Fig 1.
Dynamic patterns of H3K4me2/3 during porcine IVF and SCNT cleavage-stages embryo development.
(A) Representative images of IVF and SCNT embryos at different developmental stages immunostained with an anti-H3K4me2 antibody. Antibody was localized with an Alexa Flour 488-conjugated secondary antibody (green). DNA was stained with propidium iodide (red). Middle panels showed the merged images (yellow) between H3K4me2 signal (green) and DNA staining (red). Red dash line marks the developmental stages with different H3K4me2 signal intensity between IVF and SCNT embryos. Arrow denotes H3K4me2 signal intensity was abnormally higher in SCNT embryos than that in IVF counterparts. Arrowhead means H3K4me2 signal intensity was abnormally lower in SCNT embryos than that in IVF counterparts. (B) Representative images of IVF and SCNT embryos at different developmental stages immunostained with an anti-H3K4me3 antibody. Arrow denotes H3K4me3 signal intensity was abnormally higher in SCNT embryos than that in IVF counterparts. Scale bar = 50 μm. (C) Quantification of H3K4me2 intensity between IVF and SCNT early embryos. (D) Quantification of H3K4me3 intensity between IVF and SCNT early embryos. Blue bars denote IVF group, red bars represent SCNT group. Values are mean ± S.E.M. Different letters (a-b) on the bars indicate a statistically significant difference between IVF and SCNT groups (p < 0.05).
Fig 2.
Dynamic patterns of H3K9me2/3 during IVF and SCNT cleavage-stages embryo development.
(A) Representative images of porcine IVF and SCNT embryos at different developmental stages immunostained with an anti-H3K9me2 antibody. Antibody was localized with an Alexa Flour 488-conjugated secondary antibody (green). DNA was stained with propidium iodide (red). Middle panels showed the merged images (yellow) between H3K9me2 signal (green) and DNA staining (red). Red dash line marks the developmental stages with different H3K9me2 signal intensity between IVF and SCNT embryos. Arrow denotes H3K9me2 signal intensity was abnormally higher in SCNT embryos than that in IVF counterparts. White dash circle denotes ICM. (B) Representative images of IVF and SCNT embryos at different developmental stages immunostained with an anti-H3K9me3 antibody. Arrow denotes H3K9me3 signal intensity was abnormally higher in SCNT embryos than that in IVF counterparts. White dash circle denotes ICM. Scale bar = 50 μm. (C) Quantification of H3K9me2 intensity between IVF and SCNT early embryos. (D) Quantification of H3K9me3 intensity between IVF and SCNT early embryos. Blue bars denote IVF group, red bars represent SCNT group. Values are mean ± S.E.M. Different letters (a-b) on the bars indicate a statistically significant difference between IVF and SCNT groups (p < 0.05).
Fig 3.
Dynamic patterns of H3K27me2/3 during IVF and SCNT cleavage-stages embryo development.
(A) Representative images of porcine IVF and SCNT embryos at different developmental stages immunostained with an anti-H3K27me2 antibody. Antibody was localized with an Alexa Flour 488-conjugated secondary antibody (green). DNA was stained with propidium iodide (red). Middle panels showed the merged images (yellow) between H3K27me2 signal (green) and DNA staining (red). Red dash line marks the developmental stages with different H3K27me2 signal intensity between IVF and SCNT embryos. Arrow denotes H3K27me2 signal intensity was abnormally higher in SCNT embryos than that in IVF counterparts. Arrowhead means H3K27me2 signal intensity was abnormally lower in SCNT embryos than that in IVF counterparts. (B) Representative images of IVF and SCNT embryos at different developmental stages immunostained with an anti-H3K27me3 antibody. White dash circle denotes ICM. Scale bar = 50 μm. (C) Quantification of H3K27me2 intensity between IVF and SCNT early embryos. (D) Quantification of H3K27me3 intensity between IVF and SCNT early embryos. Blue bars denote IVF group, red bars represent SCNT group. Values are mean ± S.E.M. Different letters (a-b) on the bars indicate a statistically significant difference between IVF and SCNT groups (p < 0.05).
Fig 4.
Dynamic patterns of H3K36me2/3 during IVF and SCNT cleavage-stages embryo development.
(A) Representative images of porcine IVF and SCNT embryos at different developmental stages immunostained with an anti-H3K36me2 antibody. Antibody was localized with an Alexa Flour 488-conjugated secondary antibody (green). DNA was stained with propidium iodide (red). Middle panels showed the merged images (yellow) between H3K36me2 signal (green) and DNA staining (red). (B) Representative images of porcine IVF and SCNT embryos at different developmental stages immunostained with an anti-H3K36me2 antibody. Scale bar = 50 μm. (C) Quantification of H3K36me2 intensity between IVF and SCNT early embryos. (D) Quantification of H3K36me2 intensity between IVF and SCNT early embryos. Blue bars denote IVF group, red bars represent SCNT group. Values are mean ± S.E.M.
Fig 5.
Dynamic patterns of H3K79me2/3 during IVF and SCNT cleavage-stages embryo development.
(A) Representative images of porcine IVF and SCNT embryos at different developmental stages immunostained with an anti-H3K79me2 antibody. Antibody was localized with an Alexa Flour 488-conjugated secondary antibody (green). DNA was stained with propidium iodide (red). Middle panels showed the merged images (yellow) between H3K79me2 signal (green) and DNA staining (red). (B) Representative images of porcine IVF and SCNT embryos at different developmental stages immunostained with an anti-H3K79me3 antibody. Scale bar = 50 μm. (C) Quantification of H3K79me2 intensity between IVF and SCNT early embryos. (D) Quantification of H3K79me2 intensity between IVF and SCNT early embryos. Blue bars denote IVF group, red bars represent SCNT group. Values are mean ± S.E.M.
Fig 6.
Dynamic patterns of H4K20me2/3 during IVF and SCNT cleavage-stages embryo development.
(A) Representative images of porcine IVF and SCNT embryos at different developmental stages immunostained with an anti-H4K20me2 antibody. Antibody was localized with an Alexa Flour 488-conjugated secondary antibody (green). DNA was stained with propidium iodide (red). Middle panels showed the merged images (yellow) between H4K20me2 signal (green) and DNA staining (red). Red dash line marks the developmental stages with different H4K20me2 signal intensity between IVF and SCNT embryos. Arrow denotes H4K20me2 signal intensity was abnormally lower in SCNT embryos than that in IVF counterparts. (B) Representative images of porcine IVF and SCNT embryos at different developmental stages immunostained with an anti-H4K20me3 antibody. Arrow denotes H4K20me3 signal intensity was abnormally higher in SCNT embryos than that in IVF counterparts. White dash circle denotes ICM. Scale bar = 50 μm. (C) Quantification of H4K20me2 intensity between IVF and SCNT early embryos. (D) Quantification of H4K20me3 intensity between IVF and SCNT early embryos. Blue bars denote IVF group, red bars represent SCNT group. Values are mean ± S.E.M. Different letters (a-b) on the bars indicate a statistically significant difference between IVF and SCNT groups (p < 0.05).
Fig 7.
Dynamic reprogramming model for histone lysine methylation during porcine early embryo development.
We propose the following three distinct models about dynamic reprogramming of histone lysine methylation during porcine early embryo development. Red line indicates the first model, the signal intensity from the majority of histone lysine methylation modifications (H3K4me2/me3, H3K9me2/me3, H3K27me2/me3, H3K36me3, and H4K20me2/me3) gradually decrease from pronuclear stage to 8-cell stage and reappear at morula stage. Histone methylations at blastocyst stage show symmetric distribution pattern with similar methylation level between both cell lineages or asymmetric distribution pattern as hypermethylation in TE and hypomethylation in ICM. Green line indicates the second model, the expression level of histone methylation (H3K36me2) invariably keep constant from pronuclear stage to blastocyst stage and the signal intensity is almost consistent between both cell lineages. Blue line indicates the third model, histone methylation (H3K79me2 and H3K79me3) is quickly demethylated after fertilization and always keep low level during development to the blastocyst stage.