RES ETT ING
3 MATERIALS AND METHODS
4.4 Study of DNA methylation: Intragenic Differentially Methylated Region (DMR), methylation status of bovine preimplantation
4.4.2 Methylation status of bovine preimplantation embryos derived from different origins
The Differentially Methylated Region within exon 10 of the bovine IGF2 gene was analyzed for its methylation status in bovine embryos derived from different origins and in adult fibroblasts used to generate cloned embryos (figs. 50-55). The fragment contained 27 CpG dinucleotides and was identical to the fragment analyzed in section 4.4.1. Due to sequencing differences, CpG 1 in this experiment corresponds to CpG 2 in the experiment described in section 4.4.1. A total of 2496 sequences derived from 1248 bacterial clones were analyzed.
Differences in the methylation patterns were statistically investigated for blastocysts, cloned blastocysts and somatic donor cells, zygotes and 4-cell embryos (fig. 56).
Figure 50: Montage of representative gel bands derived from PCR amplification of bisulfite treated embryonic DNA
The intragenic DMR of the bovine IGF2 gene (within exon 10) was amplified using bisulfite treated DNA from bovine preimplantation embryos and adult fibroblasts as template. The product contained 420 bp after nested PCR.
(IVF: in vitro fertilized blastocysts; NTf: female nuclear transfer blastocysts; DCf: female donor cells; NTm: male nuclear transfer blastocysts; DCm: male donor cells; PA:
parthenogenetic blastocysts; A: androgenetic blastocysts; Vivo: in vivo collected blastocysts;
Zyg: zygotes; 4-cell: 4-cell embryos; neg.: negative control) IVF
100 bp
ladder IVF NTf DCf NTm DCm PA A Vivo Zyg 4-cell neg.
100 bp
ladder NTf DCf NTm DCm PA A Vivo Zyg 4-cell neg.
Figure 51: Methylation patterns of each group of embryos and adult fibroblasts analyzed by bisulfite sequencing
Each bar represents one CG dinucleotide within the intragenic DMR identified within exon 10
0
In vitrofertilized 4-cell embryos Methylation (%; n=2; 20 clones)
0
Female nuclear transfer expanded blastocyst Methylation (%; n=4; 56 clones)
0
In vivocollected expanded blastocyst Methylation (%; n=5; 52 clones)
0
In vitrofertilized expanded blastocyst Methylation (%; n=6; 71 clones)
0
Female fibroblasts (donor cells for NT) Methylation (%; n=3; 55 clones)
Methylation (%; n=4; 54 clones)
0
Male nuclear transfer expanded blastocyst
0
Male fibroblasts (donor cells for NT) Methylation (%; n=4; 84 clones)
0 Methylation (%; n=6; 56 clones)
0 Methylation (%; n=4; 40 clones)
0
In vitrofertilized zygotes Methylation (%; n=3; 33 clones)
0
In vitrofertilized 4-cell embryos Methylation (%; n=2; 20 clones)
0
Female nuclear transfer expanded blastocyst Methylation (%; n=4; 56 clones)
0
In vivocollected expanded blastocyst Methylation (%; n=5; 52 clones)
0
In vitrofertilized expanded blastocyst Methylation (%; n=6; 71 clones)
0
Female fibroblasts (donor cells for NT) Methylation (%; n=3; 55 clones)
Methylation (%; n=4; 54 clones)
0
Male nuclear transfer expanded blastocyst
0
Male fibroblasts (donor cells for NT) Methylation (%; n=4; 84 clones)
0 Methylation (%; n=6; 56 clones)
0 Methylation (%; n=4; 40 clones)
0
In vitrofertilized zygotes Methylation (%; n=3; 33 clones)
Figure 52: Methylation patterns of expanded blastocysts collected in vivo, generated by in vitro fertilization, parthenogenesis and androgenesis
The methylation levels of different bacterial clones are shown as in figure 51. Each row represents one bacterial clone, each circle one CpG dinucleotide. The number of different clones, that show identical methylation patterns, is written on the right side of the specific row. Open circles indicate non-methylated CpGs, whereas dark circles represent methylated
In vivoexpanded blastocyst In vitrofertilized
expanded blastocyst
In vivoexpanded blastocyst In vitrofertilized
expanded blastocyst
Figure 53: Methylation patterns of different bacterial clones are shown for female nuclear transfer derived expanded blastocysts and adult female fibroblasts used as donor cells.
Each row represents one bacterial clone, each circle one CpG dinucleotide. The number of
Female fibroblasts (donor cells for NT) Female nuclear transfer expanded blastocyst
2x Female fibroblasts (donor cells for NT)
Female nuclear transfer expanded blastocyst
2x Female nuclear transfer expanded blastocyst
2x
Figure 54: Methylation patterns of male nuclear transfer expanded blastocysts and adult male fibroblasts used as donor cells
Each row represents one bacterial clone, each circle one CpG dinucleotide. The number of different clones with the same methylation patterns is indicated on the right side of the specific row. (open circles: non-methylated cytosines; dark circles: methylated cytosines)
Male fibroblasts (donor cells for NT) Male nuclear transfer expanded blastocyst
2x Male fibroblasts (donor cells for NT)
Male nuclear transfer expanded blastocyst 2x
Figure 55: Methylation patterns of in vitro fertilized zygotes and 4-cell embryos
Each row represents one bacterial clone, each circle one CpG dinucleotide. The number of different clones with the same methylation patterns is indicated on the right side of the specific row. (open circles: non-methylated cytosines; dark circles: methylated cytosines)
The proportion of methylated CpG dinucleotides in each group of embryos (expanded blastocysts, NT blastocysts and adult fibroblasts, zygotes and 4-cell embryos) was tested for statistically significant differences (fig. 56). One way ANOVA was performed when testing for normal distribution failed.
The methylation level of androgenetic embryos (30%±2.1 SEM) was significantly increased (p≤0.05) compared to parthenogenetic embryos (2.3%±1 SEM), in vitro fertilized embryos (10.1%±0.7 SEM), female nuclear transfer embryos (12.4%±1.4) and in vivo collected embryos (10.2%±1.2). Male nuclear transfer blastocysts (22%±1.9 SEM) showed a significantly higher methylation level (p≤0.05) than their parthenogenetic, in vitro fertilized, in vivo collected and female cloned counterparts. No methylation difference was detected between androgenetic and male cloned embryos.
Statistical analyses of nuclear transfer blastocysts and donor cells revealed a significant difference (p≤0.05) in the methylation level between the donor cells (77%±2.2 SEM; 72%±2.9) and the embryos (12.4%±1.4; 22%±1.9). The methylation level of zygotes (28.4%±3.8 SEM) was significantly increased (p≤0.05) compared to 4-cell embryos (6.3%±2.2).
In vitrofertilized zygotes In vitrofertilized 4-cell embryos 6x In vitrofertilized zygotes In vitrofertilized 4-cell embryos
6x
Figure 56: Methylation level of the intragenic DMR within the bovine IGF2 gene in preimplantation embryos and adult fibroblasts
Bars represent the overall percentage of all CpGs analyzed in each group of embryos and donor cells. Statistically significant differences among treatment groups were detected by one way ANOVA: Dunn's method (a:b:c p≤0.05); Bonferroni t-test (a:b:c p≤0.05); Dunn's method (a:b p≤0.05).
In summary, the methylation level of the intragenic DMR decreased after fertilization in bovine preimplantation embryos. In vitro fertilized four-cell embryos showed only a low methylation level. Blastocysts were remethylated and no difference was detected in the methylation levels between in vivo collected, in vitro fertilized and female cloned embryos. In contrast, male nuclear transfer embryos were higher methylated than female cloned embryos and the blastocysts generated by other protocols except their androgenetic counterparts. The methylation level was low in parthenogenetic embryos and high in androgenetic embryos.
a
Vivo: in vivo; IVF: in vitrofertilization; NTf: Nucelar transfer, female; DCf: Donor cell, female;
NTm: Nuclear transfer, male; DCm: Donor cell, male; PA: parthenogenetic; A: androgenetic;
Zyg: zygotes; 4-cell: 4-cell embryos
Vivo IVF NTf NTm PA A NTf DCf NTm DCm Zyg 4-cell
Methylation (%)
Vivo: in vivo; IVF: in vitrofertilization; NTf: Nucelar transfer, female; DCf: Donor cell, female;
NTm: Nuclear transfer, male; DCm: Donor cell, male; PA: parthenogenetic; A: androgenetic;
Zyg: zygotes; 4-cell: 4-cell embryos
Vivo IVF NTf NTm PA A NTf DCf NTm DCm Zyg 4-cell
Methylation (%)
5 DISCUSSION
According to the classical model, imprinted genes are only expressed from one parental allele. However, imprinted genes are frequently bi-allelically expressed during preimplantation development (REIK and DEAN 2001). In the mouse, the genome is remethylated at the blastocyst stage shortly before implantation. In contrast, bovine embryos are remethylated from the 8-16-cell stage onwards and implantation starts around day 18 of development (DEAN et al. 2001; RÜSSE and SINOWATZ 1991). In the present study, gene expression was analyzed to determine mono-or bi-allelic gene expression levels of the bovine IGF2, IGF2R and MASH2 genes in preimplantation embryos. The Igf2, Igf2r and Mash2 genes were selected because they are known to be imprinted in the mouse (DeCHIARA et al. 1991;
BARLOW et al. 1991; GUILLEMOT et al. 1995). Recently, it was shown that the bovine IGF2 and IGF2R genes are also imprinted (KILLIAN et al. 2001; DINDOT et al. 2004). The present study made a major effort to analyze methylation patterns within the bovine IGF2 gene in gametes and preimplantation embryos of different origins. Bisulfite sequencing is a sensitive and valuable tool to detect methylation patterns in single CG dinucleotides. This technology was employed for the first time to characterize the methylation status of the IGF2 gene in bovine preimplantation embryos. Additionally, for the first time, a Differentially Methylated Region was identified in the bovine IGF2 gene. Results of this thesis contribute to a better understanding of the molecular structure of the bovine IGF2 gene and methylation reprogramming in bovine preimplantation embryos.
5.1 Blastocyst morphology and gene expression analyses of the