scriptaid improves in vitro developmental competence of ovine somatic cell nuclear transferred embryos. Theriogenology, 81, 332–339.

42.

Wilmut

, Schnieke

A.E.

, McWhir

, Kind

A.J.

, and Campbell

K.H.

(1997). Viable offspring derived from fetal and adult mammalian cells. Nature, 385, 810–813.

43.

Wolf

, Braun-Reichhart

, Streckel

, and Renner

(2014). Genetically engineered pig models for diabetes research. Transgenic Res. 23, 27–38.

44.

, Li

, Yu

, He

, Shi

, Zhou

, Liu

, and Wu

(2013). Effects of DNMT1 and HDAC inhibitors on gene-specific methylation reprogramming during porcine somatic cell nuclear transfer. PloS One, 8, e64705.

45.

Zhang

, Wang

, Sang

, Zhang

, and Hua

(2014). Combination of S-adenosylhomocysteine and scriptaid, a non-toxic epigenetic modifying reagent, modulates the reprogramming of bovine somatic-cell nuclear transfer embryos. Mol. Reprod. Dev., 81, 87–97.

46.

Zhao

, Hao

, Ross

J.W.

, Spate

L.D.

, Walters

E.M.

, Samuel

M.S.

, Rieke

, Murphy

C.N.

, and Prather

R.S.

(2010a). Histone deacetylase inhibitors improve in vitro and in vivo developmental competence of somatic cell nuclear transfer porcine embryos. Cell. Reprogram., 12, 75–83.

47.

Zhao

, Whyte

, and Prather

R.S.

(2010b). Effect of epigenetic regulation during swine embryogenesis and on cloning by nuclear transfer. Cell Tissue Res. 341, 13–21.

48.

Zhou

, Huang

, Xie

, Song

, Ji

, Zhang

Y.P.

, Ouyang

H.S.

, Lai

L.X.

, Pang

D.X.

, and Tang

X.C.

(2013). Scriptaid affects histone acetylation and the expression of development-related genes at different stages of porcine somatic cell nuclear transfer embryo during early development. Chinese Sci. Bull., 58, 2044–2052.