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Reproductive Technology |
Department of Animal Science/Center for Regenerative Biology,4 University of Connecticut, Storrs, Connecticut 06269
Laboratory of Cell Genetics and Embryo Transfer,5 Kagoshima Prefectural Cattle Breeding Institute, Kagoshima 899-8212, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, embryo
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A DNA demethylation agent, 5-aza-2'-deoxycytidine (5-aza-dC) has previously been shown to induce overexpression of imprinted genes in mouse embryonic fibroblast cells by lowering DNA methylation levels [11]. A histone-deacetylase inhibitor, trichostatin A (TSA) enhances the pool of acetylated histones and also overexpresses imprinted genes in embryonic stem cells [12, 13]. The link between histone acetylation and DNA methylation has been established in imprinted genes as well as in the inactivated X chromosomes in females, but it is unknown if global levels of DNA methylation or histone acetylation are altered by these epigenetic-modifying compounds in somatic cells [14]. Furthermore, the effects on development of embryos cloned from donor cells subjected to treatment by these agents have not been fully tested.
The objectives of this research were as follows: 1) to evaluate the effects of 5-aza-dC and TSA on global epigenetic changes in somatic cells, 2) to investigate the link between global levels of histone acetylation and DNA methylation in somatic cells, and 3) to determine if treatment of somatic cells with TSA or 5-aza-dC results in altered development of cloned embryos.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult Somatic Cell Collection and Culture
Adult fibroblast cells used in the present study were derived from a 15-yr-old Holstein cow that has been previously cloned [9]. Briefly, a skin biopsy from the ear of the donor animal was cut into small pieces (2–3 mm2), which were cultured as tissue explants in Dulbecco modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and antibiotics (penicillin, 100 IU/ml) at 37.5°C in a humidified atmosphere of 5% CO2. After 7 days in culture, fibroblast cell monolayers had formed around the tissue explants. The explants were then removed, and the fibroblast cells were cultured to confluence. To passage cells, confluent cells were disaggregated by incubation in 0.1% trypsin and 0.02% EDTA solution and allocated to three dishes. Normally, each cell passage was equivalent to approximately two cell-doublings and was sustained for 4 days. Cells were grown to confluence at passage 5 and subsequently seeded into DMEM plus 10% FBS containing 0, 0.08, 0.3, 1.25, or 5.0 µM of either TSA or 5-aza-dC. These levels were selected to produce dose-response curves, because to our knowledge, the dose-response effects of these drugs have not been tested in fibroblast cells for NT. Cells were subsequently subjected to analysis for histone acetylation and DNA methylation levels by flow cytometry or used for NT.
Quantification of Total Levels of Cellular DNA Methylation and Histone Acetylation
The levels of total cellular histone acetylation and DNA methylation were quantified by flow cytometry through measuring the fluorescent levels of cells after incubation in antihistone H3 (lysine 18; Upstate Biotechnologies, Lake Placid, NY) or anti-5-methyl cytosine (Maine Biotechnologies, Portland, ME) primary antibodies and fluorescent-conjugated secondary antibodies. The procedures for flow cytometry were followed as described previously [15, 16] with the following modifications: Cells were trypsinized and resuspended in cold "saline GM" (6.1 mM glucose, 137 mM NaCl, 5.4 mM KCl, 1.5 mM Na2HPO47H2O, 0.9 mM KH2PO4, and 0.5 mM EDTA), followed by fixing in 100% ethanol. To measure levels of acetyl histone H3 or 5-methyl cytosine, cells were incubated in the primary antibody (1:100 dilution), then washed in PBS plus 5% FBS and subsequently in fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:100 dilution) for 30 min at 37°C. To stain for DNA, cells were incubated in PBS containing 30 µg/ml of propidium iodide (PI) and 0.3 mg/ml of RNase A. To eliminate multicell aggregates, cells were filtered through a 30-µm nylon mesh (Spectrum, Los Angeles, CA). Ten thousand cells were collected with a fluorescence-activated cell sorter (FACS Caliber; Becton Dickinson, San Jose, CA) and were analyzed using CELL QUEST 3.1 software (Becton Dickinson). Appropriate controls for autofluorescence and nonspecific binding by the secondary antibodies were included. Flow cytometric analysis of cells was carried out in duplicate and repeated three times.
In Vitro Fertilization, NT, and Embryo Culture
Fertilization and culture of oocytes were performed as described previously [17]. Briefly, sperm were washed in a modified Brackett-Oliphant medium (supplemented with 10 mM caffeine and 4 mg/ml of BSA), and following 6 h of sperm-oocyte coincubation, oocytes were cultured in CR1aa medium. After 48 h of culture, cumulus cells were removed from presumptive zygotes. The cleaved embryos were cultured further in CR1aa media supplemented with 5% FBS with cumulus cell coculture for another 5 days at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Embryos that developed to the blastocyst stage on Day 7 postfertilization were fixed in 4% formaldehyde.
Nuclear transfer was carried out as previously described [9]. Briefly, treated and untreated donor cells were trypsinized, washed by centrifugation (200 x g, 5 min), and resuspended in PBS. Cells with an approximate diameter of 10–15 µm were transferred to the perivitelline space of the enucleated recipient cytoplast. Following transfer, the cell-cytoplast complexes were induced to fuse with two pulses of direct current at 2.5 kV/cm for 10 µsec each by an Electrocell Manipulator 200 (BTX, San Diego, CA). Fusion was then confirmed by microscopic analysis. All fused embryos were activated by culturing with cycloheximide (10 µg/ml) in CR1aa media for 5 h.
Following activation, fused couplets were cultured for 48 h in CR1aa medium at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Cleavage rates were recorded, and embryos were cultured further in CR1aa medium supplemented with 5% FBS with cumulus cell coculture for an additional 5 days. Reconstructed embryos that had reached the blastocyst stage on Day 7 were fixed in 4% formaldehyde. Nuclear transfer experiments were replicated three times.
Confocal Microscopy
Fixed blastocysts were washed in 0.9% saline, followed by subsequent blocking for nonspecific antibody binding (PBS plus 1% BSA plus 0.5% Triton X-100). Embryos were then incubated in acetyl histone H3 (lysine 18) primary antibody (1:100 dilution), followed by incubation in FITC-conjugated secondary antibody (1:100 dilution). Embryos were mounted in PBS containing 10 µg/ml of PI, which stains for DNA, and observed with a confocal microscope (TCSSP2 True scanning; Leica Microsystems, Heidelberg, Germany). For quantification of acetyl histone H3, the mean FITC fluorescence amplitude of each embryo was recorded using the region-of-interest function. Appropriate controls for autofluorescence and nonspecific binding by the secondary antibody were included.
Statistical Analysis
Statistical analysis was performed using the general linear models (GLM) procedure of the Statistical Analysis System [18]. Flow cytometric data were analyzed within cell cycle and treatment type, with a main effect of treatment dose. The main effects of data on cloned embryo development were treatment type (5-aza-dC or TSA) and treatment dose. The main effect for immunostaining and cell count data was treatment type. When the main effects or interactions were significant (P < 0.05), the predicted difference (PDIFF) function of the GLM procedure was used to compare individual least-squares means.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fluorescence-activated cell sorting analysis was used to assess the cell cycle-stage distribution and levels of histone acetylation and DNA methylation of donor cells. Overall, 85% of TSA-treated and 78.8% of 5-aza-dC-treated cells were at the G0/G1 phase of the cell cycle. For these cells, the total levels of DNA methylation were significantly reduced by treatment with 5-aza-dC, a DNA demethylation reagent. Interestingly, this was associated with increased levels of histone H3 acetylation in these cells, although these changes were not significant until the level of 5-aza-dC reached the highest dose used, 5 µM (Fig. 1A). The total acetylated histone H3 levels in G0/G1 cells were significantly increased following treatment with TSA, a histone-deacetylase inhibitor, at both 1.25 and 5 µM (Fig. 2A). However, TSA had no significant effect on DNA methylation in these cells at all doses tested (Fig. 2A).
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For cells at the G2/M phase of the cell cycle, neither methylation nor acetylation levels were altered by treatment with 5-aza-dC (Fig. 1B). However, the total acetylated histone H3 levels in these cells were increased following treatment with TSA at 1.25 and 5 µM (Fig. 2B), whereas no changes were observed for DNA methylation levels following TSA treatment. The changes in histone acetylation were also confirmed at the maximum dose of TSA by Western blot analysis (data not shown).
Although flow cytometry did not detect changes in methylation or acetylation levels of cells treated with 0.08 µM 5-aza-dC or TSA, an alteration was observed in cell morphology after treatment with this low dose of these drugs (Fig. 3). Untreated cells had a compact cytoplasm and appeared slightly concave in shape, whereas cells treated with TSA or 5-aza-dC, even at the lowest dose of 0.08 µM, appeared flattened and elongated (Fig. 3). These observed changes in cell morphology became more pronounced as the concentration of TSA or 5-aza-dC increased (Fig. 3).
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Development of Embryos Cloned from Cells Treated with TSA or 5-aza-dC
The lowest doses that caused DNA hypomethylation or histone hyperacetylation were 0.31 µM 5-aza-dC or 1.25 µM TSA; therefore, these were selected for treatment of donor cells for NT. Although neither drug at 0.08 µM caused significant changes in the levels of methylation or acetylation, significant morphological changes were induced. For this reason, 5-aza-dC and TSA at 0.08 µM were also selected to treat donor cells for NT.
Treatment of donor cells with either TSA or 5-aza-dC affected development of embryos produced from these cells (Table 1). Donor cells treated with 0.08 or 0.31 µM 5-aza-dC resulted in decreased rates of embryo development in a dose-related fashion. However, the speed of early embryo development to the 2-cell stage was increased in embryos cloned from 5-aza-dC-treated donor cells. In contrast to the results with 5-aza-dC treatment, donor cells treated with 0.08 µM TSA showed improved development to blastocyst versus control (35.1% vs. 25.1%, respectively). Embryos cloned from cells treated with a higher dose of TSA (1.25 µM), however, had lower blastocyst development than controls, suggesting an overdose effect.
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To our surprise, embryos cloned from TSA-treated donor cells had less histone acetylation than control embryos cloned from untreated cells or embryos produced by in vitro fertilization (Fig. 4 and Table 2). Furthermore, the number of cells in embryos derived from the TSA-treated cells was similar to that in untreated controls (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both 5-aza-dC and TSA are widely used in imprinting studies and have been shown to enhance expression of the transcriptionally silent alleles of imprinted genes [11, 19, 20]. However, the roles of these two drugs in global DNA methylation and histone acetylation of somatic cells have not been clearly established. Furthermore, to our knowledge, the interaction between DNA methylation and histone acetylation has not been characterized in somatic cells. In the present study, we employed a method that was established for the measurement of global levels of methylation and acetylation [15], and we tested the effects of a wide range of TSA or 5-aza-dC concentrations on cells destined for NT. We found that 5-aza-dC was effective not only in inducing global demethylation but also in inducing global histone hyperacetylation. The effect of 5-aza-dC on histone acetylation likely is indirect, because increased DNA methylation is closely associated with decreased histone acetylation. It has been shown that methylated DNA attracts a methyl-CpG-binding protein (MeCP2), which recruits histone deacetylase, and maintains a low level of acetylation [14]. Therefore, reducing DNA methylation by 5-aza-dC caused dissociation of methyl-binding proteins such as MeCP2, which in turn removed histone deacetylase and resulted in histone hyperacetylation.
We also found that a relatively high concentration (1.25 or 5 µM) of TSA induced global histone hyperacetylation in G0/G1- and G2/M-stage cells but had no effect on DNA methylation, at least at the levels tested. This may be caused by the specificity of TSA, which inhibits histone deacetylase but does not affect DNA methyl transferases, and therefore DNA methylation levels were not modified [13].
Our results showing that global epigenetic marks can be modified by treatment with TSA or 5-aza-dC led us to test if these agents could be used to reduce DNA methylation and/or increase histone acetylation in NT donor cells. Thus, making the epigenetic characteristics of the donor cell genome similar to those of gametes or blastomeres may make it more amenable to reprogramming [21–24]. However, we found that treating donor cells with 5-aza-dC reduced blastocyst formation of cloned embryos. At the lowest concentration found to be effective in reducing global DNA methylation (0.31 µM), we observed a nearly 6-fold decrease (from 25.1% to 4.2%) in blastocyst development. However, at a concentration that caused morphological changes but no DNA hypomethylation in the donor cells (0.08 µM), we observed a 2.5-fold decrease (from 25.1% to 9.7%) in blastocyst development. The use of 5-aza-dC for treating donor cells has been reported previously in both cow and mouse [25, 26]. However, the detrimental effects of 5-aza-dC on blastocyst development were much less severe in those studies. Jones et al. [25] treated bovine fetal fibroblast cells with much higher doses of 5-aza-dC (1 or 5 µM) and found that blastocyst development of cloned embryos was 5%, compared to 15.5% and 7.9% for those from donor cells subjected to serum starvation or serum feeding, respectively, although none of these differences was significant. According to Zhou et al. [26], mouse stem cells treated with 5-aza-dC resulted in decreased cloned blastocyst development (from 30% to 18%). The difference between these previous studies and the present results may be explained by the treatment of the cells; these observations may provide insight regarding the design of future experiments. It is known that 5-aza-dC blocks DNA methylation in newly replicated DNA molecules [27]. To induce DNA demethylation, cells were treated with 5-aza-dC for 72 h, thereby allowing three rounds of DNA duplication with little or no DNA methylation (cell population doubling time is 
24 h). This ensured that sufficient demethylation would occur in the donor cells used in the present study. In the study of Jones et al. [25], the method of donor cell treatment with 5-aza-dC was not described. Therefore, the degree of DNA demethylation is not directly comparable between the previous and the present study. However, the consensus from these studies [25, 26, present study] suggests that lowering the levels of DNA methylation in donor cells does always not improve development of cloned embryos. At high concentrations, 5-aza-dC may have been cytotoxic to the donor cells. Additionally, prolonged treatment at lower concentration may have caused severe hypomethylation, which resulted in disrupted expression of essential genes important for embryo development. Therefore, further experiments are required to test lower concentrations and shorter durations of 5-aza-dC treatment on donor cells.
Treating donor cells with TSA, by contrast, improved NT embryo development. Previous reports indicated that treatment of mouse stem cells with TSA reduced development of cloned embryos [26]. The difference between these and the present results may be caused by differences in the concentrations of TSA used. Before NT, we treated donor cells with a wide range of TSA concentrations and identified the lowest effective concentration that was capable of inducing histone hyperacetylation (1.25 µM). The lowest concentration tested (0.08 µM) did not cause hyperacetylation but resulted in observable morphological changes similar to those described previously [28]. It is this lower concentration of TSA (0.08 µM) that improved development of cloned embryos in the present study, whereas the higher concentration (1.25 µM) inhibited embryo development. The detrimental effect of a higher dose of TSA on embryo development, as seen in the present study and in that of Zhou et al. [26], may be explained by the fact that treatment of cells with high concentrations of TSA causes chromatin breaks and apoptosis [29]. However, TSA at 0.08 µM caused sufficient changes to allow the donor cells to be more easily reprogrammed without adverse effects. These changes, despite being below the detection limit, likely are histone hyperacetylation, because the effect of TSA is specific [13]. Thus, the improved development of cloned embryos observed in the present study may have resulted from the erasure of preexisting epigenetic marks in the donor cells before NT.
The data from the present study indicate that concentrations of 5-aza-dC and TSA used in imprinting studies cannot be applied directly to treat donor cells in NT studies without further evaluation [11, 19, 20]. This is because cells in imprinting studies are intended for DNA/RNA extraction and expression analysis, and their DNA integrity or subsequent viability is not critical. However, for NT, the donor nucleus is expected to remain viable and capable of directing development of the cloned individual. Therefore, it is important to identify the optimal concentrations of TSA and 5-aza-dC for NT studies by using embryo development as an indicator.
It is surprising that NT embryos reconstructed from TSA-treated cells had lower levels of histone acetylation than embryos from untreated donor cells or from those produced by in vitro fertilization. This may have been caused by a rebound effect from TSA treatment, because the effect of TSA is reversible [29]. However, it is unclear whether the observation that cloned embryos have less acetylation is significant, because 1) NT embryos appeared morphologically normal, 2) development of embryos cloned from TSA-treated donor cells was higher than that of control embryos, and 3) the mean cell numbers of embryos cloned from TSA treated and nontreated donor cells were similar and within the allowable ranges for further embryo development [30]. All of these observations indicate that TSA at low doses is nontoxic to donor cells. To further understand these observations, embryos cloned from TSA-treated donor cells should be subjected to the in vivo development test, which is the scope of our future studies.
In conclusion, we tested a wide range of concentrations of two drugs that alter the epigenetic status of cells for the specific purpose of NT and assessed the effects of these two drugs on histone acetylation both before and after the cloning procedure. The principal finding of the present study is that cloning efficiency, as judged by embryo in vitro development, can be improved by artificially modifying the acetylation level of histone in donor cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: X.C. Tian, Agricultural Biotechnology Laboratory, 1392 Storrs Road, U 4243, University of Connecticut, Storrs, CT 06269. FAX: 860 486 8809; xtian{at}canr.uconn.edu
3 These authors contributed equally to this work
Received: 7 April 2003.
First decision: 1 May 2003.
Accepted: 8 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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