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Methylation of cytosine base in the CpG dinucleotide sequence (DNA methylation) is a major epigenetic modification of the genome and plays an important role in gene expression. Recently, global DNA methylation in genome was studied by using a restriction landmark genomic scanning (RLGS) method and/or a representational difference analysis (RDA) method. However, these methods are complicated and need to use restriction enzymes. Therefore, the information derived from those methods is restricted to the region of the DNA sequence which is able to be cleaved by restriction enzymes. In this study, to establish a simple method to estimate global DNA methylation level in bovine spermatozoa, we tried to develop the DNA methylation analyzing method by using immunostaining of 5-methylcytosine. The immunostaining method for 5-methylcytosine in this study was based on the method developed by Benchaib et al. (2003 Fertil. Steril. 80, 947-952) for human spermatozoa. Because of the species difference, we modified some treatments to apply to bovine spermatozoa. Frozen-thawed bovine spermatozoa were washed by using 30 and 45% Percoll gradient solutions. After washing, spermatozoa were treated with 0.25 dithicthreitol M (DTT) and 1% sodium dodecyl sulfate (SDS) at room temperature (RT). Then, treated spermatozoa were spread on a slide glass with Cytospin4 (30g, 5 _ 104 cells/mL) and air-dried at RT. Air-dried bovine spermatozoa specimens were fixed in methanol: glacial acetic acid (3:1) solution at RT and treated with 1% Triton X and 1% SDS at RT; DNA was denatured with 6 N HCl at RT. After the denaturation, 5-methylcytosine in sperm DNA was analyzed by immunofluorescence technique with mouse anti 5-methylcytosine antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti mouse IgG antibody. The total sperm DNA was counterstained with propidium iodide (PI). Stained samples were observed with a confocal laser scanning microscope and obtained images were analyzed with fluorescence image analysis software. The area that was clearly stained with PI in each sperm head was designated and measured as the area of total sperm DNA, and the number of the dots that showed FITC fluorescence within the total sperm DNA area was designated and measured as the area of 5-methylcytosine in total sperm DNA. The area measurement was performed with fixed light strength. Three bovine spermatozoa samples derived from different bulls, used daily for calf production by AI, were examined. The ratio of the mean total area of the 5-methylcytosine in sperm DNA to the mean total area of the sperm DNA was 34.1% in bull A (9.13 � 5.66 _m2, 26.75 � 5.29 _m2, n = 57), 45.2% in bull B (16.60 � 3.79 _m2, 36.74 � 5.95 _m2, n = 41) and 43.9% in bull C (14.66 � 4.27 _m2, 33.45 � 7.13 _m2, n = 22). There was significant difference in the ratio between bull A and bulls B and C (P < 0.01). More research is required to evaluate the meaning of this individual difference of DNA methylation between bulls.<fn_group><fn>This work was supported by Wakayama Prefecture Collaboration of Regional Entities for the Advanced of Technological Excellence, Japan, and by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan.</fn></fn_group> |
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