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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
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* Embryo Technology and Stem Cell Research Center and School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand;
and
Center for Regenerative Biology and Department of Animal Science, University of Connecticut, Storrs 06269
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: DNA methylation • epigenetics • histone acetylation • nuclear transfer • preimplantation embryo • swamp buffalo
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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reported successful creation of swamp buffalo cloned blastocysts. However, to date only 1 live-born cloned swamp buffalo, which died a few days after birth, has only been reported in the news (http://news.xinhuanet.com/english/2005-03/21/content_2724026.htm; last accessed 6 February, 2006). This low efficiency seems to be a common trait of SCNT in all species in which live animals have been obtained. The success of SCNT has increased significantly in species such as cattle because of the large number of studies conducted to improve the nuclear transfer procedure in the bovine. However, there have been very few studies with the swamp buffalo because of the limited population distribution of the species.
It is known that SCNT can reprogram a differentiated somatic cell to a totipotent state because cloned animals are born with all tissue types (Kikyo et al., 2000; Wade and Kikyo, 2002). However, incomplete nuclear reprogramming is believed to be the cause of developmental failure in cloned animals. Nuclear reprogramming is brought about by epigenetic mechanisms, such as DNA methylation and histone acetylation, which do not involve changes in the DNA sequence. The assessment of the epigenetic status in cloned embryos of different species is valuable to learn more about its role in early mammalian development and determine any relationship with low cloning efficiency. In the current study, we report for the first time DNA methylation and histone acetylation patterns in preimplantation, swamp buffalo SCNT embryos compared with in vitro-fertilized (IVF) embryos.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Donor Cell Preparation
Ear skin was biopsied from a swamp buffalo at a local abattoir and kept in modified Dulbeccco’s phosphate buffered saline (Invitrogen, Carlsbad, CA) at 4° C during transportation to the laboratory. Skin tissues were removed from cartilage and cut into small pieces (about 1 mm2) before being placed in a 60-mm culture dish (Nunc GmbH & Co. KG, Wiesbaden, Germany) and covered with a glass slide. Five milliliters of alpha modified Eagle’s medium (
MEM, Sigma, St. Louis, MO) plus 10% fetal bovine serum (FBS, Invitrogen) was added to the dish, and the tissue was cultured under a humidified atmosphere of 5% CO2 in air at 37.0° C.
Fibroblast outgrowths from the ear skin tissue were harvested and seeded into a 25-cm2 culture flask (Nunc, GmbH & Co. KG) in MEM plus 10% FBS. To preserve these cells, skin fibroblasts were frozen with 10% di-methyl sulfoxide (Sigma) in MEM plus 20% FBS at the third cell culture passage and stored in liquid nitrogen. For SCNT, frozen-thawed fibroblasts were cultured for 4 to 8 passages in MEM plus 10% FBS and used as nuclear donor cells. The subconfluent donor cells were resuspended in Emcare medium (ICPBio, Auckland, New Zealand) before injection and were not subjected to serum starvation.
Somatic Cell Nuclear Transfer
Swamp buffalo oocytes derived from abattoir-collected ovaries were matured in vitro for 22 h (Parnpai et al., 1999). Cumulus cells were mechanically removed by repeated pipetting in 0.2% hyaluronidase using a fine-tip pipette, and the oocytes were subsequently washed 5 times in Emcare medium. For enucleation, matured oocytes were placed into culture medium containing 5 µg of cytochalasin B (Sigma)/mL for 15 min. The zona pellucida above the first polar body was cut with a glass needle, and a small volume (about 10%) of the cytoplasm was extracted. Complete enucleation was confirmed by staining with Hoechst 33342 (Sigma).
To transfer the donor cells, individual fibroblasts were injected into the perivitelline space of the enucleated oocytes, and fusion was initiated by placing the couplets into Zimmerman fusion medium (Zimmermann and Vienken, 1982) followed by electrical stimulation using 2 DC pulses at 26 V, for 17 µs using a SUT F-1 stimulator (Suranaree University of Technology, Nakorn Ratchasima, Thailand). The reconstructed embryos were activated in 7% ethanol for 5 min and then cultured in modified synthetic oviductal fluid culture media (mSOF; Gardner et al., 1994) containing 3 mg of BSA (Sigma)/mL, 1.25 µg of cytochalasin D (Sigma)/mL and 10 µg of cycloheximide (Sigma)/mL for 5 h.
Activated embryos were cultured for 2 d in 100-µL droplets of mSOF under a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 38.5° C. The 8-cell stage embryos were selected and cocultured for another 5 d in 100-µL droplets of mSOF with bovine oviductal epithelial cells under a humidified atmosphere of 5% CO2 in air at 38.5° C. Embryos at 2-, 4-, and 8-cell, morula, and blastocyst stages were collected at 22, 28, 34, 108, and 132 h postactivation, respectively. All embryos at the 2-, 4-, 8-cell, morula, and blastocyst stages were fixed in 4% paraformaldehyde and stored at 4° C until further analysis.
In Vitro Fertilization
In vitro-fertilized embryos were selected as control embryos in this study because in vivo swamp buffalo embryos are very difficult to obtain due to the lack of experimental animals and the lack of information on superovulation and nonsurgical flushing methods. Although IVF embryos have less developmental competence than those produced in vivo, they do not suffer the extreme embryonic/fetal loss that is commonly seen in SCNT (Heyman et al., 2002). Additionally, in bovine SCNT studies, IVF embryos are commonly compared with SCNT embryos, even though in vivo-produced embryos are more readily attained (Daniels et al., 2000; Bourc’his et al., 2001; Wrenzycki et al., 2002). Therefore, whereas the use of in vivo-derived embryos is ideal, comparing IVF embryos with those produced by SCNT is a meaningful and well-accepted alternative.
To control for variation from genetic differences, buffalo semen from the same bull was used for the production of IVF embryos. Frozen semen was thawed at 37° C and washed twice by centrifugation at 500 x g for 7 min in Brackett and Oliphant (BO) medium (Brackett and Oliphant, 1975) without BSA but supplemented with 10 mM caffeine (caffeine-BO; Sigma). The pellet was resuspended in caffeine-BO at the concentration of 8 x 106 sperm/mL and then diluted with an equal volume of BO medium supplemented with 20 mg of BSA/mL and 20 µg of heparin (Sigma)/mL.
After 22 h of maturation, the cumulus oocyte complexes were transferred into 100-µL droplets of sperm suspension under mineral oil (Sigma) and incubated for 2 d under a humidified atmosphere of 5% CO2, 5% O2, 90% N2 at 38.5° C. Eight-cell stage embryos were selected and cocultured for another 5 d in 100-µL droplets of mSOF with bovine oviductal epithelial cells under humidified atmosphere of 5% CO2 in air at 38.5° C. Embryo collection and fixation were as described for SCNT embryos.
Immunostaining of Preimplantation Buffalo Embryos
Fixed embryos were washed in PBS before permeabilization in 0.5% Triton X-100 (Sigma). Embryonic DNA was denatured by incubation of the fixed embryos in 4 N HCl for 1 h at 37° C, and then to prevent nonspecific binding the fixed embryos were incubated in PBS containing 2% BSA (PBS-BSA 2%) for 1 h. Embryos were stained with a 1:50 dilution of primary mouse monoclonal antibody against 5-Methylcytosine (Eurogen-tech, San Diego, CA) and a 1:50 dilution of primary rabbit monoclonal antibody against acetylated histone H3 lysine 18 (H3K18; Cell Signaling-Technologies Inc., Boston, MA). Embryos were subsequently incubated in fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin G (IgG, 1:100; Jackson ImmunoResearch, West Grove, PA) and in Texas Red isothiocyanate (TRITC)-conjugated anti-rabbit IgG (1:100; Jackson ImmunoResearch), respectively. Individual embryos were mounted on slides by using 50% glycerol.
Confocal Microscopy
The immunostained embryos were observed with confocal microscopy (TCSSP2 True scanning, Leica Microsystems, Heidelberg, Germany). The DNA methylation and histone acetylation on H3K18 were analyzed and quantified by manually outlining a limited area of each individual nucleus at the brightest focal plane for the emission frequencies of FITC and TRITC. Emission intensities of all individual nuclei of embryos at the 2-, 4-, and 8-cell stages, and 20 nuclei per morula and 30 nuclei per blastocyst were captured and automatically converted to an arbitrary fluorescence unit and recorded. Arbitrary fluorescence intensity of each nucleus in an embryo was used to represent the DNA methylation/histone acetylation of that nucleus, and the level of global DNA methylation/histone acetylation of an embryo was represented by the sum of all analyzed nuclei in that embryo. Experiments were performed in 4 replicates per stage with 3 embryos per replicate (n = 12) for each embryo type. Appropriate controls for autofluorescence and nonspecific binding by the secondary antibodies were used to subtract the background from the arbitrary fluorescence measurements.
Blastocyst Counter-Stain and Cell Count
To distinguish cells of the inner cell mass (ICM) and trophectoderm (TE), embryos were counterstained. Briefly, the zona pellucidas of blastocysts at 6.5 d postactivation/insemination were removed by 0.5% protease (Sigma) and washed in SOF medium. The zona-free blastocysts were incubated in 10% rabbit anti-buffalo spleenocyte antibodies generated in our lab (Iwasaki et al., 1990) for 45 min before subsequent transfer into a mixture of 10% guinea pig complement (Sigma), 75 µg of propidium iodide (Sigma)/mL, and 100 µg of Hoechst 33258 (Bis-benzimide; Sigma)/mL for 45 min. The ICM cells (blue) and TE (red) cells were counted under UV light-fluorescence microscopy.
Statistical Analysis
Data analyses for differences in embryonic development, and for DNA methylation and histone acetylation, as assessed by relative amounts of arbitrary FITC and TRITC emission signals, respectively, were carried out by ANOVA using the GLM procedure of SAS (version 9.0, SAS Inst. Inc., Cary, NC). Main effects were embryo type (SCNT or IVF), stage of embryo development, and day of analysis. The interaction of embryo type and stage also was included in the model. We tested the normality and homogeneity of variance assumptions of all data sets before ANOVA. In an attempt to satisfy these assumptions, the histone acetylation and DNA methylation data were log transformed. The transformation was effective for all data sets except for the global DNA methylation, for which the normality was only slightly improved.
The predicted difference function of the GLM procedure was used to compare least squares means among the developmental stages and between the embryo types. A P value of < 0.05 was considered significant. To determine any relationship between DNA methylation and histone acetylation on a per embryo basis (sum of intensities from each nucleus within an embryo) and a per developmental stage basis (sum of intensities of all nuclei from the 12 embryos per stage within an embryo type), Pearson correlation was used.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Stable losses were seen throughout embryo development in both IVF and SCNT groups. In most cases, developmental rates for embryos derived from SCNT were less than those for embryos derived from IVF. A significant difference (P < 0.05) in development to the blastocyst stage was observed between the 2 types of embryos. Interestingly, the most dramatic loss of SCNT embryos, as much as 42%, occurred during development from the 8-cell to the morula stage. In the blastocysts, there were 3 times as many cells in the TE as in the ICM in embryos from both groups. No differences, however, were observed in the number of cells in ICM or TE, or the proportion of ICM over total cells between blastocysts from SCNT or IVF.
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. To compare with previously published data, we obtained and analyzed data for global DNA methylation of the entire embryo because these data were the only criterion used in studies of methylation status of SCNT embryos in other species. Additionally, we also obtained and analyzed data from individual nuclei in each embryo because the DNA methylation status in a particular nucleus is independent of that in another nucleus of the same embryo. Furthermore, mosaic DNA methylation of different nuclei in SCNT embryos has been observed (Dean et al., 2001). The DNA methylation in each nucleus, as represented by the means of arbitrary fluorescence intensities of nuclei of SCNT and IVF embryos at various embryonic developmental stages, is shown in Figure 1B. In both types of embryos, the relative levels of DNA methylation per nuclei decreased from the 2-cell stage until the 8-cell stage, where they reached a nadir. Then, levels of DNA methylation/nucleus began to increase at the morula stage and reached the greatest level at the blastocyst stage. Additionally, DNA methylation levels of nuclei from SCNT embryos were much more variable than those in IVF embryos, from 102 to 7,795 vs. 78 to 3,894 arbitrary units of FITC emission intensity. This demonstrates the highly heterogenic nature of DNA methylation in cloned embryos.
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), as represented by the sum of arbitrary fluorescence intensities of all analyzed nuclei in the embryo, was also greater (P < 0.05) in embryos derived from SCNT at the 4-cell, 8-cell, and morula stages.
Histone Acetylation in Preimplantation Swamp Buffalo Embryo
Representative images of SCNT and IVF buffalo embryos immunostained for acetylated histones are shown in Figure 2A. Mean relative levels of histone acetylation from each nuclei of SCNT and IVF embryos at various stages of embryonic development are shown in Figure 2B. Overall, we observed similar patterns in the relative levels of histone acetylation in nuclei of SCNT and IVF embryos throughout preimplantation development. In embryos of both types, histone acetylation increased moderately from 2- to 4-cell stages, which was followed by a decrease to a nadir at the morula stage. Subsequently, histone acetylation/nucleus increased again to the greatest level at the blastocyst stage. Similar to the DNA methylation data, the relative levels of histone acetylation of individual nuclei of the SCNT embryos were also highly variable, from 45 to 3,279 in the SCNT embryos vs. 43 to 2,889 arbitrary units of TRITC emission intensity in the IVF embryos. Globally, greater relative levels of histone acetylation were observed in the SCNT embryos than in the IVF embryos at the 4-(P < 0.01) and 8-cell (P < 0.02) stages (Figure 2C).
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; Kharroubi et al., 2001). Therefore, we analyzed correlations of DNA methylation and histone acetylation in buffalo embryos in the current study. Significant positive correlations were found between relative levels of DNA methylation and histone acetylation in IVF (r = 0.65) and SCNT embryos (r = 0.87) at the 2-cell stage. At the 4-cell stage, the correlation was significant (P < 0.05) in the SCNT embryos (r = 0.85) but not for the IVF embryos (r = 0.46). Interestingly for the 8-cell embryos, correlations between global histone acetylation and DNA methylation were reversed in the 2 embryo types, i.e., a significant positive correlation (r = 0.76) was found in the IVF embryos but not in the SCNT group (r = –0.1). When correlations in individual embryos were examined at this stage, 10 of 12 of the IVF embryos had significant positive correlations (r > 0.75), whereas most of SCNT embryos (9 of 12) showed low or negative correlations (–0.3 < r < 0.3). At the blastocyst stage, little correlation between DNA methylation and histone acetylation was found among the IVF or SCNT embryos (r < 0.3). |
DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Santos et al., 2003; Beaujean et al., 2004). There is evidence that aberrant DNA methylation leads to loss of imprinting (Li, 2002), which plays an essential role in fetal development. Furthermore, DNA methylation cooperates with histone deacetylation and heterochromatic proteins to cause the heterochromatin configuration and global gene silencing.
The DNA methylation has been studied in cloned embryos from numerous species. With the exception of the pig, all species have exhibited global DNA hypermethylation (Dean et al., 2001; Kang et al., 2001). Additionally, aberrant gene expression was also reported in cloned embryos (Daniels et al., 2001; Wrenzycki et al., 2001). The latter observation may be related to DNA methylation, which can directly and indirectly block transcription leading to aberrant gene expression and possible subsequent fetal abortion and developmental abnormalities in cloned animals.
During in vitro culture of SCNT and IVF buffalo embryos, a greater developmental arrest rate was found in SCNT embryos, and this developmental arrest was mostly observed during the 8-cell to morula stages. This observation is in agreement with a report by Kitiyanant et al. (2001), who reported a 33% blastocyst rate after a decline from a 77% cleavage rate in swamp buffalos embryos during in vitro production. This arrest in embryonic development is correlated with the beginning of de novo DNA methylation as shown in bovine IVF embryos at this stage (Dean et al., 2001). The sharp decline in the development rate of the SCNT embryos at this stage observed in this study corresponds with the drastic changes in DNA methylation. Because DNA methylation has been shown to suppress gene expression, it is plausible that the hypermethylation may cause improper expression of developmentally important genes, which in turn leads to the developmental failure of the SCNT embryo.
Although there are no prior data for comparison in swamp buffalo SCNT or IVF embryos, the hypermethylation of DNA in SCNT swamp buffalo embryos and the dynamic changes of DNA methylation during embryo development observed in the current study are consistent with previous work in bovine SCNT embryos and other species (Dean et al., 2001; Kang et al., 2001; Beaujean et al., 2004). In bovine SCNT embryos, it was shown that both greater DNA de novo methylation and insufficient DNA demethylation account for the aberrant hypermethylation of DNA (Dean et al., 2001). It was also shown that greater levels of maternal DNA methylatransferase 1 (DNMT1), which is responsible for maintaining DNA methylation, were present in SCNT bovine embryos (Wrenzycki et al., 2001). Our previous work showed that DNMT1, DNMT3A, and DNMT3B (genes responsible for de novo DNA methylation) were expressed at greater levels at the 8-cell and blastocyst stages in swamp buffalo SCNT embryos as compared with IVF embryos (Suteevun et al., 2005). Taken together, the hypermethylation observed here could result from lesser levels of demethylation and greater levels of de novo methylation in SCNT embryos. Additionally, the greater variation of DNA methylation in different nuclei of each SCNT buffalo embryo observed here may indicate that nuclear reprogramming occurs throughout preimplantation development and is incomplete in a proportion of the cells in the embryos.
Histone acetylation neutralizes the histone positive charge through binding of the acetyl group on lysine residue at histone N-terminus and allows chromatin decondensation and gene activation (Kikyo et al., 2000; Wade and Kikyo, 2002; Jaskelioff and Peterson, 2003). Enright et al. (2003) indicated that histone acetylation patterns in somatic cells can be remodeled in culture and that the lysine 18 on histone H3 (H3K18) is a common acetylated lysine position that is acetylated throughout the bovine embryonic stages. Therefore, we also chose to use the same antibody to detect histone acetylation status and found moderate histone hyperacetylation among the swamp buffalo SCNT embryos at all preimplantation stages except for the blastocyst stage. Additionally, when the global levels of histone acetylation were compared between SCNT and IVF embryos, we found significant hyperacetylation at the 4-cell and 8-cell stages. These findings agree with the study by Santos et al. (2003), who reported hyperacetylation of H3K9 in bovine SCNT embryos at all embryonic stages examined. Surprisingly, we observed the least histone acetylation level at the morula stage among all preimplantation stages analyzed. The significance of this phenomenon in embryo development is unclear; however, it is possible that the pattern observed here is unique to swamp buffalo embryos.
Previously, negative correlations between the levels of DNA methylation and histone acetylation were found on chromatin of individual genes subjected to epigenetic regulation (Goto and Monk, 1998). We analyzed correlations between these 2 chromatin modifications on a whole cell and whole embryo basis. Also, for the first time, we studied correlations during many different stages of preimplantation development, whereas previous studies were limited to studying bovine blastocysts (Enright et al., 2003; Santos et al., 2003), which were found to be both hypermethylated and hyperacetylated. We found that swamp buffalo embryos at different stages of development have different correlations between global DNA methylation and histone acetylation, suggesting dramatic changes in chromatin modification during the early preimplantation period. Inconsistent correlations between DNA methylation and histone acetylation at the 4- and 8-cell stages in the IVF vs. SCNT embryos indicates that the interaction of these epigenetic mechanisms influences early embryonic development.
In summary, we generated SCNT swamp buffalo embryos and compared their epigenetic status with that of IVF embryos. We found that the SCNT embryos are not only hypermethylated and hyperacetylated but are also more heterogeneous in DNA methylation and histone acetylation among different cells of the same embryo than those in IVF embryos. Additionally, the anomalous correlations between DNA methylation and histone acetylation may contribute to the developmental failure seen in SCNT embryos.
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Footnotes |
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2 Corresponding author: xiuchun.tian{at}uconn.edu
Received for publication December 1, 2005. Accepted for publication March 1, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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