Trichostatin A Modified Histone Covalent Pattern and Enhanced Expression of Pluripotent Genes in Interspecies Black-Footed Cat Cloned Embryos But Did Not Improve In Vitro and In Vivo Viability

Abstract

The black-footed cat (BFC; Felis nigripes), one of the smallest wild cats, is listed as threatened. Interspecies somatic cell nuclear transfer (Is-SCNT) offers the possibility of preserving endangered species. Development to term of interspecies BFC (Is-BFC) cloned embryos has not been obtained, possibly due to abnormal epigenetic reprogramming. Treatment of intraspecies cloned embryos with TSA improves nuclear reprogramming and in vitro and in vivo viability. In this study, we evaluated (1) whether covalent histone modifications differ between Is-BFC cloned embryos and their IVF counterparts, (2) the optimal TSA concentration and exposure times to modify the covalent histone patterns, (3) if TSA enhances in vitro and in vivo developmental competence of cloned embryos, and (4) expression of pluripotent genes. Results indicated that the covalent histone modifications of Is-BFC cloned embryos aberrantly differ from their DSH-IVF counterpart embryos. Aberrant epigenetic events may be due partially to the inability of the DSH cytoplasm to modify the restrictive epigenetic marks of the BFC nuclei after somatic cell nuclear transfer (SCNT). Incomplete remodeling of the histone H3K9me2 in Is-BFC cloned embryos possibly contributes to abnormal expression of pluripotent genes and low embryonic development. Treatment of Is-BFC cloned embryos with TSA remodeled the covalent pattern in H3K9ac and H3K9me2, resembling epigenetic patterns in IVF counterpart embryos, and resulted in activation of some pluripotent genes. However, genomic reprogramming of Is-BFC cloned blastocysts did not follow the same reprogramming pattern observed in DSH-IVF embryos, and in vitro and in vivo developmental competence was not enhanced.

Introduction

Most of the 36 species of wild felids are classified as threatened, vulnerable, or endangered. As an example, the black-footed cat (BFC; Felis nigripes), one of the smallest wild cats, found only in parts of South Africa, Botswana, Namibia, and Southern Angola, is listed in CITES Appendix I as “threatened” largely due to indiscriminate use of pest control products by local farmers. Much progress has been made toward applying assisted reproductive technologies for aiding in the conservation of wild felids. In particular, somatic cell nuclear transfer (SCNT) is a valuable tool for retaining genetic variability and offers the possibility of species continuation rather than extinction. In fact, we have demonstrated the potential of using SCNT for preserving wild felids with the birth of African wildcat (Felis silvestris lybica) and sand cat (Felis margarita margarita) cloned kittens after the transfer of interspecies cloned embryos into domestic cat (DSH) recipients (Gómez et al., 2004, 2006, 2008). The effectiveness and efficiency of SCNT depends on a variety of factors, but the currently low success rate appears to be associated with epigenetic errors, such as abnormal DNA methylation (Dean et al., 2001; Kang et al., 2001). An abnormal methylation pattern during SCNT perturbs the expression of imprinted and pluripotent-related genes (Bortvin et al., 2003; Mann et al., 2003) that, consequently, may result in fetal and neonatal abnormalities. We have observed fetal abnormalities in African wildcat (Gómez et al., 2006) and sand cat (Gómez et al., 2008) cloned fetuses that may be associated with deregulation of several genes (Murrell et al., 2004) and epigenetic alterations in donor cells (Gómez et al., 2008). In fact, sand cat cloned embryos reconstructed with hypoacetylated donor cells showed aberrant or nonexpression of the Oct-4 gene that resulted in fetal and neonatal abnormalities (Gómez et al., 2008). In addition, pregnancies after the transfer of interspecies BFC cloned embryos into DSH recipients were established, but no implanted embryos developed to term (Gómez et al., 2009). Thus, we suggest that abnormal epigenetic reprogramming in BFC cloned embryos may contribute to their low viability.

It has been reported that treatment of intraspecies bovine, mouse, pig, and rabbit cloned embryos with an inhibitor of histone deacetylases activity (trichostatin A; TSA) regulated acetylation levels of certain histone residues, to levels that were similar to those of their IVF counterparts (Lager et al., 2008), improved embryo development to the blastocyst stage (Beebe et al., 2009; Costa-Borges et al., 2010; Kishigami et al., 2006, 2007; Lager et al., 2008; Li et al., 2008a; Shao et al., 2009; Shi et al., 2008; Zhang et al., 2007; Zhao et al., 2010), increased the number of live offspring after transfer to foster mothers (Costa-Borges et al., 2010; Maalouf et al., 2009; Rybouchkin et al., 2006; Wakayama et al., 2007), and reduced abnormal phenotypes, with the exception of placental overgrowth in mice (Kishigami et al., 2006). Even though improvements in cloning efficiency were observed in intraspecies cloned embryos treated with TSA, conflicting results have been reported when interspecies cloned embryos were treated with TSA (Shi et al., 2008; Srirattana et al., 2009). Other than the two latter studies, there is no additional information on using TSA in interspecies cloned embryos. Therefore, we explored whether treating interspecies BFC cloned embryos with TSA enhanced in vitro and in vivo developmental competence.

After SCNT, the somatic donor cell must be reverted to an embryonic-like epigenetic state. Consequently, efficiency of reprogramming after SCNT and the effect of TSA treatment on cloned embryos can be measured by analyzing epigenetic markers for active or silent chromatin. It is known that lysine 9 in histone 3 (H3K9) can be targeted for acetylation and methylation modifications, most likely in different biological circumstances (Rice and Allis, 2001). In effect, acetylation in H3K9 is linked to active transcription (Shahbazian and Grunstein, 2007), while methylation in H3K9 is linked to repressive transcription (Rice and Allis, 2001). Analysis of the covalent pattern in H3K9 is therefore a useful epigenetic marker for elucidating modifications in the chromatin of the donor cell after SCNT. Also, nuclear reprogramming efficiency can be evaluated by measuring the expression of pluripotent genes, which are silent in somatic cells but active in embryonic cells, and by determining the frequency of embryos that develop to the blastocyst stage and survive to birth after implantation. Thus, in the current study, we first determined the covalent pattern of acetylation and dimethylation in H3K9 (H3K9ac and H3K9me2; respectively) in interspecies BFC cloned embryos in relation to that of IVF counterpart embryos. Then, we evaluated the optimal TSA concentration and exposure times to modify the covalent pattern of H3K9ac and H3K9me2 of interspecies BFC cloned embryos to levels that resembled their IVF counterparts, and last, we investigated the influence of TSA on in vitro and in vivo developmental competence and the expression levels of pluripotent Oct-4, Nanog, and Sox-2 gene transcripts and the proto-oncogene C-Myc at four developmental stages (2–8 cells, 9–20 cells, morula, and blastocyst) of interspecies BFC cloned embryos.

Materials and Methods

Subjects

Domestic cats used as oocyte donors and embryo recipients were group housed in environmentally controlled rooms at the Audubon Center for Research of Endangered Species (ACRES). The BFC male was housed individually in an outdoor enclosure at the Freeport MacMoRan Species Survival Center. Fresh food was provided daily with water always available. All animal procedures were approved by the Institutional Animal Care and Use Committee of ACRES as required by the Health Research Extension Act of 1985 (Public Law 99-1580).

Chemicals

All chemicals were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO. USA) unless otherwise stated.

Establishment and culture of donor fibroblasts

Fibroblast cell cultures were generated from skin tissue collected by biopsy of one adult BFC male in 2002 and 2005 before his death in 2009, and from one adult DSH male in 2009 after his death. Each tissue sample was processed as previously described (Gómez et al., 2003) and cultured in Glasgow Minimal Essential Medium (GMEM) supplemented with 50 μg/mL of gentamicin, 2.4 mM glutamine, 2.4 mM sodium pyruvate, 2.5 μg/mL amphotericin B, and 15% (v/v) fetal bovine serum (FBS; Hyclone, Logan, UT, USA) at 38°C in 5% CO₂/air. When monolayer outgrowths with fibroblastic-like morphology reached 100% confluence, cells were disaggregated with 2.5 mg/mL of trypsin and resuspended in GMEM with 10% FBS and 10% (v/v) dimethyl sulphoxide and cooled at 1.0°C/min to −80°C (Mr. Frosty; Nalgene, Rochester, NY, USA) before storage in liquid nitrogen (LN2). Fibroblast cells frozen at passage 1 (P1) were thawed, plated equally into tissue culture tubes, and synchronized in the G₀/G₁ phase by contact inhibition, that consisted of continue culture of fibroblast cells until the cells reach 100% of confluence, followed by an additional 3 to 5 days in culture, during which time the culture medium was replaced every other day. Then, synchronized fibroblast cells were dissociated from culture tubes with 2.5 mg/mL of pronase and used immediately for SCNT.

Oocyte maturation

For collecting in vivo matured oocytes, DSH were treated with a total of 3 to 5 IU of porcine-follicle stimulating hormone (p-FSH, #915 Sioux Biochemical, Sioux Center, IA, USA) given in decreasing daily doses (s.c.) for 4 days followed by 3 IU of porcine-luteinizing hormone (p-LH, #925 Sioux Biochemical) on the fifth day. At 24 to 26 h after LH injection (i.m.), oocytes were collected by laparoscopic aspiration of preovulatory ovarian follicles (Gómez et al. 2000).

Embryo production by IVF or SCNT

Semen was collected from a DSH and a BFC male using an artificial vagina or standard electroejaculation techniques, respectively (Pope et al., 2006). An aliquot of each fresh semen sample was extended separately in HEPES Buffered Tyrode's solution (for DSH) or in TEST yolk buffer (for BFC; #9972, Irving Scientific, Santa Ana, CA, USA). Black-footed cat semen was frozen in TEST yolk buffer containing 6% glycerol (#9971, Irving Scientific) and thawed as previously described (Pope et al., 2006). For IVF, DSH oocytes were coincubated with 1 × 10⁶ motile fresh DSH or frozen/thawed BFC sperm/mL in modified Tyrode's solution containing 6 mg/mL bovine serum albumin (BSA) (IVF medium) with an overlay of mineral oil (Sage BioPharma, Bedminster, NJ, USA) in 5% CO₂ in air at 38°C for 4–5 h. Oocytes were then washed in IVF medium and cultured in modified Tyrode's solution containing 1% MEM nonessential amino acids, 3 mg/mL BSA, 10 ng/mL EGF (IVC-1 medium; Pope et al., 2009) and cultured for 2, 6, or 20 h before fixation.

SCNT was conducted according to the method previously reported by Gómez et al. (2003). Briefly, 1–2 h after retrieval, mature oocytes were denuded of cumulus and corona cells and enucleated (the metaphase spindle and first polar body were visualized after exposing oocytes to 25 μg/mL Hoechst 33342). After enucleation, a single synchronized fibroblast cell was introduced into the perivitelline space and fusion was induced by applying two electrical pulses (3-sec AC of 19 V, 1 MHz; followed by a 30-μsec DC of 21 V) delivered with two stainless steel electrodes (LF-101; Nepa Gene, Tokyo, Japan) attached to micromanipulators. After 30 min of culture, fusion was confirmed visually. Fused couplets were cultured with or without TSA. After 2 h, activation was induced electrically by exposing cybrids to two 60-μsec DC pulses of 120 V/mm, followed by incubation for 4 h in a droplet (30 μL) of IVC-1 medium supplemented with 10 μg/mL cycloheximide and 5 μg/mL cytochalasin B with or without TSA at 38°C in 5% CO₂ and air under mineral oil. After activation, reconstructed couplets were cultured in 500 μL of IVC-1 medium with or without TSA (1) for 4 or 18 h before fixation, (2) for 18 h before being transferred to oviducts of domestic cat females on day 1, or (3) cultured in vitro until day 9 at which time development to the blastocyst stage was recorded.

Covalent modifications of histone H3K9

The mean nuclear intensity of histone H3 in oocytes, fibroblasts, and embryos was assessed by scanning confocal microscopy after incubation with primary rabbit anti-acetyl-Histone H3 (lys9) (H3K9ac; Cat# 07-352; Upstate Biotechnology/Millipore, Billerica, MA, USA) and mouse antihistone H3-dimethyl K9 (H3K9me2; cat# ab1220; Abcam Inc. Cambridge, MA, USA) according to the method previously reported by Rybouchkin et al. (2006) with minor modifications. Briefly, after removal of the zona pellucida, embryos were fixed for 24 h in phosphate buffered saline (PBS) containing 4% paraformaldehyde at 4°C. After fixation, embryos were permeabilized with 1% BSA and 0.5% Triton X-100 in PBS for 20 min at room temperature and blocked overnight at 4°C in 1% BSA, 5% goat serum, and glycine (0.1 M) in PBS. After blocking, embryos were incubated with anti-H3K9ac (1:600 dilution) and H3K9me2 (1:83 dilution) antibodies overnight at 4°C. Finally, embryos were washed in PBS with 0.2% Tween 20 and incubated with secondary donkey antirabbit IgG-Alexa Fluor® 568 (1:600; Cat# A10042, Molecular Probes/Invitrogen, Carlsbad, CA, USA) and goat antimouse IgG2-Alexa Fluor® 488 (1:300; Molecular Probes/Invitrogen) antibodies for 4 h at room temperature and counterstained with DAPI. Control for nonspecific binding was performed by incubating embryos with the secondary conjugated antibody only, but not primary antibody.

The fluorescence signals for H3K9ac and H3K9me2 were detected with a laser scanning confocal system (Bioradiance 2000; BioRad, Hercules, CA, USA) mounted in a microscope (Nikon Eclipse TE3000). The fluorescence signal was quantified as mean nuclear intensity with LaserSharp 2000 software (BioRad; version 3.1). All embryos at each exposure time after IVF or SCNT were stained concurrently and exposed to the same light intensity to reduce experimental variability.

Detection of cat gene transcripts by RT-qPCR

The expression of cat pluripotent transcripts Oct-4, Nanog, Sox-2, the proto-oncogene C- Myc and the internal standard GADPH gene, which has been widely used as a housekeeping gene in various species of mammalian embryos, were detected by qRT-PCR performed as described previously (Gómez et al., 2010). Total mRNA was isolated from interspecies BFC cloned embryos, intraspecies DSH-IVF, and DSH fibroblasts, and cDNA was produced by using a Cells-to-cDNA™ II Kit (Ambion, Inc., Austin, TX, USA) according to manufacturer's directions. Briefly, each sample containing between 1 to 13 embryos or 2000 to 8000 fibroblast cells was washed in PBS, placed in RNAse-free tubes containing 10 μL of cell lysis II buffer, and heated at 75°C for 10 min. Then, 0.06 U/μL of DNase was added to the crude cell lysate and heated at 37°C for 15–20 min to degrade genomic DNA and heated again at 75°C for 5 min to inactivate DNase. Cell lysate was subsequently reverse transcribed into cDNA in the same tube using a two step RT-PCR. One microliter of each isolate was used for negative RT controls (reverse transcriptase omitted), then each sample was mixed with 4 μL dNTP mix, 2 μL random oligonucleotides, and nuclease-free water to a total volume of 16 μL, and denatured at 70°C for 3 min and placed on ice. Cooled samples were mixed with 1 μL RNase inhibitor and 1 μL M-MLV reverse transcriptase or 1 μL of water for the M-MLV reverse transcriptase negative RT controls, and incubated at 42°C for 60 min with a final RTase inactivation step of 95°C for 10 min. cDNA was frozen immediately and stored at −80°C until RT-qPCR. The sequences, dynamic ranges, amplicon sizes, efficiencies, r² values, and GenBank accession numbers of the primers used for amplification of the target genes are described in Table 1. Primers were designed based upon proposed primers using Beacon Designer Software (versions 5.0-7.5, PREMIER Biosoft International, Palo Alto, CA) and BLAST analysis. Primers for each target gene were tested for efficiency and amplification specificity by standard curve analysis, melt curve, and sequencing of PCR products; all primers showed similar amplification efficiency and single product amplification. RT-qPCR reactions were achieved by the addition of 1.75 μL of cDNA to 40 μL Mastermix containing 30 μL 2X iQ SybrGreen Supermix (BioRad) and 300 nM each of forward and reverse primer, with nuclease-free water. Reactions were performed in single clear tubes in duplicates of 25 μL each, with negative RT controls to verify absence of genomic contamination, and a nontemplate control for each sample and gene, respectively. The cycling conditions were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 10 sec, 54.2°C for 45 sec, and a final step of 72°C for 5 min using a BioRad iQ™ 5 multicolor real-time PCR system. A melt curve was carried out after each run to ensure single product amplification.

Table 1.

Primer Sequences and Conditions for Transcription Detection of Cat Genes

Primer sets	Primer sequences	Amplicon size (bp)	Linear range (Ct)	R² value	Accession no. (ref)
GAPDH	F: 5′-catcaagaaggtggtgaagca-3′ R: 5′-aaggtggaagagtgggtgtc-3′	114	17.7–36	0.995	NM001009307
OCT-4	F: 5′-tgcagctcagtttcaagaaca-3′ R: 5′-acaagtgtctctgctttgcata-3′	112	20–30	0.993	EU366914 (Yu et al., 2009)
NANOG	F: 5′-cctgaacatacagcctgaagtc-3′ R: 5′- aaggcagagatagaggacccta-3′	113	21.1–29.7	0.990	EU366913 (Yu et al., 2009)
SOX-2	F: 5′-catcacccgcagcaaatga-3′ R: 5′-aagaagtccaggatctctcataaa-3′	111	25.5–31.6	0.982	HQ385804 (Gómez et al., 2010)
C-MYC	F: 5′-ctcctctggcgttccaaga-3′ R: 5′- agatcagcaacaaccgcaaa-3′	97	24.9–30.78	0.998	HQ846918 (Gómez et al., 2010)

Embryo transfer and pregnancy detection

On day 1, interspecies BFC cloned embryos were transferred by laparoscopy into the oviducts of gonadotropin-treated DSH recipients after induced ovulation or oocyte aspiration (Gómez et al., 2004). Recipients were examined by ultrasonography on day 21 after embryo transfer to determine pregnancy status. A recipient was classified as pregnant if one or more gestational sacs were observed.

Statistical analysis

The ratio of H3K9ac/DNA or H3K9me2/DNA signals was compared by one-way ANOVA using SigmaStat (version 3.1.1, Systat software, Inc., Point Richmond, CA, USA). Main effects were embryo type (intraspecies DSH-IVF vs. interspecies BFC-Hybrid vs. interspecies BFC cloned embryos not treated with TSA), TSA concentration (0 vs. 50 vs. 100 vs. 500 nM) and exposure time to TSA (2, 6, or 20 h after IVF or SCNT). Pairwise Multiple Comparison Procedure Dunn's Method was used to determine differences between two means after ANOVA. Correlation between acetylation and dimethylation in H3K9 of interspecies BFC cloned embryos treated with TSA were investigated using the Pearson correlation test. Data on embryo cleavage, blastocyst development, pregnancies, and number of embryos implanting and reabsorbing were analyzed by chi-square.

RT-qPCR analysis

The delta-delta Ct method was used for real-time PCR data evaluation. Data collected using iQ5 Optical System Software (version 1.0, BioRad) from baseline subtracted curve fit analysis was normalized for different amounts of input cDNA using ΔCt (Ct for the GAPDH reference gene derived at each embryonic developmental stage) − (Ct for the gene of interest). Next, ΔΔCt was calculated by subtracting the ΔCt of each sample from the ΔCt of a reference cDNA sample. The n-fold increase or decrease in expression levels of each gene at each developmental stage in intraspecies DSH-IVF, interspecies BFC cloned embryos treated with 50 or 100 nM and not treated (0 nM) with TSA, and fibroblast cells was calculated using the formula 2^−ΔΔCt. We used one-way ANOVA and a t-test to determine differences between the 2^−ΔΔCt value for each gene at each developmental stage and embryo type [intraspecies DSH-IVF vs. interspecies BFC cloned embryos not treated (0 nM) vs. treated with 50 nM vs. 100 nM of TSA] A p-value of <0.05 was considered significant. Any samples for which signal was detected in the negative RT control or no template control or in which the PCR cycling was interrupted were omitted from the results.

Experimental design

Experiment 1

Covalent modification in histones of cloned embryos has been classified as normal or aberrant depending on their resemblance to that of IVF counterpart embryos (Lager et al., 2008; Wee et al., 2006). However, interspecies cloned embryos are produced with recipient cytoplast and donor cells that are from two different species. It is not clear, however, whether histone modification patterns in interspecies felid cloned embryos are similar to the patterns found in intraspecies or interspecies IVF embryos. If we assume that histone modification patterns differ between intraspecies and interspecies IVF embryos, then using the histone modification pattern of intraspecies IVF embryos as a “control” for the covalent modification pattern in interspecies BFC cloned embryos is inadequate.

To identify similarities or differences between embryos, we examined the average profiles represented as the ratio of H3K9ac/DNA or H3K9me2/DNA signals in interspecies BFC cloned embryos produced by fusion of BFC fibroblasts with DSH cytoplasts compared to the ratio of H3K9ac/DNA or H3K9me2/DNA signals in cat embryos produced by IVF of DSH oocytes with DSH spermatozoa (intraspecies DSH-IVF) or BFC spermatozoa (BFC-hybrid-IVF). Fluorescence intensities in H3K9ac and H3K9me2 were detected at 2, 6, and 20 h post-IVF or at 2 h after electrofusion and before activation (delayed activation; DA), and at 6 and 20 h after electrofusion for SCNT embryos. The ratio of H3K9ac/DNA or H3K9me2/DNA signals in chromosomes of DSH metaphase II oocytes and in interphase nuclei of BFC fibroblasts were used as the initial parameter for covalent modifications in IVF and cloned embryos, respectively. Two to twelve oocytes/embryos were examined in each treatment.

Experiment 2

To test whether the covalent pattern of H3K9ac and H3K9me2 of interspecies BFC cloned embryos could be modified to levels that resemble their IVF counterparts, we treated interspecies BFC cloned embryos with different TSA concentrations (50, 100, and 500 nM) and exposure times (2, 6, and 20 h after fusion). Control embryos were not treated (0 nM). Continuous deacetylation of mouse cloned embryos with TSA before and after activation improved in vitro developmental competence (Rybouchkin et al., 2006). Therefore, interspecies BFC cloned embryos were treated with TSA immediately after electrofusion and before activation. The fluorescence intensities in H3K9ac and H3K9me2 were analyzed at 2 (DA), 6, and 20 h after SCNT and the average profiles represented as the ratio of H3K9ac/DNA or H3K9me2/DNA signals were used to determine differences between treatments. Two to eight embryos were examined in each treatment.

Experiment 3

It has been proposed that “histone hyperacetylation improves nuclear reprogramming,” and that TSA treatment of intraspecies cloned embryos from different mammalian species results in improved in vitro and in vivo embryo development. To determine if TSA enhances in vitro and in vivo developmental competence of cloned embryos, interspecies BFC cloned embryos were treated with various TSA concentrations (50, 100, and 500 nM) for 20 h after SCNT and compared to interspecies BFC cloned embryos not treated (0 nM) with TSA and intraspecies DSH cloned embryos. Cleavage and development to the blastocyst stage were evaluated on day 2 and 9, respectively. The number of embryos from each treatment was derived from two to eight replicates. In vivo developmental competence of intraspecies BFC cloned embryos not (0 nM) or treated with TSA (50, 100, and 500 nM) and intraspecies DSH cloned embryos was evaluated by transferring cloned embryos (day 1) into the oviducts of synchronized DSH recipients. Two to seven recipient cats received cloned embryos from each TSA treatment.

Experiment 4

Increasing histone acetylation levels in specific residues of different histones by using an inhibitor of deacetylases (TSA) results in higher expression levels of some genes associated with pluripotency (Li et al., 2008b). Thus, to determine if TSA influences the expression of genes in interspecies BFC cloned embryos, we detected the expression of developmental pluripotent genes (Oct-4, Nanog, Sox-2) and the proto-oncogene C-Myc in: (1) interspecies BFC cloned embryos treated with 50 nM and 100 nM of TSA for 20 h after SCNT, (2) interspecies BFC cloned embryos not treated with TSA (0 nM; control for TSA), and (3) intraspecies DSH-IVF embryos (control for gene expression in cat embryos). To identify the timing of gene expression and determine if the limited progression to the blastocyst stage was due to a failure of reactivation of pluripotent genes in SCNT embryos, we evaluated the transcript abundance levels for each of the four genes at various stages of development: (1) 2–8 cells (pool of 9–12 embryos), (2) 9–20 cells (pool of 3–9 embryos), (3) morula (pool of 3 embryos), and (4) blastocyst stage (single embryos). Transcript levels in DSH fibroblasts were used as a negative control for the gene expression. Two replicates per treatment at each developmental stage were carried out and each qRT-PCR was performed in duplicate.

In this experiment, we did not analyze or include: (1) interspecies BFC cloned embryos treated with 500 nM of TSA because they did not develop to the blastocyst stage or implant after being transferred into DSH recipients (see Experiment 3), and (2) interspecies BFC cloned blastocysts not treated with TSA (0 nM) because we were not able to produce enough blastocysts for analysis.

Results

Experiment 1. Histone acetylation of interspecies BFC cloned embryos partially resembled the covalent patterns of interspecies BFC-hybrid-IVF embryos, while the covalent pattern of histone methylation was aberrant in comparison to IVF counterpart embryos

At 2 h after IVF, DSH oocytes were at MII and DSH and BFC spermatozoa were decondensed and had formed pronuclei (PN). The male PN was smaller than the female PN, so, the ratio signals for male and female PN at 2 h after IVF were analyzed separately. At 6 and 20 h after IVF, both male and female PN were similar in size and had similar ratio of H3K9ac/DNA or H3K9me2/DNA signals. Thus, ratio signals for male and female PN at the two later time points were pooled for statistical analyses.

Confocal microphotographs of interspecies BFC cloned embryos, intraspecies DSH-IVF, and BFC-hybrid-IVF embryos after immunocytochemistry with specific antibodies against H3K9ac and H3K9me2 are shown in Figure 1A. Fluorescence signals of both H3K9ac and H3K9me2 were observed in the chromosomes of MII stage oocytes and BFC fibroblasts. Nuclear intensities of H3K9ac in interphase PN of intraspecies DSH-IVF embryos at 2 h were detected and significantly increased in comparison to the intensity levels in MII oocytes (p < 0.001), whereas, at 6 h levels were deacetylated to similar intensities in MII oocytes. At 20 h, nuclear intensities for H3K9ac were increased again to similar intensity levels in interphase PN at 2 h after IVF (Fig. 1B). Likewise, nuclear intensities for H3K9ac in the PN of BFC-hybrid-IVF and interspecies BFC cloned embryos at 2 h after IVF or SCNT, respectively, increased the acetylation levels and were similar to that of interphase PN of intraspecies DSH-IVF embryos. However, at 6 h, PN of BFC-hybrid-IVF and interspecies BFC cloned embryos were markedly hyperacetylated in comparison to the intensity levels in interphase PN of intraspecies DSH-IVF embryos (p < 0.05; Fig. 1B); whereas, acetylation levels between interspecies BFC cloned embryos and BFC-hybrid-IVF embryos were similar (Fig. 1B). At 20 h, PN of BFC-hybrid-IVF and interspecies BFC cloned embryos were deacetylated, but acetylation levels in PNs of BFC-hybrid-IVF embryos were similar to interphase PNs in intraspecies DSH-IVF embryos, whereas levels in interspecies BFC cloned embryos were significantly lower and different than intraspecies DSH-IVF and BFC-hybrid-IVF embryos (Fig. 1A and 1B). Although acetylation levels in intraspecies DSH-IVF embryos were significantly increased at 2 and 20 h after IVF, the nuclear intensities in H3K9me2 of intraspecies DSH-IVF embryos markedly decreased at 2, 6, and 20 h after IVF in comparison to H3K9me2 intensities in MII oocytes (p < 0.05; Fig. 1C). Some background fluorescence was detected in the nonspecific binding control in which embryos were incubated only with the secondary conjugated antibody at 6 and 20 h after IVF, indicating that fluorescence in H3K9me2 of PNs of intraspecies DSH-IVF and interspecies BFC-hybrid-IVF embryos at 6 and 20 h after IVF was due to nonspecific binding of the conjugated secondary antibody and that PN were completely hypomethylated (Fig. 1A and C). Nonetheless, nuclear intensities in female and male PN at 2 h after IVF were higher than fluorescence intensities detected in the nonspecific binding control embryos at 2 h after IVF (p < 0.05). These observations indicate that domestic cat PN have a low level of methylation at 2 h in relation to staining controls, but still were globally hypomethylated in comparison to H3K9me2 intensities in MII oocytes. It seems unlikely that the complete loss of signal in the interphase of PN at 6 and 20 h after IVF was an artifact caused by the changes in structure of the chromatin because the interphase nuclei of BFC fibroblasts and PNs of BFC cloned embryos stained against H3K9me2. A similar trend in the fluorescence signals of H3K9me2 was observed in PN of BFC-hybrid-IVF embryos and intraspecies DSH-IVF embryos at 2, 6, and 20 h after IVF (Fig. 1A and 1C).

FIG. 1.

(A) Confocal microscopy images of the covalent histone modification pattern in acetylation (H3K9ac) and dimethylation (H3K9me2) of intraspecies DSH-IVF, BFC-hybrid-IVF and interspecies BFC cloned embryos not treated with TSA at 2, 6, and 20 h post-IVF or SCNT. Embryos were labeled for acetylated lysine 9 of H3 (H3K9ac; red), dimethylated lysine 9 of H3 (H3K9me2; green), and DNA (DAPI). Graphs showing the average profiles represented as the ratio of H3K9ac/DNA (B) and H3K9me2/DNA (C) signals of BFC cells, DSH oocytes, BFC clones not treated with TSA at 2 h after fusion and before activation (2 DA), female pronuclei (2 FP), and male pronuclei (2 MP) of DSH-IVF and BFC-hybrid-IVF embryos at 2 h after IVF, and pronuclei of DSH-IVF, BFC-hybrid-IVF, and BFC clones at 6 and 20 h after SCNT. Fluorescence detected below the lines in the lower part of graph (C) represents nonspecific binding control. Fluorescence of H3K9me2 in PNs of DSH-IVF and BFC-hybrid-IVF embryos at 6 and 20 h after IVF was due to nonspecific binding of the conjugated secondary antibody and PN's were completely hypomethylated. Numbers indicated the number of embryos analyzed. Different superscripts within 2 FP, 2 MP, 6 and 20 h differ significantly (p < 0.05).

The covalent pattern of H3K9me2 in interspecies BFC cloned embryos showed aberrant expression. In fact, nuclear intensities in H3K9me2 of interspecies BFC cloned embryos significantly increased at 2 h after SCNT (Fig. 1C), to higher levels than that of male or female PN in BFC-hybrid-IVF or intraspecies DSH-IVF embryos (p < 0.05; Fig.e 1A and C). After activation, PNs of BFC cloned embryos were slightly hypomethylated, but levels still were significantly higher than H3K9me2 levels in BFC-hybrid and intraspecies DSH-IVF embryos (Fig. 1A and 1C). These data present direct evidence that covalent modifications in H3K9ac of interspecies BFC cloned embryos partially resembles the covalent patterns of BFC hybrid-IVF embryos but differ from the intraspecies DSH-IVF embryos, and the covalent modifications in H3K9me2 were deviant to counterpart intraspecies DSH-IVF and BFC-hybrid-IVF embryos.

Experiment 2. TSA modifies histone acetylation and methylation patterns in H3K9 of interspecies BFC cloned embryos to levels that resemble their IVF counterparts

Confocal microscopy of immunocytochemistry with specific antibodies against H3K9ac and H3K9me2 in interspecies BFC cloned embryos treated or not with TSA is shown in Figure 2A. The fluorescence intensity in H3K9ac of interspecies BFC cloned embryos treated with 50, 100, and 500 nM of TSA gradually increased at 2 and 6 h, in comparison to the fluorescence intensity in BFC fibroblasts, but were similar to the levels of interspecies BFC cloned embryos not treated with TSA (0 nM). Exceptionally, in embryos treated with 100 nM of TSA, levels of H3K9ac were hypoacetylated at 6 h and had similar nuclear intensities as those of intraspecies DSH-IVF embryos (Fig. 2A and B). At 20 h after SCNT, fluorescence intensity of H3K9 was hyperacetylated in all BFC cloned embryos treated with TSA in comparison to that of nontreated embryos (0 nM; p < 0.05; Fig. 2A and B). Conversely, dimethylation levels in H3K9 of intraspecies BFC cloned embryos treated with 50, 100, and 500 nM of TSA gradually decreased at 2, 6, and 20 h (Fig. 2A and C). Although marked reduction of dimethylation in H3K9 was observed at all TSA concentrations, only embryos treated with 100 nM of TSA showed a dimethylation pattern that resembled their IVF counterparts. Moreover, an inverse correlation was observed between the mean nuclear intensities in H3K9ac and H3K9me2 (r = −0.45; p = 0.001). BFC cloned embryos that had the highest acetylation levels of H3K9 after TSA treatment for 20 h had the lowest dimethylation levels of H3K9, indicating that TSA also modified the methylation covalent pattern on histones. Collectively, these results indicate that TSA exposure for 20 h is required to modify the covalent pattern in H3K9ac and H3K9me2 of interspecies BFC cloned embryos. However, 100 nM of TSA was the optimal concentration for modifying the covalent pattern of H3K9ac and H3K9me2 to levels that resembled their IVF counterparts (Fig. 3A and B).

FIG. 2.

(A) Confocal microscopy images of the covalent histone modification pattern in acetylation (H3K9ac) and dimethylation (H3K9me2) of interspecies BFC cloned embryos not treated (0 nM) or treated with 50, 100, and 500 nM of TSA at 2, 6, and 20 h after SCNT. Embryos were labeled for acetylated lysine 9 of H3 (H3K9ac; red), dimethylated lysine 9 of H3 (H3K9me2; green), and DNA (DAPI). Graphs showing the average profiles represented as the ratio of H3K9ac/DNA (B) and H3K9me2/DNA (C) signals of interspecies BFC cloned embryos not treated (0 nM) or treated with 50, 100, and 500 nM of TSA at 2, 6, and 20 h after SCNT. Any fluorescence detected below the lines in the lower part of the graph (C) represents nonspecific binding control. Fluorescence in H3K9me2 of PNs of interspecies BFC cloned embryos treated with 100 nM of TSA at 6 and 20 h, and in embryos treated with 50 and 500 nM of TSA at 20 h was due to nonspecific binding of the conjugated secondary antibody and PN's were completely hypomethylated. Numbers indicated the number of embryos analyzed. Different superscripts within 2, 6, or 20 h differ significantly (p < 0.05).

FIG. 3.

Graphs showing the average profiles represented as the ratio of H3K9me2/DNA (A) and H3K9ac/DNA (B) signals of interspecies BFC cloned embryos treated with 100 nM of TSA (BFC-100 nM) and intraspecies DSH-IVF (DSH-IVF) and interspecies BFC-hybrid-IVF (BFC-hybrid-IVF) embryos at 2, 6, and 20 h after SCNT or IVF. Any fluorescence detected below the lines in the lower part of the graph (A) represents nonspecific binding control. All embryos were hypomethylated at 6 and 20 h after IVF or SCNT. Embryos with different superscripts within 2, 6, or 20 h differ significantly (p < 0.05).

Experiment 3. TSA did not enhance in vitro and in vivo developmental competence of interspecies BFC cloned embryos

The observation that TSA increases the global acetylation levels in H3K9 and modifies the acetyl- and dimethyl-covalent patterns (at 100 nM) in H3K9 to those that resemble their IVF counterparts (Experiment 2) led us to hypothesize that TSA induced hyperacetylation of the core histones in BFC chromatin might result in an open chromatin arrangement that is accessible to transcription factors and, consequently, enhance in vitro and in vivo developmental competence of interspecies BFC cloned embryos. However, in contrast to our hypothesis, we found that cleavage and development to the blastocyst stage of cloned embryos was not enhanced by TSA treatment (Table 2), regardless of concentration. Cleavage rates of interspecies BFC cloned embryos treated with 50, 100, or 500 nM of TSA (91 vs. 93 vs. 90%, respectively) were similar to the rates observed in BFC cloned embryos not treated with TSA (0 nM; 90%) and intraspecies DSH cloned embryos (control; 88%). Likewise, development to the blastocyst stage was similar between embryos treated with 0, 50, or 100 nM of TSA (3 vs. 3 vs. 1%, respectively), but significantly lower than that of intraspecies DSH cloned embryos (31%; p < 0.05). None of the embryos treated with 500 nM of TSA developed to the blastocyst stage (Table 2).

Table 2.

Embryo Cleavage (Day 2), Development to Blastocyst Stage (Day 9), Number of Pregnancies, Embryo Implantation, and Fetuses Reabsorbed of Interspecies Black-Footed Cat Cloned Embryos (BFC-NT) Nontreated (0 nM) or Treated with TSA (50, 100, or 500 nM) for 20 h After SCNT, and Intraspecies Domestic Cat Cloned Embryos (DSH-NT; Control)

Species	TSA concentration nM	Replicates n	Cleavage n/total fused (%)	Blastocysts n/total cleaved (%)	DSH recipients,		Embryos transferred,			Fetus reabsorbed n (%)	Live offspring from total embryos transferred n (%)
					Total n	Pregnant n (%)	Total n	Per recipient ± SD	Implanted n (%)
Interspecies BFC-NT	0	5	179/199 (90)	6 (3.3)^a	7	4 (57)	213	37 ± 7.5	9 (4.2)	9 (100)	0
	50	3	64/70 (91)	2 (3.3)^a	7	1 (29)	261	37 ± 8.8	2 (0.9)	2 (100)	0
	100	8	138/151 (91)	2 (1.4)^a	2	1 (50)	68	34 ± 7.0	3 (4.4)	3 (100)	0
	500	4	74/82 (90)	0 (0)^a	2	0 (0)	70	35 ± 0.0	—	—	—
IntraspeciesDSH-NT	(control)	2	51/58 (88)	16 (31)^b	3	1 (67)	121	40 ± 5.5	5 (4.1)	1 (20)	4 (3.3)

a,b

Different superscripts within the same column indicate significant differences (p<0.05).

We then tested the influence of treating interspecies BFC cloned embryos with TSA on pregnancy, embryo implantation, and rate of live offspring by transferring cloned embryos not treated (0 nM) or treated with TSA (50, 100, and 500 nM) and intraspecies DSH cloned embryos into the oviduct of DSH recipients. Similarly, in vivo developmental competence was not enhanced by TSA. Pregnancies in recipient cats were established after transfer of interspecies BFC cloned embryos treated with 50 (1/7; 29%) or 100 nM (1/2; 50%) of TSA at similar rates to those obtained from cloned embryos not treated with TSA (4/7; 57%) or with intraspecies DSH cloned embryos (2/3; 67%) (Table 2). However, none of the transferred embryos treated with 500 nM of TSA established a pregnancy (0/2; 0%) (Table 2). The number of embryos that implanted after the transfer of interspecies BFC cloned embryos treated with 50 and 100 nM (1 vs. 4%; respectively) was similar to the implantation rates observed after the transfer of interspecies BFC cloned embryos not treated with TSA (4%) or intraspecies DSH cloned embryos (4%) (Table 2).

Similar to a previous study (Gómez et al., 2009), ultrasound analyses of all interspecies BFC cloned embryos treated or not treated with TSA that implanted into the uterus of DSH recipients revealed an amorphous fetal mass with no heartbeat inside the gestational sac that had started reabsorbing by day 30 of gestation. However, four out of five (80%) intraspecies DSH cloned embryos that implanted into the uterus of DSH recipients showed normal fetal mass and heartbeat. Of the two pregnant recipients carrying intraspecies DSH-cloned fetuses, one (50%) delivered a total of four kittens. The average birth weight of kittens was 83.4 ± 15.6 g. Phenotypically, all cloned kittens were identical to the male cell donor. Subsequent analysis of 20 cat-specific microsatellite loci confirmed that they were identical to DNA of the cell donor male and not to the recipient domestic cat.

Experiment 4. TSA upregulated the expression of pluripotent and proto-oncogene genes in interspecies BFC-cloned embryos

Relative transcript abundance for Oct-4, Nanog, Sox-2, and C-Myc at various stages of development in interspecies BFC cloned embryos treated (50 and 100 nM) or not (0 nM) with TSA, intraspecies DSH-IVF embryos and BFC fibroblast cells are shown in Figure 4. The RT-qPCR analysis revealed that intraspecies DSH-IVF embryos expressed the pluripotent genes Oct-4, Nanog, and Sox-2 and the proto-oncogene C-Myc gene at all stages of development with a significant increase from the 2–8-cell to the morula stage, followed by a decrease transcription at the blastocyst stage (D9). Similarly, expression of the four genes in interspecies BFC cloned embryos treated or nontreated with TSA increased from the two- to eight-cell stage to the morula stage and significantly decreased at the blastocyst stage. Nonetheless, the level of expression of each gene in interspecies BFC cloned embryos varied between treatments at each developmental stage. For instance, at 2–8-cell and 9–16-cell stages the expression levels of Oct-4, Nanog, C-Myc, and Sox-2 were similar between interspecies BFC cloned embryos and intraspecies DSH-IVF embryos, except that Sox-2 that was not expressed by interspecies BFC cloned embryos treated with 50 and 100 nM TSA at the 2–8-cell stage, and BFC embryos treated with 100 nM TSA at the 9–16-cell stage. Moreover, at the morula stage, interspecies BFC cloned embryos expressed all four genes with the exemption of embryos treated with 50 nM TSA that only expressed C-Myc. The most important differences between intraspecies DSH-IVF and interspecies BFC cloned embryos treated with TSA were observed at the blastocyst stage, where interspecies BFC cloned embryos treated with 100 nM TSA only transcribed the Oct-4 gene, and embryos treated with 50 nM TSA transcribed Oct-4 and C-Myc.

FIG. 4.

Relative transcript abundance of Oct-4 (A), Nanog (B), Sox-2 (C), and C-Myc (D) genes in intraspecies domestic cat-IVF embryos (DSH-IVF), interspecies BFC cloned embryos not treated (BFC-0 nM), or treated with 50 nM (BFC-50 nM) or 100 nM of TSA (BFC-100 nM) at four stages of embryonic development (2–8 cells, 9–16 cells, morula, and blastocyst), and BFC fibroblast cells (BFC-cells). The Oct-4, Nanog, Sox-2, and C-Myc PCR signals were normalized with their corresponding GAPDH signals at each developmental stage, and results are presented as a relative change in ratio among groups. A value of zero represents no change in transcript abundance. Abundance levels were expressed an n-fold differences relative to blastocysts which was set at 1. Values greater than zero represent an increase in transcript abundance in DSH-IVF, BFC-0 nM, BFC-50 nM, BFC-100, and BFC-cells relative to DSH-IVF blastocysts, and values less than zero represent a decrease in DSH-IVF, BFC-0 nM, BFC-50 nM, BFC-100, and BFC-cells relative to DSH-IVF blastocysts. Different letters denote samples that differed significantly within each developmental stage (p < 0.05). Jagged arrow = values are greater than the ones showed in graph. N/A = no data available.

In addition, our results confirmed our hypothesis that TSA enhanced the expression of developmental pluripotent genes (Oct-4, Nanog, Sox-2) and the proto-oncogene, C-Myc, in interspecies BFC cloned embryos treated with TSA; but the enhancement was primarily observed at the 100 nM concentration. Indeed, at the 9–16-cell stage, interspecies cloned embryos treated with 100 nM TSA had a moderate increase in the expression of Oct-4, Nanog, Sox-2, and C-Myc, but at the morula stage, the expression of Oct-4 (∼57.3-fold, ∼65.0-fold, ∼41.9-fold), Nanog (∼972.0-fold, ∼1010.2-fold, ∼954.9-fold), Sox-2 (∼38.8-fold, ∼48.0-fold, ∼33.7-fold), and C-Myc (∼102.4-fold, ∼88.1-fold, ∼87.1-fold) was significantly higher than the expression in not treated (0 nM) or TSA treated (50 nM) and in intraspecies DSH-IVF embryos, respectively (p < 0.05). Even though not all genes were expressed in BFC cloned blastocysts treated with TSA, expression of Oct-4 in embryos treated with 100 nM TSA and Oct-4 and C-Myc in embryos treated with 50 nM TSA were ∼15.9-fold, ∼22.4-fold, and ∼11.8-fold higher, respectively, than the levels in intraspecies DSH-IVF blastocysts. These results give additional credence to the concept that the morula stage is an important developmental step for cat embryos and reveal that, although 100 nM of TSA enhanced the expression of pluripotent and proto-oncogene genes at the morula stage, downregulation was observed, particularly at the blastocyst stage.

Discussion

Epigenetic reprogramming is essential for normal development and restoration of totipotency (Reik et al., 2001) and, during SCNT, the epigenetic marks of the differentiated state of a somatic donor cell must be removed and an epigenetic state that resembles the embryonic pattern must be acquired. Aberrant epigenetic reprogramming of intraspecies bovine cloned embryos that exhibited hypermethylation in H3K9, which was significantly higher than and different from that of their IVF counterparts showed a lower proportion of development to the blastocyst stage (Santos et al., 2003). Similarly, in this study, we observed that the covalent modification pattern in H3K9me2 of interspecies BFC cloned embryos aberrantly differed from their intraspecies DSH-IVF and BFC-hybrid-IVF counterpart embryos, and embryos with an aberrant hypermethylation in H3K9 had lower in vitro development. Although it is not clear how global chromatin histone demethylation occurs in a donor cell after SCNT, it has been suggested that factors present in the recipient cytoplasm are responsible for inducing DNA demethylation of the transferred nucleus and the process is not species-dependent (Chen et al., 2006; Wang et al., 2009). Therefore, we suggest that DSH cytoplasm had enough demethylation activity to induce demethylation in H3K9 of the male and female pronucleus of intraspecies DSH-IVF and hybrid-BFC-IVF embryos, but was unable to induce demethylation in chromatin of the BFC donor cell. Demethylation differences between both types of BFC chromatin may be due to dissimilar chromatin configuration, where BFC somatic cell chromatin is more rigid and transcriptionally repressive than the BFC sperm chromatin configuration (Rideout et al., 2001). Consequently, we concluded that (1) aberrant histone demethylation of interspecies BFC cloned embryos was due, in part, to the inability of the DSH cytoplasm to modify the restrictive epigenetic marks of BFC chromatin after SCNT, and was not due to the absence of enzyme activities in DSH cytoplasm that are responsible for inducing demethylation in H3K9 (Armstrong et al., 2006), and (2) the lower in vitro development of interspecies BFC cloned embryos may be in part due to aberrant histone modifications during reprogramming.

There is growing evidence suggesting that proper histone acetylation may be a critical element in SCNT success. Indeed, it has been proposed that “histone hyperacetylation improves nuclear reprogramming.” Frozen/thawed sand cat fibroblasts that showed a higher level of acetylation in H3K9 supported a higher rate of embryo implantation, had lower fetal loss, and resulted in a higher number of live offspring after SCNT than cells with a reduced level of acetylation (Gómez et al., 2008). Recently, it was reported that treating cat leopard cells with TSA before SCNT, significantly increased the level of acetylation in H3K9 donor cells and in the pronucleus of leopard cat cloned embryos reconstructed with domestic cat oocytes, and, as well, increased the total cell numbers of leopard cat cloned blastocysts cultured in the oviduct of domestic cat recipients (Lee et al., 2010). Likewise, we observed that global acetylation in H3K9 of interspecies BFC cloned embryos was substantially increased when embryos were treated for 20 h after SCNT, regardless of TSA concentration, and histone acetylation levels were higher than non-TSA treated cloned embryos or IVF counterparts. Moreover, methylation levels in H3K9me2 were significantly decreased at 20 h after SCNT in all TSA-treated embryos; but, only BFC cloned embryos treated with 100 nM of TSA had “normalized” the pattern of acetylation inH3K9ac and global losses of methylation in H3K9me2 to levels that resembled their IVF counterparts (see Fig. 3A and B). These results plainly indicated that histone deacetylases in DSH cytoplasm were effectively inhibited by TSA treatment, thereby inducing a significant increase in global levels at H3K9ac, whereas inhibition of histone methyl transferases activities in the DSH cytoplasm induced hypomethylation in H3K9, which was directly affected by the concentration of TSA. Regardless of the positive effect of TSA on inducing histone hyperacetylation, and “normalization” of the histone methylation pattern in H3K9me2 at 100 nM, we did not observe an improvement in in vitro and in vivo developmental competence of treated cloned embryos.

It is not clear why TSA did not enhance development of interspecies BFC cloned embryos, but developmental failure may have been caused by an abnormal epigenetic covalent pattern of histone acetylation. Some studies have suggested that the oocyte cytoplasm initializes a program of gene expression by deacetylating histones (Kim et al., 2003). In fact, a histone deacetylation and reacetylation pattern has been reported in normally fertilized mouse embryos, in which chromatin of both gametes became highly acetylated after fertilization. Similarly, levels of acetylation of several lysine residues of histone H3 and H4 of mouse cloned embryos were reduced drastically at 2 h after SCNT and before activation, and then the reacetylation occurred after oocyte activation (Kim et al., 2003). Recently, Shi et al. (2008) evaluated the histone acetylation pattern of interspecies rabbit–human and intraspecies rabbit cloned embryos and its influences on in vitro development. The authors reported that marked differences in deacetylation/reacetylation patterns exist between interspecies and intraspecies cloned embryos, where intraspecies rabbit cloned embryos followed a similar deacetylation/reacetylation pattern in some lysine residues of histone H3 and H4, whereas interspecies rabbit–human cloned embryos failed to deacetylate prior to activation, and deacetylation was only observed in non-TSA treated embryos later after activation (3–6 h). After evaluating development of intraspecies and interspecies cloned embryos, the authors concluded that the lower in vitro development of interspecies rabbit–human cloned embryos was due to the inability of the histone deacetylases in the rabbit oocytes to deacetylate the human chromatin prior to reacetylation (Shi et al., 2008). Similarly, we did not observe deacetylation in H3K9 prior to activation and deacetylation was observed in non-TSA-treated embryos later after activation (18 h). Although, there are acute differences between the Shi et al. (2008) study and ours, in which a higher phylogenetic distance between the rabbit and the human, and closer phylogenetic distance between the DSH and the BFC exist, our results coincide with their results, where, in our study, factors in the cytoplasm of the DSH oocyte could not deacetylate the chromatin of BFC prior to activation. Even though we did not observe deacetylation in the BFC chromatin before activation, we observed a deacetylation/reacetylation pattern after activation in embryos treated with 100 nM of TSA that resembled the histone covalent pattern of intraspecies DSH-IVF embryos. Previously, it has been demonstrated that deacetylation of somatic histones prior to activation (at least in embryos treated with TSA) is not important for nuclear reprogramming (Rybouckin et al., 2006). For these reasons, and contrary to the results reported in rabbit–human cloned embryos, we conclude that inhibition of deacetylation in interspecies BFC cloned embryos prior to activation with TSA may not be the cause of the lower in vitro development and, instead, a deacetylation/reacetylation pattern after fertilization in cat embryos is probably required for normal embryonic development. It is probable that normalization of the covalent pattern in H3K9ac by treatment with TSA increases the possibility of nuclear remodeling and reprogramming of the transplanted nucleus, but by itself is not sufficient to inhibit embryo developmental failure.

A positive influence of TSA has been reported on the transcription expression of genes associated with pluripotency. In fact, mouse cloned embryos treated with TSA showed an increase in the expression of Sox-2 and C-Myc genes, and these two pluripotent genes were significantly higher than the expression in cloned embryos not treated with TSA (Li et al., 2008b). Similarly, in the present study, we observed that TSA increased the expression levels of pluripotent genes in interspecies BFC cloned embryos in comparison to embryos not treated with TSA. The expression of Oct-4, Nanog, Sox-2, and C-Myc genes were substantially upregulated after the 9–16-cell stage, preferentially in cloned embryos that were treated with 100 nM of TSA. Although, the mechanism(s) of how TSA increases the expression of pluripotent genes in cloned embryos has not been elucidated, previous studies had indicated that inhibitors of deacetylases (1) in somatic cells increased histone acetylation levels in amino acid residues, resulting in decondensation of dense chromatin regions (Tóth et al., 2004), and, (2) in cloned embryos, remodeled the constitutive heterochromatin into a zygotic-like organization (Maalouf et al., 2009). As a result, the chromatin structure relaxed after SCNT was exposed to oocyte proteins such RNA polymerases and transcription factors (Shogren-Knaak et al., 2006; Van Thuan et al., 2009; Zlatanova et al., 2000) that promote transcription of genes associated with pluripotency (Li et al., 2008b). Although, we did not examine the physical structural changes in the BFC chromatin, we observed that TSA induced epigenetic modifications in the chromatin of interspecies BFC cloned embryos, as characterized by an increased acetylation in H3K9 and global losses of methylation in H3K9me2. Thus, we suggest that histone acetylation modulates structural changes in the BFC chromatin allowing certain proteins to bind to the DNA and induce upregulation of proto-oncogene and pluripotent genes. It is clear that the analysis of an individual lysine in a specific histone does not confirm that gene upregulation was a consequence of histone hyperacetylation. Nonetheless, studies analyzing the genome-wide acetylation state of 11 lysines in the four core of histones of Saccharomyces cerevisiae showed that hyperacetylation of histone H3K9/18/27 and hypoacetylation of H4K16 and H2BK11/16 were correlated with transcription status, suggesting that the pattern of acetylation, rather than individual lysines, may be more closely correlated with gene transcription (Kurdistani et al., 2004). Additional studies that analyze the acetylation pattern in several histones and their relationship with transcription of certain groups of genes may provide additional information on how epigenetic modifications in histones affect gene transcription in cloned embryos treated with TSA.

The molecular mechanism by which histone acetylation and dimethylation affect transcription and upregulate the expression of certain genes is not clear, but two major epigenetic events may take place. Previous studies had indicated that erasure of DNA methylation and loss of histone modifications are involved in genome-wide epigenetic reprogramming during mammalian embryonic development, and these epigenetic events are closely linked to changes in the chromatin structure and the establishment of a chromatin signature that resemble totipotency. For instance, global losses of methylation in H3K9me2 and enrichment of acetylation in H3K9 are observed in mouse primordial germ cells (PGCs) (Hajkova et al., 2008) after demethylation of DNA (Hajkova et al., 2002), resulting in expression of pluripotent related genes (Hajkova et al., 2008; Surani et al., 2004). Similarly, reactivation of Oct-4 transcription in embryonic stem cells is dependent on demethylation of DNA, and hyperacetylation and hypomethylation of H3K9 (Kimura et al., 2004). Moreover, DNA methylation has an important role in the control of gene expression, where it is often correlated with transcriptional repression (Bird, 2002), whereas DNA demethylation is associated with the erasure of epigenetic modifications inherited from the gametes (Santos et al., 2002). In the present study, we observed that interspecies BFC cloned embryos treated with TSA had an increase in acetylation in H3K9 that was inversely related to methylation levels in H3K9me2, in which cloned embryos with the highest acetylation levels in H3K9 after TSA treatment for 20 h had the lowest methylation levels in H3K9me2. Also, interspecies BFC cloned embryos treated with 100 nM TSA had global losses of methylation in H3K9me2 that resembled the levels of their IVF counterparts. Although we did not evaluate DNA methylation levels, it is known that an increase in histone acetylation is associated with a decrease in global DNA methylation (Jackson et al., 2004; Ou et al., 2007), and demethylation in the paternal pronucleus is associated with global losses of methylation in H3K9me2 and H3K9me3 (Santos et al., 2005). Therefore, we suggest that global histone acetylation and losses of methylation in H3K9me2 may be associated with a decrease in global DNA methylation, and these epigenetic events are directly linked in remodeling the chromatin structure and upregulation of all four genes that were analyzed. Furthermore, in mouse PGCs, global losses of methylation in H3K9me2 coincides with the activation of the pluripotent gene Nanog (Yamaguchi et al., 2005); thus, marked global losses of methylation in H3K9me2 of interspecies BFC cloned embryos treated with 100 nM of TSA may, in part, explain the higher transcription levels of Nanog. Consequently, from our data we strongly suggest that TSA induces corrective chromatin changes, possibly by the erasure/removal of repressive epigenetic marks, such as DNA methylation and dimethylation in H3K9 that influence upregulation of proto-oncogene and pluripotent genes. Nevertheless, inducing hyperacetylation and hypomethylation in H3K9 by treating cloned embryos with TSA was not enough to reactivate some of the pluripotent genes at the blastocyst stage. Therefore, additional approaches to induce epigenetic modifications in the chromatin of BFC cloned embryos for improving nuclear reprogramming are required.

The feasibility of producing viable endangered felid and domestic cat offspring by SCNT has been demonstrated both in our laboratory (Gómez et al., 2004, 2008, 2009) and others (Shin et al., 2002; Yin et al., 2005, 2007). However, overall efficiency remains low, especially when differentiated nuclei are used as donor cells. Fetal losses and abnormalities have been suggested to be due to abnormal gene expression (Bortvin et al., 2003; Nichols et al., 1998; Park et al., 2010). Aberrant gene expression has been reported in interspecies cat cloned embryos, which was related to lower in vitro development to the blastocyst stage (Imsoonthornrusksa et al., 2010). Moreover, early fetal losses of interspecies sand cat cloned embryos were associated with abnormal expression of the Oct-4 gene (Gómez et al., 2008). Likewise, in the present study, we observed abnormal gene expression of Sox-2 and Nanog in interspecies BFC cloned blastocysts treated with 50 or 100 nM TSA, and all interspecies BFC cloned embryos treated or not with TSA that implanted were reabsorbed, regardless of the upregulation of the Oct-4 gene, whereas intraspecies DSH cloned embryos that expressed all four pluripotent genes produced normal live offspring after their transfer into domestic cat recipients.

Therefore, it is possible that the early fetal losses observed in the present study were associated with abnormal or nonexpression of pluripotent genes. In fact, it is known that the presence of Nanog is required for maintenance of epiblast in mouse blastocysts (Mitsui et al., 2003). Nanog-deficient mouse embryos died soon after implantation and Nanog-deficient embryonic stem cells differentiated into parietal–visceral and endodermal lineages. Sox-2-deficient mouse embryos can develop to the blastocyst stage, but they died after implantation with an abnormal formation of trophoblast giant cells and extra-embryonic endoderm cells (Avilion et al., 2003). Proper expression of Nanog and Oct-4 is required for establishing a pluripotential cell-specific epigenotype (Hatano et al., 2005), and correspondingly, the presence of Oct-4 and Sox-2 is required for maintenance of epiblast after implantation (Avilion et al., 2003; Nichols et al., 1998). Moreover, overexpression of Oct-4 in embryonic stem cells results in differentiation into extra-embryonic endoderm cells (Niwa et al., 2000), whereas Oct-4-deficient mouse embryos formed empty deciduas that contained trophoblast cells that were devoid of yolk sac or embryonic structures and died during the peri-implantation period (Nichols et al., 1998). Therefore, we suggest that upregulation of Oct-4 in interspecies BFC cloned embryos treated with TSA promoted formation of blastocysts that were able to implant after transfer. However, downregulation of Sox-2 and Nanog inhibited the maintenance of the epiblast in a pluripotent state and, instead, extra-embryonic endoderm cells were formed, as demonstrated by (1) the presence of an amorphous mass of fetal tissue with no heartbeat inside the gestational vesicle, and (2) the later death and initiation of reabsorption at ∼30 days postimplantation. We concluded that fetal losses observed in this study are a consequence of the failure of reactivation of Sox-2 and Nanog that, along with the Oct-4 gene, are required and must be expressed for (1) forming a molecular network to maintain a pluripotent epiblast, and (2) subsequent expression of other important genomic or imprinted genes.

In summary, these results showed that aberrant epigenetic events in interspecies BFC cloned embryos may be partially due to the inability of DSH cytoplasm to modify the restrictive epigenetic marks of BFC nuclei after SCNT, suggesting a certain degree of nucleocytoplasmic incompatibilities between the two species. We also demonstrated that incomplete remodeling of histone H3K9me2 in BFC cloned embryos possibly contributes to abnormal expression of pluripotent genes and low embryonic development. Treatment of interspecies BFC cloned embryos with TSA remodeled the covalent acetylation and methylation pattern in H3K9 to resemble epigenetic patterns in IVF counterpart embryos, and resulted in activation of some pluripotent genes that are important for embryonic development. However, genomic reprogramming of interspecies BFC cloned blastocysts did not follow the same reprogramming pattern observed in intraspecies DSH-embryos, and in vitro and in vivo developmental competence was not enhanced. We suggest that this specific cell line has rigid and restrictive epigenetic marks that cannot be modified by factors present in the cytoplasm of DSH oocytes, and epigenetic modifications in the BFC chromatin induced by TSA alone were not sufficient to improve nuclear reprogramming. Alternative methods to overcome some of the problems associated with abnormal nuclear reprogramming by using “epigenetic modifying compounds” in both cells and embryos may help to erase some epigenetic marks, and in turn, determine a new gene expression program that facilitates nuclear reprogramming and improves SCNT success rates.

Footnotes

Acknowledgments

We are grateful to Dr. Bob MacLean and Amanda Franklin for the surgical procedures and to animal care personnel at ACRES for the care of domestic cats. The assistance of Dr. Luis Marrero and Jennifer Simkin with the confocal microscopy photography is appreciated. This study was funded by a grant from ACRES and Louisiana State University System collaborative projects.

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

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