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Comparison of DNA methylation levels of repetitive loci during bovine development.

Kaneda M, Akagi S, Watanabe S, Nagai T - BMC Proc (2011)

Bottom Line: These results suggest that there is a dynamic change of DNA methylation during embryonic development and spermatogenesis in cattle.However, art2 sequences are not de-methylated during spermatogenesis, suggesting that this region is not reprogrammed during germ cell development.These results show dynamic changes of DNA methylation levels during bovine embryogenesis, especially genome-wide reprogramming in germ cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Reproductive Biology and Technology Research Team, National Institute of Livestock and Grassland Science (NILGS), National Agriculture and Food Research Organization (NARO), 2 Ikenodai, Tsukuba, Ibaraki 305-0901, Japan. taku@affrc.go.jp.

ABSTRACT

Background: DNA methylation of cytosine residues in CpG dinucleotide controls gene expression and dramatically changes during development. Its pattern is disrupted in cloned animals suggesting incomplete reprogramming during somatic cell nuclear transfer (the first reprogramming). However, the second reprogramming occurs in the germ cells and epigenetic errors in somatic cells of cloned animals should be erased. To analyze the DNA methylation changes on the spermatogenesis of bulls, we measured DNA methylation levels of three repetitive elements in blastocysts, blood and sperm.

Methods: DNA from PBLs (peripheral blood leukocytes), sperm and individual IVF (in vitro fertilized) and parthenogenetic blastocysts was isolated and bisulfite converted. Three repetitive elements; Satellite I, Satellite II and art2 sequences were amplified by PCR with specific pairs of primers. The PCR product was then cut by restriction enzymes and analyzed by agarose gel electrophoresis for determining the DNA methylation levels.

Results: Both Satellite I and Satellite II sequences were highly methylated in PBLs, whereas hypo-methylated in sperm and blastocysts. The art2 sequence was half methylated both in PBLs and sperm but less methylated in blastocysts. There was no difference in DNA methylation levels between IVF and parthenogenetic blastocysts.

Conclusions: These results suggest that there is a dynamic change of DNA methylation during embryonic development and spermatogenesis in cattle. Satellite I and Satellite II regions are methylated during embryogenesis and then de-methylated during spermatogenesis. However, art2 sequences are not de-methylated during spermatogenesis, suggesting that this region is not reprogrammed during germ cell development. These results show dynamic changes of DNA methylation levels during bovine embryogenesis, especially genome-wide reprogramming in germ cells.

No MeSH data available.


DNA methylation analysis in PBLs, sperm and blastocysts. Bulls #1-#9 shows each number of analyzed bulls. Uncut band indicates the PCR product not cut by restriction enzymes. (A) Satellite I regions were cut by AciI. (B) Satellite II regions were cut by AccII. (C) art2 sequences were cut by TaqI.
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Figure 1: DNA methylation analysis in PBLs, sperm and blastocysts. Bulls #1-#9 shows each number of analyzed bulls. Uncut band indicates the PCR product not cut by restriction enzymes. (A) Satellite I regions were cut by AciI. (B) Satellite II regions were cut by AccII. (C) art2 sequences were cut by TaqI.

Mentions: We analyzed genomic DNA methylation patterns to monitor the changes of epigenetic patterns during bovine embryogenesis and spermatogenesis. We chosen three repetitive regions; Satellite I, Satellite II and art2 sequences. Satellite sequences are repetitive sequences at the peri-/centromeric regions of the chromosomes, whereas art2 sequences are Alu-like short interspersed nuclear elements (SINEs). First, we analyzed genomic DNA of bull blood (peripheral blood leukocytes, PBLs) and sperm for methylation status of the same regions. We found a large difference in methylation status using restriction enzyme analysis (Figure 1A-C). Satellite I sequences were highly methylated in PBLs (almost PCR fragments were cut by AciI), whereas hypo-methylated in sperm (almost PCR fragments were not cut by AciI) (Figure 1A). Satellite II sequences were also hyper-methylated in PBLs but hypo-methylated in sperm (Figure 1B). However, there were no differences in art2 sequence methylation levels between PBLs and sperm (Figure 1C). These results clearly indicated that both Satellite I and Satellite II sequences, which are located on the centromeric heterochromatic regions, were de-methylated during spermatogenesis, whereas art2 sequences, which are located on euchromatic regions, were not methylated/de-methylated during spermatogenesis. Of nine bulls analyzed, there were no differences in DNA methylation patterns of three repetitive elements among individuals and breeds (bulls #1 and #6-9 are Japanese Black, bulls #3-5 are Japanese Brown and Bull #2 is Holstein).


Comparison of DNA methylation levels of repetitive loci during bovine development.

Kaneda M, Akagi S, Watanabe S, Nagai T - BMC Proc (2011)

DNA methylation analysis in PBLs, sperm and blastocysts. Bulls #1-#9 shows each number of analyzed bulls. Uncut band indicates the PCR product not cut by restriction enzymes. (A) Satellite I regions were cut by AciI. (B) Satellite II regions were cut by AccII. (C) art2 sequences were cut by TaqI.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108225&req=5

Figure 1: DNA methylation analysis in PBLs, sperm and blastocysts. Bulls #1-#9 shows each number of analyzed bulls. Uncut band indicates the PCR product not cut by restriction enzymes. (A) Satellite I regions were cut by AciI. (B) Satellite II regions were cut by AccII. (C) art2 sequences were cut by TaqI.
Mentions: We analyzed genomic DNA methylation patterns to monitor the changes of epigenetic patterns during bovine embryogenesis and spermatogenesis. We chosen three repetitive regions; Satellite I, Satellite II and art2 sequences. Satellite sequences are repetitive sequences at the peri-/centromeric regions of the chromosomes, whereas art2 sequences are Alu-like short interspersed nuclear elements (SINEs). First, we analyzed genomic DNA of bull blood (peripheral blood leukocytes, PBLs) and sperm for methylation status of the same regions. We found a large difference in methylation status using restriction enzyme analysis (Figure 1A-C). Satellite I sequences were highly methylated in PBLs (almost PCR fragments were cut by AciI), whereas hypo-methylated in sperm (almost PCR fragments were not cut by AciI) (Figure 1A). Satellite II sequences were also hyper-methylated in PBLs but hypo-methylated in sperm (Figure 1B). However, there were no differences in art2 sequence methylation levels between PBLs and sperm (Figure 1C). These results clearly indicated that both Satellite I and Satellite II sequences, which are located on the centromeric heterochromatic regions, were de-methylated during spermatogenesis, whereas art2 sequences, which are located on euchromatic regions, were not methylated/de-methylated during spermatogenesis. Of nine bulls analyzed, there were no differences in DNA methylation patterns of three repetitive elements among individuals and breeds (bulls #1 and #6-9 are Japanese Black, bulls #3-5 are Japanese Brown and Bull #2 is Holstein).

Bottom Line: These results suggest that there is a dynamic change of DNA methylation during embryonic development and spermatogenesis in cattle.However, art2 sequences are not de-methylated during spermatogenesis, suggesting that this region is not reprogrammed during germ cell development.These results show dynamic changes of DNA methylation levels during bovine embryogenesis, especially genome-wide reprogramming in germ cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Reproductive Biology and Technology Research Team, National Institute of Livestock and Grassland Science (NILGS), National Agriculture and Food Research Organization (NARO), 2 Ikenodai, Tsukuba, Ibaraki 305-0901, Japan. taku@affrc.go.jp.

ABSTRACT

Background: DNA methylation of cytosine residues in CpG dinucleotide controls gene expression and dramatically changes during development. Its pattern is disrupted in cloned animals suggesting incomplete reprogramming during somatic cell nuclear transfer (the first reprogramming). However, the second reprogramming occurs in the germ cells and epigenetic errors in somatic cells of cloned animals should be erased. To analyze the DNA methylation changes on the spermatogenesis of bulls, we measured DNA methylation levels of three repetitive elements in blastocysts, blood and sperm.

Methods: DNA from PBLs (peripheral blood leukocytes), sperm and individual IVF (in vitro fertilized) and parthenogenetic blastocysts was isolated and bisulfite converted. Three repetitive elements; Satellite I, Satellite II and art2 sequences were amplified by PCR with specific pairs of primers. The PCR product was then cut by restriction enzymes and analyzed by agarose gel electrophoresis for determining the DNA methylation levels.

Results: Both Satellite I and Satellite II sequences were highly methylated in PBLs, whereas hypo-methylated in sperm and blastocysts. The art2 sequence was half methylated both in PBLs and sperm but less methylated in blastocysts. There was no difference in DNA methylation levels between IVF and parthenogenetic blastocysts.

Conclusions: These results suggest that there is a dynamic change of DNA methylation during embryonic development and spermatogenesis in cattle. Satellite I and Satellite II regions are methylated during embryogenesis and then de-methylated during spermatogenesis. However, art2 sequences are not de-methylated during spermatogenesis, suggesting that this region is not reprogrammed during germ cell development. These results show dynamic changes of DNA methylation levels during bovine embryogenesis, especially genome-wide reprogramming in germ cells.

No MeSH data available.