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Reproductive Technology |
Department of Animal Science/Connecticut Center for Regenerative Biology, University of Connecticut, Storrs, Connecticut 06269
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, cumulus cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most genes in differentiated cells are regulated through epigenetic modifications (i.e., changes in the chromosomes that do not alter the DNA sequences) [7]. These include DNA methylation and histone acetylation [8]. In the mammalian genome, the cytosine residue (C) 5' to a guanine residue (G) can be methylated and form mCG. This epigenetic modification of DNA in the promoter regions of genes is correlated to gene silencing [9]. Closely associated with DNA methylation is the acetylation of nucleosomal histone molecules at numerous lysine residues (for review, see [10]). Acetylated histone molecules in chromatin are associated with increased gene expression [11]. Additionally, histone H1, a linker histone that associates with internucleosomal DNA, plays an important role in regulating chromatin structure and transcriptional activity [10].
Cloned embryos have higher levels of DNA methylation than embryos from natural reproduction [12–14]. This may result from an incomplete erasure of preexisting methylation in the donor cells. It is possible that in somatic cell cloning, donor cells with less epigenetic modifications, such as lower levels of methylated DNA and/or higher levels of histone acetylation, may be reprogrammed more easily or more completely and, thus, give rise to improved cloning efficiency.
To our knowledge, there have been no reports on the assessment of preexisting epigenetic levels in donor cells from different tissues, passage numbers, or cell-cycle stages. In the present study, we developed a method to measure the levels of histone acetylation in individual donor cells, specifically to determine the histone acetylation levels of skin fibroblast and ovarian cumulus cells at different cell-cycle stages and following various periods of culture.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult somatic cells used in the present study were skin fibroblast and ovarian cumulus cells. Both types of cell were derived from a 15-yr-old Holstein cow that had previously been cloned successfully. Fibroblast cells were collected and cultured as described previously [5]. Briefly, a skin biopsy specimen from the ear of the donor animal was cut into small pieces (2–3 mm2), which were then cultured as tissue explants in Dulbecco modified Eagle medium (DMEM; all reagents were purchased from Sigma Chemical Co., St. Louis, MO, unless otherwise stated) 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 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 for 1 min at 37°C and then allocated to three dishes. Normally, each cell passage was equivalent to approximately two cell doublings and was sustained for 4 days.
Cumulus cells were derived from cumulus-oocyte complexes aspirated from the same donor cow. Briefly, oocytes were denuded by pipetting, and the cells were placed in culture medium containing DMEM plus 20% FBS and antibiotics at 37.5°C in a humidified atmosphere of 5% CO2. After 4 days in culture, cumulus cell monolayers formed and were cultured to confluence. Cumulus cells were passaged as described above for fibroblast cells.
Fibroblast and cumulus cells were each grown to confluent passages 5, 10, and 15 and were subjected to the following analyses for histone acetylation levels.
Antibodies and Western Blot Analysis
The specificity of antibodies to histone H1, acetylated histone H3 at lysine 18, and acetylated histone H4 at lysine 8 was determined by Western blot analysis with enhanced chemiluminescence. Western blot analysis was carried out as previously described [15] with the following modifications: Cells were grown to confluence and counted in a hemocytometer. Laemmli sample buffer was added so that each cell sample vial contained 7.5 x 106 cells/ml; sample vials were then stored at -20°C until use. After cell lysis and protein denaturation by boiling for 5 min, samples of cell lysate with the equivalent of 7.5 x 104 cells per lane were loaded onto a 15% SDS-polyacrylamide gel and then transferred onto polyvinylidene fluoride membrane (Millipore, Bedford, MA). The membrane was probed in a sealed plastic bag at 4°C overnight with either anti-histone H1, anti-acetyl histone H3 (lysine 18) (Cell Signaling-Technologies, Inc., Boston, MA), or anti-acetyl histone H4 (lysine 8) antibodies (Upstate Biotechnologies, Lake Placid, NY). All antibodies were generated in either rabbit or mouse against human antigens. The antibody dilutions for anti-histone H1, anti-acetyl histone H3, and anti-acetyl histone H4 were 1:2000, 1:2000, and 1:1000, respectively. The blot was subsequently incubated with horseradish peroxidase-conjugated immunoglobulin G secondary antibody and subjected to enhanced chemiluminescence (PerkinElmer, Boston, MA) according to the manufacturer's protocol.
Confocal Microscopy
Confocal microscopy was used to confirm that the antibody binding to donor cells occurred specifically to nuclear histones. Briefly, cells were grown to confluence on chamber slides (Fisher Scientific, Pittsburgh, PA) and fixed in 2% formaldehyde solution (Fisher). Cells were then incubated in the primary antibody to acetylated histones at a dilution of 1:100 for 12 h at 4°C and subsequently in the fluorescein isothiocyanate (FITC)-conjugated secondary antibody at a dilution of 1:50 for 1 h at room temperature. Cells were mounted in 50% glycerol and observed under a confocal microscope (TCSSP2 True Scanning; Leica Microsystems, Heidelberg, Germany). Appropriate controls for autofluorescence and nonspecific binding by the secondary antibody were included.
Flow Cytometry
The histone acetylation levels for individual skin fibroblast and ovarian cumulus cells were quantified with flow cytometry by measuring the fluorescence of cells following incubation in histone-specific primary antibodies and FITC-conjugated secondary antibodies. The procedures for flow cytometry and DNA staining were described previously [16]. For flow cytometric analysis, confluent cells at passages 5, 10, and 15 were trypsinized and resuspended in cold "saline GM" (6.1 mM glucose, 137 mM NaCl, 5.4 mM KCl, 1.5 mM Na2HPO4·7H2O, 0.9 mM KH2PO4, and 0.5 mM EDTA) and then fixed in 100% ethanol. To measure levels of histone H1 or acetylated histone H3 and H4, cells were incubated in the primary antibodies at 1:100 dilution for 30 min, washed in PBS plus 5% FBS, and subsequently resuspended in FITC-conjugated secondary antibody (1:50 dilution) for 30 min at 37°C. To stain for DNA, cells were incubated in PBS containing 30 µg/ml of propidium iodide 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). The percentage of cells at each cell-cycle stage was determined by their DNA content. Appropriate controls for autofluorescence and nonspecific binding by the secondary antibodies were included. The experiments described above were replicated three times. On each occasion, two samples of each treatment were analyzed.
Statistical Analysis
Statistical analysis was carried out using the general linear models procedure of the Statistical Analysis System [17]. The main effects were cell type, cell cycle, and cell passage number, which were all treated as fixed effects. Least-significant-difference (LSD) means were used to test the differences among the cell cycle by cell type treatment means, for percentage of cells, and for relative levels of each histone type. The LSD values were also used to test the differences among the cell passage number by cell type treatment means and for relative levels of histone type within the G0/G1 and G2/M stages. A probability value of P < 0.05 was considered to be significant. The values presented are mean ± SEM unless otherwise stated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The distribution of bovine fibroblast and cumulus cells in each cell-cycle stage was determined by flow cytometry through quantification of their DNA (Fig. 1A). As expected, the majority of cells (>80%) in confluent cultures were at the G0/G1 phase, regardless of their passage number (Fig. 1B). The proportion of cells, regardless of cell type, at the S phase was minimal (<6%). A consistent difference, however, was observed between fibroblast cells and cumulus cells in the proportion of cells at the G0/G1 vs. the G2/M phase. More of the cumulus cells were in the G0/G1 phase compared to fibroblast cells (88% vs. 82%, respectively). This was consistent with, and perhaps resulted from, the fact that fewer cumulus cells were at the G2/M phase than fibroblast cells (6.3% vs. 12.3%, respectively).
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Histone and Histone Acetylation Levels
The levels of histone H1 and acetylated histone H3 and H4 in cells were measured by flow cytometric quantification of the fluorescence of cells caused by binding of the appropriate antibodies. To confirm the specificity of the primary antibodies to histone H1 and acetylated histone H3 and H4, Western blot analysis was conducted using both cell types. Figure 2 shows Western blots of confluent fibroblast and cumulus cells at passage 5 probed for histone H1, acetyl histone H3, and acetyl histone H4. A single band for histone H1 and acetyl histone H3 was observed, indicating that the binding of these antibodies was highly specific. This confirmed that in FACS analyses, the fluorescence staining for histone H1 and acetyl histone H3 was attributable to specific binding of antibody to these proteins. For acetyl histone H4, a minor band was detected above the expected band for this protein. However, the band indicating acetyl histone H4 comprised more than 75% of the total signal. Furthermore, the acetyl histone H4 antibody used in the present study has been widely used in the literature [18–20]. The minor nonspecific protein is located at the same position as acetyl histone H3, suggesting that the acetyl histone H4 antibody may cross-react somewhat with acetyl histone H3. Western blot analysis for acetyl histone H2B at lysine 20 was also conducted. However, the primary antibody was found to have high levels of cross-reactivity with other proteins and was not used in this evaluation.
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Immunocytochemistry was subsequently performed to confirm that the binding of antibodies in fixed cells was restricted to the nucleus, where the histones are located. According to staining patterns, all histones were colocalized with the DNA (Fig. 3), indicating that the antibodies specifically labeled the histone molecules on the nucleosomes. As shown in Figure 3, cells in culture were acetylated, to varying amounts, at histone H4 and H3, as determined by the intensities of the green label. Histone H1 was also immunolabeled to varying amounts (Fig. 3). For each of the acetyl histones, we consistently observed that a proportion of the cells was highly acetylated, whereas others had low levels of acetylation. No fluorescence was detected from control cells incubated in primary or secondary antibody alone (data not shown).
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After confirming the degree of specificity and the localization of the antibodies binding to histones, we determined the amount of histones in fibroblast versus cumulus cells at various cell-cycle stages relative to autofluorescence of the cells in each treatment group. We found that histone H1 as well as histone acetylation levels were different at different cell-cycle stages in both cell types examined (Fig. 4). Acetyl histone H3 and acetyl histone H4 levels (Fig. 4, A and B) increased with progression through the cell cycle from G0/G1 to S to G2/M phase in both cumulus and fibroblast cells. This demonstrated the linear nature of the fluorescent signal, because G0/G1-phase cells (2n of DNA) have approximately half the amount of histone antigen and fluorescent signal when compared to G2/M-phase cells (4n of DNA) (Fig. 4)
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Different cell types also appear to differ in their acetylation levels for each histone subtype. A significant main effect on cell type was detected on acetyl histone H3 and H4 as well as histone H1. Cumulus cells were more acetylated on H4 (Fig. 4A) but less acetylated on H3 than fibroblast cells (Fig. 4B) during their progression from the G0/G1 to the G2/M phase of the cell cycle. The most dramatic difference between these two cell types was found in the levels of histone H1; here, cumulus cells had almost twice as much histone H1 as did fibroblast cells at all cell-cycle stages examined (Fig. 4C).
Significant changes in histone acetylation levels were induced during cell culture. At passage 15 in G0/G1- and G2/M-phase cell populations, both fibroblast and cumulus cells had higher levels of acetyl histone H4 (Fig. 5, A and D) and/or acetyl histone H3 (Fig. 5, B and E) than they did at passage 5. Furthermore, the percentage of G0/G1-phase cells that were bound to acetyl histone H4 increased from passage 5 to passage 15 in cumulus (53% to 88% ± 9%; P < 0.05) (Fig. 6) and from passage 5 to passage 10 in fibroblast (59% to 86% ± 8%; P < 0.05; data not graphed) cells. This "culture effect" on the levels of histone H1, however, appeared only in cumulus cells and when progressing from passage 5 to passage 10 at both G0/G1 and G2/M phases of their cell cycle (Fig. 5, C and F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells at the G0/G1 phase of the cell cycle are commonly considered to be at the ideal stage for NT [22, 23]. Our results indicate that more than 80% of both fibroblast and cumulus cells were in the G0/G1 stage of the cell cycle in both short- and long-term cultures. This is consistent with data regarding porcine cell cycles, in which 85% of fetal fibroblast cells were at the G0/G1 phase at confluence [16].
Gene expression is generally correlated to the amount of acetylation of lysine residues on histone N-terminal tails within nucleosomes [9]. Using flow cytometry, we quantified the histone H1 and histone acetylation status of confluent fibroblast and cumulus cells based on their cell-cycle characteristics. Our results show that histone H1 and histone acetylation of both cumulus and fibroblast cells increase during their progression from the G0/G1 to G2/M phase of the cell cycle. This corresponds to the different amount of chromatin in cells at these cell-cycle stages and demonstrates that the fluorescent signal increases with the amount of antigen in the cell.
In the present study, we observed that cumulus and fibroblast cells are different in their levels of acetylated histones and amount of histone H1. These data suggest that these cells may have different chromatin organizations; however, further research is required to determine if these differences affect their potential to be reprogrammed by NT. Results from our laboratory [24] and those of others [25] indicate that embryos cloned from cumulus cells had higher blastocyst development and, furthermore, that the clones from these embryos had an improved survival rate compared to those derived from fibroblast cells. The data in the present study may provide a possible explanation for these observations.
Fibroblast and cumulus cells both have an increased level of histone acetylation, either in H3, H4, or both, after long-term culture (passage 15 vs. passage 5). This observation is consistent with previous data stating that cells in culture have high levels of gene expression [26]. Furthermore, we have shown that treating donor cells with a histone acetylation-modifying drug affects subsequent development of cloned embryos [27]. The "cell culture effect" may help to elucidate why cells at later passages differ in their capacity to be reprogrammed following NT. Indeed, previous data from our laboratory indicate that adult somatic cells at early passages had lower developmental rates than those derived from later passages [5]. However, because the majority of NT studies using fibroblasts as donor cells have used early passages (less than nine) [22, 23, 28], further studies are required to confirm the effect that cell passage number may have on the outcome of NT.
In the present study, the epigenetic characteristics of adult cells used as donors in somatic cell NT were examined; specifically, we determined the overall histone acetylation levels. To our knowledge, this is the first description of epigenetic properties of cumulus and fibroblast cells at different cell passages and cell-cycle stages. The use of flow cytometry allowed for the division of a heterogeneous cell population into subsets of specific subpopulations for analysis, such as G0/G1. Particularly, the use of flow cytometry allows for the quantification of cells showing strong histone acetylation, which may prove to be vitally important information for successful NT. For instance, we found that the percentage of G0/G1-phase cumulus cells with high levels of histone acetylation increased from 53% to 88% from passage 5 to passage 15. This indicates that utilizing G0/G1-phase somatic cells from passage 15 for NT will assure a greater probability of selecting a cell that has higher levels of histone acetylation and, thus, that is potentially easier to be reprogrammed [27]. Therefore, the method described here is useful for characterizing donor cells used in cloning, because a subset of the cells can be fixed and analyzed whereas the remaining cells can be used for NT. The results from cell characterization and embryo development can then be correlated for the improvement of future NT efforts.
In summary, the primary value of the present study is that we established a quantitative method for determining the overall histone acetylation levels of cultured cells for NT and showed that cells of different origin retain different epigenetic characteristics. Further development of such cell-sorting methods may also allow us to use a more uniform cell population after sorting. Furthermore, these characteristics change with cell passage and cell-cycle stage. The differences found in the histone acetylation levels of donor cells may function as a guideline in future studies of donor cell reprogrammability.
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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 06269-4243. FAX: 860 486 8809; xtian{at}canr.uconn.edu
Received: 15 February 2003.
First decision: 7 March 2003.
Accepted: 5 June 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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