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Methylation and Acetylation Characteristics of Cloned Bovine Embryos from Donor Cells Treated with 5-aza-2'-Deoxycytidine¹

Department of Animal Science/Center for Regenerative Biology,⁴ University of Connecticut, Storrs, Connecticut 06269

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Differentiated somatic cells and embryos cloned from somatic cells by nuclear transfer (NT) have higher levels of DNA methylation than gametes and early embryos produced in vivo. Reducing DNA methylation in donor cells before NT by treating them with chemicals such as the DNA methyl-transferase inhibitor (5-aza-2'-deoxycytidine; 5-aza-dC) may improve cloning efficiency of NT embryos by providing donor cells with similar epigenetic characteristics as in vivo embryos. Previously, high levels of this reagent were used to treat donor cells, and decreased development of cloned embryos was observed. In this study, we tested a lower range (0.005 to 0.08 µM) of this drug and used cell cycle distribution changes as an indicator of changes in the characteristics of donor cells. We found that at 0.01 µM 5-aza-dC induced changes in the cycle stage distribution of donor cells, increased the fusion rate of NT embryos, and had no deleterious effect on the percentage of blastocyst development. Levels of 5-aza-dC greater than 0.01 µM significantly decreased embryo development. Embryos cloned from donor cells treated with a low dose of 5-aza-dC had higher levels of DNA methylation than embryos produced by in vitro fertilization, but they also had higher levels of histone acetylation. Although 5-aza-dC at 0.04 µM or higher reduced DNA methylation and histone acetylation levels to those of in vitro-fertilized embryos, development to blastocyst was reduced, suggesting that this concentration of the drug was detrimental. In summary, 5-aza-dC at 0.01 µM altered donor cell characteristics while showing no deleterious effects on embryos cloned from treated cells.

assisted reproductive technology

	INTRODUCTION

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The successful reprogramming of a differentiated donor cell back to a totipotent stage, by somatic cell nuclear transfer (NT), has been demonstrated in several species. However, in all cases, although the efficiency of development to blastocyst can be comparable or even higher than that by in vitro fertilization (IVF) at least in the bovine, fetal loss during gestation remains elevated. Even if the cloned fetuses are carried to term, incidences of developmental anomalies and neonatal deaths are commonplace. These cloning failures may be explained by incomplete reprogramming of the donor cell genome. Although differentiated cells contain complete genetic information, they are modified epigenetically (by methylation of DNA and acetylation of histones) according to tissue specificity. Any faults in a donor cell will be amplified in subsequent development, as failure to alter its epigenetic marks will lead to an embryo not fully totipotent and affect further development and differentiation [1, 2].

In natural reproduction, relatively low levels of DNA methylation exist in male and female gametes, which are further demethylated during early development [3, 4]. Somatic cells, however, have been shown to have much higher levels of DNA methylation than either gametes or early embryos, due to tissue differentiation. In nuclear transfer (NT), highly methylated somatic donor cells are used to generate cloned embryos, which in turn, have been shown to be abnormally hypermethylated [1, 5–7]. Therefore, alteration of the epigenetic marks in donor cells before NT may improve the ability of the donor cell to become fully reprogrammed by the recipient karyoplast. Previously, we conducted studies in which the preexisting epigenetic markers were reduced by treating donor cells with either 5-aza-2'-deoxycytidine (5-aza-dC), which reduces DNA methylation, or trichostatin A (TSA), which increases histone acetylation [8]. We found that the lowest level of 5-aza-dC that could elicit detectable changes in donor a cell's methylation status as measured by immunostaining was deleterious to early embryo growth. This would suggest that the method used to quantify the changes in DNA methylation was not sensitive enough to detect the alterations in donor cells. In this study, we aimed to reduce the effective levels of 5-aza-dC in donor cell treatment. More sensitive detection methods, such as changes in the distribution of donor cells throughout the cell cycle stages, as well as assessing DNA replication by bromodeoxyuracil (BRDU) incorporation, were used because 5-aza-dC has been shown to lower the proportion of cycling cells in culture. The objectives of this research were as follows: 1) to ascertain the lowest effective dose of 5-aza-dC for treatment of donor cells that does not decease development of cloned embryos to the blastocyst stage, and 2) to investigate the epigenetic changes in embryos cloned from treated donor cells.

	MATERIALS AND METHODS

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All reagents were purchased from Sigma Chemical Co (St. Louis, MO) unless otherwise stated.

Adult Somatic Cell Collection and Culture

This project was approved by the Institutional Animal Care and Use Committee of the University of Connecticut. Adult fibroblast cells used in this study were derived from a 15-yr-old Holstein cow that had previously been cloned [9]. Procedures for cell culture were described previously [8] with the following modifications: cells were grown to confluence at passage 5 and subsequently seeded into Dulbecco modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS) containing 0.005, 0.01, 0.02, 0.04, or 0.08 µM 5-aza-dC and were cultured for an additional 3 days. To monitor changes caused by the 5-aza-dC treatment, two parameters were used: 1) DNA replication analyzed by BRDU labeling and 2) changes in the distribution of cells throughout the cell cycle analyzed by flow cytometry.

Cell Proliferation and Cell Cycle Analysis

To assess the effects of 5-aza-dC on DNA replication, cells were incubated with 5 µg/ml BRDU in DMEM plus 10% FBS for 2 h in a humidified atmosphere at 37°C. Cells were then fixed in 100% methanol and incubated with anti-BRDU primary antibody (1:100 dilution), washed in PBS plus 5% FBS, and subsequently incubated in fluorescein isothiocyanate-(FITC) conjugated secondary antibody (1:100 dilution). To stain for DNA, cells were incubated in PBS containing 30 µg/ml propidium iodide and 0.3 mg/ml RNase A.

Cells were filtered through 30-µm nylon mesh (Spectrum, Los Angeles, CA) to remove multicell aggregates. 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). Flow cytometric analysis of cells was performed in duplicate and repeated three times, including appropriate controls for autofluorescence and nonspecific binding by the secondary antibody.

Vitro Fertilization, NT, and Embryo Culture

All oocytes used in this study were purchased from Bomed Inc. (Madison, WI). The procedures for fertilization and culture of oocytes were similar to those described previously with embryos that developed to the blastocyst stage on Day 7 postfertilization fixed in 4% formaldehyde [8]. Nuclear transfer was performed as previously described [8], and embryos that reached the blastocyst stage on Day 7 postactivation were fixed in 4% formaldehyde. Nuclear transfer experiments were replicated three times.

Confocal Microscopy

Fixed blastocysts were washed in PBS, permeabilized in 0.5% Triton X-100, followed by incubation in 4 M hydrochloric acid. Embryos were subsequently washed in PBS plus 0.05% Tween-20 (wash solution), and blocked in 2% BSA. Next, the embryos were incubated in 5-methyl cytosine primary antibody (Eurogentec, Philadelphia, PA; 1:100 dilution), washed, and then incubated in acetyl histone H3 (lysine 18) primary antibody (Upstate Biotechnologies, Lake Placid, NY; 1:100 dilution).

Embryos were subsequently incubated in FITC (1:200 dilution), then rhodamine (TRTIC; 1:200 dilution)-conjugated secondary antibodies, mounted in PBS plus 50% glycerol, and observed with a confocal microscope (TCSSP2 True scanning; Leica Microsystems, Heidelberg, Germany). For quantification of acetyl histone H3 and 5-methyl cytosine, the mean FITC and TRITC fluorescence amplitudes of each embryo were recorded using the region of interest function. Each slide contained all the 5-aza-dC groups and an IVF group. The fluorescence of each NT embryo was adjusted to the control IVF embryos on that day. Appropriate staining controls were included for autofluorescence of each of the primary antibodies alone and nonspecific binding of each of the secondary antibodies without the primary antibodies. Additional controls were also included for primary and secondary antibody cross-reactivity (i.e., primary antibody 1 with secondary antibody 2, and primary antibody 2 with secondary antibody 1, etc.).

Statistical Analysis

Statistical analysis was performed using the general linear model (GLM) procedure of the Statistical Analysis System [10]. Flow cytometry data were analyzed within cell cycle and treatment type, with a main effect of 5-aza-dC dose. The main effect of data on cloned embryo development and immunostaining was 5-aza-dC treatment dose. When the main effect was significant (P < 0.05), the predicted difference function (PDIFF) function of the GLM procedure was used to compare individual least-square means. Data presented in tables are least square means and pooled SEM for each category.

	RESULTS

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Effects of 5-aza-dC on Donor Cells

The effect of 5-aza-dC on donor cells was measured by changes in cell cycle stage distribution and BRDU incorporation. These were conducted so that the lowest effective dose of 5-aza-dC could be determined using methods more sensitive than immunostaining. For donor cell cycle stage distribution, we observed a significant decrease in the number of cells in the S phase with drug treatment, starting from 0.04 µM of 5-aza-dC compared with nontreated controls (Table 1). This is associated with an increase in the proportion of cells at G₀/G₁, suggesting that 5-aza-dC at this level was effective in preventing cells from entering into the DNA replication stage of the cell cycle. An even more sensitive detection was the observation of the proportion of cells in G₂/M phase, which was significantly altered by 0.005 µM of 5-aza-dC (Table 1). However, due to large variations, the effect of 5-aza-dC treatment on DNA replication as measured by BRDU incorporation could not be detected at these levels (data not shown). This suggests that cell cycle changes are a very sensitive indicator of cell proliferation. Additionally, changes in the dose of 5-aza-dC also elicited morphological changes in cells, in that untreated cells had a more uniform cytoplasm and significant connection to the substratum compared with treated cells, which appeared elongated and less connected (Fig. 1).

View larger version (120K): [in this window] [in a new window]   FIG. 1. Morphology of donor cells treated with 0, 0.005, 0.01, 0.02, 0.04, or 0.08 µM 5-aza-dC. Images were magnified x100

Development of Embryos Cloned from 5-aza-dC Treated Donor Cells

A concentration of 0.08 µM 5-aza-dC was included as a control because this was the lowest level used in our previous study, and it had resulted in significant decreases in blastocyst development. As we found previously, donor cells treated with 0.08 µM 5-aza-dC resulted in a significant decrease in blastocyst development rate (8.8%) compared with that of untreated control donor cells. Donor cells treated with 0.01 µM 5-aza-dC showed increased fusion, cleavage, and blastocyst rates, although the changes in the latter two parameters were not significant. An even lower dose of 0.005 µM 5-aza-dC induced cell cycle changes, suggesting that 5-aza-dC treatment in donor cells elicited changes that were continued in the subsequent development of cloned embryos (Table 2). In the present study, there was also a trend toward decreased embryo development when the 5-aza-dC dose was greater than 0.01 µM. The sensitivities of donor cells and cloned embryos to 5-aza-dC treatment as detected by cell cycle distribution changes, or by blastocyst development, were different. When comparing the treatment response of donor cells with that of the cloned embryos, the embryos appeared to be less sensitive than donor cells, with the former showing a measurable response only when 0.02 µM or higher concentrations of 5-aza-dC were used.

Although we were not able to quantify the changes in levels of methylation in donor cells using immunostaining or FACS analysis at a low level of 5-aza-dC treatment, all embryos cloned from donor cells treated with 5-aza-dC had reduced levels of methylation compared with NT embryos cloned from untreated cells (Table 3, Fig. 2). These changes in DNA methylation were not significant until 0.04 µM of 5-aza-dC or a higher dose was used. When donor cells were treated with 0.04 µM or higher of 5-aza-dC, the resulting NT embryos also had decreased histone acetylation levels compared with that of NT controls. Thus, the levels of DNA methylation and histone acetylation were positively linked in bovine embryos cloned from 5-aza-dC treated donor cells. As we found previously, the levels of DNA methylation and histone acetylation were significantly lower in IVF embryos than embryos cloned with untreated donor cells. Although we were able to bring DNA methylation and histone acetylation levels to those comparable with IVF embryos using 0.08 µM 5-aza-dC, embryo development and cell number were significantly reduced.

View larger version (56K): [in this window] [in a new window]   FIG. 2. Confocal micrographs of Day 7 blastocysts from in vitro fertilization (A), cloned from untreated donor cells (B), cloned from donor cells treated with 0.005 µM (C), 0.01 µM (D), 0.02 µM (E), 0.04 µM (F), and 0.08 µM 5-aza-dC (G). The embryos were labeled for acetyl histone H3 (red) and 5-methyl cytosine (green). The merged images of labeled histone H3 and 5-methyl cytosine appear yellow. Images were magnified x400

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the present study, our objective was to expand our previous findings using 5-aza-dC for the reduction of DNA methylation levels in donor cells, and to identify the lowest effective dose resulting in detectable changes in donor cells, while causing no deleterious effects to the development of embryos cloned from these cells.

The DNA demethylating reagent, 5-aza-dC, has been widely used to enhance the expression of transcriptionally silent alleles of imprinted genes [11–13]. It has also been tested for the pretreatment of somatic donor cells in NT in four studies published previously [8, 14–16]. In all these prior studies, however, treatment of donor cells with 5-aza-dC inhibited development of embryos cloned from the treated donor cells. This is because extremely high levels (1 µM or higher) of this drug were used [14–16]. These doses are frequently used in imprinting studies to reactivate genes, but are not suitable for NT studies because the health of the donor cells are critical to the success of NT embryo development [8]. At high concentrations, 5-aza-dC may be cytotoxic to the donor cells. Although in our previous study we systematically tested the dose effect of 5-aza-dC on donor cells by measuring DNA methylation changes through immunostaining and changes in cell morphology before using them for NT [8]. The lowest doses that induced methylation changes (0.31 µM) or morphological changes (0.08 µM), however, were still too high, and treating donor cells with these levels of 5-aza-dC caused a significant reduction in embryo development [8]. Therefore, we tested the effects of an even lower range of 5-aza-dC concentrations, based on results from our prior investigations [8]. In the present study, we may have avoided the cytotoxic effect of 5-aza-dC by using prolonged treatment of donor cells with lower drug concentrations of 0.005 to 0.08 µM. This strategy may have been effective via passive demethylation of DNA (demethylating DNA by preventing its methylation during DNA replication), thus causing alterations in donor cell characteristics. Although the two parameters used to monitor donor cell changes—BRDU labeling and cell cycle alterations—do not provide specific information on the alteration of DNA methylation in donor cells, we surmise that, because 5-aza-dC is known to specifically induce DNA demethylation, the cellular changes we observed were likely associated with DNA methylation changes. We found that 5-aza-dC induced detectable variations at a very low level, 0.005 µM, measurable in donor cells as cell cycle distribution changes. Of interest, the treatment of donor cells at 0.01 µM significantly increased the fusion rate of NT embryos. This is also the level at which deleterious effects on cloned embryo development were not observed, suggesting that epigenetic changes in donor cells are sensitive to low levels of a DNA methylation-modifying drug, which can be carried over in the cloned embryos. Previously, we found the immunostaining of donor cells to be a relatively insensitive method to detect methylation changes, and it is possible that methylation changes already occurred in embryos cloned from donor cells treated with lower doses of 5-aza-dC, but we were unable to detect the difference by immunostaining.

When the level of 5-aza-dC was increased to 0.02 µM, we found that blastocyst formation of cloned embryos decreased. Previously, we found that TSA, which increases histone acetylation, increased development of blastocysts cloned from TSA-treated donor cells. In this study, although we did not increase development of embryos cloned from treated donor cells, the use of very low levels of 5-aza-dC did not decrease development of cloned embryos, suggesting that these levels of 5-aza-dC may be suitable for donor cell pretreatment. Despite achieving a reduction in DNA methylation in embryos cloned from donor cells treated with 0.04 µM or higher concentrations of 5-aza-dC, this treatment also significantly decreased blastocyst development, and is likely not a suitable level for use in NT to improve nuclear reprogramming.

Cloned embryos reconstructed from both untreated and treated donor cells had levels of histone acetylation and DNA methylation positively correlated, even though these epigenetic states are inversely linked in donor cells [8]. That the modifications of these two parameters are closely and positively linked may be a unique feature of cloned embryos, rather than being due to donor cell treatment with 5-aza-dC, before NT. Regardless of the 5-aza-dC doses used in this study, including 0 µM, NT embryos had higher histone acetylation and DNA methylation levels compared those of IVF-produced embryos. Hence, this unlinking of histone acetylation and DNA methylation was not due to 5-aza-dC alone. Prior knowledge of the inverse relationship of histone acetylation and DNA methylation was gained from studies of somatic cells, and it is possible that histone deacetylases/acetyltransferases and DNA methyltransferases function differently in NT embryos. This abnormality of epigenetics in cloned embryos may also explain their lower development rates. A study by Gao and coworkers [17] also found that cloned embryos had donor cell-specific characteristics and that genome silencing was either incomplete or occurred progressively during preimplantation development.

In this study, we examined the effects of 5-aza-dC, a DNA methyl transferase inhibitor, on the epigenetic status of donor cells and on the outcome of NT. The main findings indicate that this reagent affects the donor cell cycle and the subsequent development of NT embryos in a dose-related manner. While treatment of the donor cell with 5-aza-dC can reduce histone acetylation and DNA methylation in cloned embryos to levels that are similar to those found in IVF-derived embryos, the developmental potential of these cloned embryos is also reduced, suggesting that complete DNA methylation reprogramming in cloned embryos may not be necessary.

	FOOTNOTES

 
¹ This manuscript is a scientific contribution of the Storrs Agricultural Experimental Station at the University of Connecticut and was supported by grants to X.Y. and X.C.T.

² Correspondence: X.C. Tian, Agricultural Biotechnology Laboratory, 1392 Storrs Road, Storrs, CT 06269-4243. FAX: 860 486 8809; xtian{at}canr.uconn.edu

³ Current address: National Institutes of Health/National Cancer Institute, Bethesda, MD 20892

Received: 17 June 2004.

First decision: 15 July 2004.

Accepted: 8 December 2004.

	REFERENCES

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This Article Abstract Full Text (PDF) An addition or correction has been published All Versions of this Article: 72/4/944    most recent biolreprod.104.033225v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Enright, B.P. Articles by Tian, X.C. Search for Related Content PubMed PubMed Citation Articles by Enright, B.P. Articles by Tian, X.C. Agricola Articles by Enright, B.P. Articles by Tian, X.C.