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DNA methylation dynamics at imprinted genes during bovine pre-implantation embryo development.

O'Doherty AM, Magee DA, O'Shea LC, Forde N, Beltman ME, Mamo S, Fair T - BMC Dev. Biol. (2015)

Bottom Line: For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05).Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization.The findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.

View Article: PubMed Central - PubMed

Affiliation: School of Agriculture and Food Science, University College Dublin, Belfield, Dublin, Ireland. alan.odoherty@ucd.ie.

ABSTRACT

Background: In mammals, maternal differentially methylated regions (DMRs) acquire DNA methylation during the postnatal growth stage of oogenesis, with paternal DMRs acquiring DNA methylation in the perinatal prospermatagonia. Following fusion of the male and female gametes, it is widely accepted that murine DNA methylation marks at the DMRs of imprinted genes are stable through embryogenesis and early development, until they are reprogrammed in primordial germ cells. However, the DNA methylation dynamics at DMRs of bovine imprinted genes during early stages of development remains largely unknown. The objective of this investigation was to analyse the methylation dynamics at imprinted gene DMRs during bovine embryo development, from blastocyst stage until implantation.

Results: To this end, pyrosequencing technology was used to quantify DNA methylation at DMR-associated CpG dinucleotides of six imprinted bovine genes (SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19) using bisulfite-modified genomic DNA isolated from individual blastocysts (Day 7); ovoid embryos (Day 14); filamentous embryos (Day 17) and implanting conceptuses (Day 25). For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05). Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization.

Conclusion: The findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.

No MeSH data available.


Related in: MedlinePlus

Pyrosequencing analysis of DNA methylation at six bovine imprinted differentially methylated regions during early embryogenesis. Bisulfite PCR and pyrosequencing was performed using genomic DNA isolated from embryos (n = 8 - 10) at four separate time points; blastocyst (Day 7), hatched ovoid embryos (Day 14), filamentous embryos (Day 17) and implanting conceptuses (Day 25). At Day 17, trophectoderm embryonic regions adjacent (TE) and peripheral to the embryonic disc (TP) were analysed. Trophectoderm samples at Day 25 were also included in the analysis. An overall trend of imprint stabilization can be observed for the six genes with increasing Days of embryonic development, post blastocyst. (A) Methylation values for SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19 at Day 7 had the most significant differences, when compared to Days 14, 17 or 25. (B) Top Panel; Standard deviations were calculated using individual methylation values (n = 4 - 9) for all genes in each group. Overall, standard deviations are greatest in group 1 (Day 7 blastocyst) with the smallest degree of variation occurring in the Day 25 implanting concepti (group 6) and Day 25 implanting concepti extra embryonic region. Bottom panel; Average methylation values, plotted with standard deviation values, for embryos isolated 7, 14, 17 and 25 Days post insemination (groups 1-3 and 6 from top panel).
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Fig2: Pyrosequencing analysis of DNA methylation at six bovine imprinted differentially methylated regions during early embryogenesis. Bisulfite PCR and pyrosequencing was performed using genomic DNA isolated from embryos (n = 8 - 10) at four separate time points; blastocyst (Day 7), hatched ovoid embryos (Day 14), filamentous embryos (Day 17) and implanting conceptuses (Day 25). At Day 17, trophectoderm embryonic regions adjacent (TE) and peripheral to the embryonic disc (TP) were analysed. Trophectoderm samples at Day 25 were also included in the analysis. An overall trend of imprint stabilization can be observed for the six genes with increasing Days of embryonic development, post blastocyst. (A) Methylation values for SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19 at Day 7 had the most significant differences, when compared to Days 14, 17 or 25. (B) Top Panel; Standard deviations were calculated using individual methylation values (n = 4 - 9) for all genes in each group. Overall, standard deviations are greatest in group 1 (Day 7 blastocyst) with the smallest degree of variation occurring in the Day 25 implanting concepti (group 6) and Day 25 implanting concepti extra embryonic region. Bottom panel; Average methylation values, plotted with standard deviation values, for embryos isolated 7, 14, 17 and 25 Days post insemination (groups 1-3 and 6 from top panel).

Mentions: Comparison across the different embryonic stages revealed that the greatest range in methylation values occurred at the Day 7 blastocyst stage (3-61% [SNRPN]; 7-59% [MEST]; 13-44% [IGF2R]; 12-64% [PLAGL1]; 7-59% [PEG10] and 20-32% [H19]), followed by the Day 14 hatched ovoid embryos (27-45% [SNRPN]; 31-36% [MEST]; 28-89% [IGF2R]; 31-42% [PLAGL1]; 22-37% [PEG10] and 21-31% [H19]). Notably, the range of methylation values for all six genes analysed were narrower at the Day 17 and Day 25 embryonic stages (Figure 2).Figure 2


DNA methylation dynamics at imprinted genes during bovine pre-implantation embryo development.

O'Doherty AM, Magee DA, O'Shea LC, Forde N, Beltman ME, Mamo S, Fair T - BMC Dev. Biol. (2015)

Pyrosequencing analysis of DNA methylation at six bovine imprinted differentially methylated regions during early embryogenesis. Bisulfite PCR and pyrosequencing was performed using genomic DNA isolated from embryos (n = 8 - 10) at four separate time points; blastocyst (Day 7), hatched ovoid embryos (Day 14), filamentous embryos (Day 17) and implanting conceptuses (Day 25). At Day 17, trophectoderm embryonic regions adjacent (TE) and peripheral to the embryonic disc (TP) were analysed. Trophectoderm samples at Day 25 were also included in the analysis. An overall trend of imprint stabilization can be observed for the six genes with increasing Days of embryonic development, post blastocyst. (A) Methylation values for SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19 at Day 7 had the most significant differences, when compared to Days 14, 17 or 25. (B) Top Panel; Standard deviations were calculated using individual methylation values (n = 4 - 9) for all genes in each group. Overall, standard deviations are greatest in group 1 (Day 7 blastocyst) with the smallest degree of variation occurring in the Day 25 implanting concepti (group 6) and Day 25 implanting concepti extra embryonic region. Bottom panel; Average methylation values, plotted with standard deviation values, for embryos isolated 7, 14, 17 and 25 Days post insemination (groups 1-3 and 6 from top panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Pyrosequencing analysis of DNA methylation at six bovine imprinted differentially methylated regions during early embryogenesis. Bisulfite PCR and pyrosequencing was performed using genomic DNA isolated from embryos (n = 8 - 10) at four separate time points; blastocyst (Day 7), hatched ovoid embryos (Day 14), filamentous embryos (Day 17) and implanting conceptuses (Day 25). At Day 17, trophectoderm embryonic regions adjacent (TE) and peripheral to the embryonic disc (TP) were analysed. Trophectoderm samples at Day 25 were also included in the analysis. An overall trend of imprint stabilization can be observed for the six genes with increasing Days of embryonic development, post blastocyst. (A) Methylation values for SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19 at Day 7 had the most significant differences, when compared to Days 14, 17 or 25. (B) Top Panel; Standard deviations were calculated using individual methylation values (n = 4 - 9) for all genes in each group. Overall, standard deviations are greatest in group 1 (Day 7 blastocyst) with the smallest degree of variation occurring in the Day 25 implanting concepti (group 6) and Day 25 implanting concepti extra embryonic region. Bottom panel; Average methylation values, plotted with standard deviation values, for embryos isolated 7, 14, 17 and 25 Days post insemination (groups 1-3 and 6 from top panel).
Mentions: Comparison across the different embryonic stages revealed that the greatest range in methylation values occurred at the Day 7 blastocyst stage (3-61% [SNRPN]; 7-59% [MEST]; 13-44% [IGF2R]; 12-64% [PLAGL1]; 7-59% [PEG10] and 20-32% [H19]), followed by the Day 14 hatched ovoid embryos (27-45% [SNRPN]; 31-36% [MEST]; 28-89% [IGF2R]; 31-42% [PLAGL1]; 22-37% [PEG10] and 21-31% [H19]). Notably, the range of methylation values for all six genes analysed were narrower at the Day 17 and Day 25 embryonic stages (Figure 2).Figure 2

Bottom Line: For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05).Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization.The findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.

View Article: PubMed Central - PubMed

Affiliation: School of Agriculture and Food Science, University College Dublin, Belfield, Dublin, Ireland. alan.odoherty@ucd.ie.

ABSTRACT

Background: In mammals, maternal differentially methylated regions (DMRs) acquire DNA methylation during the postnatal growth stage of oogenesis, with paternal DMRs acquiring DNA methylation in the perinatal prospermatagonia. Following fusion of the male and female gametes, it is widely accepted that murine DNA methylation marks at the DMRs of imprinted genes are stable through embryogenesis and early development, until they are reprogrammed in primordial germ cells. However, the DNA methylation dynamics at DMRs of bovine imprinted genes during early stages of development remains largely unknown. The objective of this investigation was to analyse the methylation dynamics at imprinted gene DMRs during bovine embryo development, from blastocyst stage until implantation.

Results: To this end, pyrosequencing technology was used to quantify DNA methylation at DMR-associated CpG dinucleotides of six imprinted bovine genes (SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19) using bisulfite-modified genomic DNA isolated from individual blastocysts (Day 7); ovoid embryos (Day 14); filamentous embryos (Day 17) and implanting conceptuses (Day 25). For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05). Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization.

Conclusion: The findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.

No MeSH data available.


Related in: MedlinePlus