Figure 2

Cl-amidine blocks embryonic development in vitro at the 2 to 4 cell stage. A. Immunofluorescent confocal images showing that Cl-amidine reduces citrullination at H4R3 in 4-cell embryos. PN stage zygotes were cultured for ~42 hours or ~68 hours in KSOM medium supplemented without or with 250 μM of Cl-amidine. Four-cell embryos were fixed and immunostained with anti-H4Cit3 antibodies (red). Phalloidin and DAPI were employed to detect F-actin (red) and DNA (blue). Arrows highlight H4Cit3 staining on chromatin. DIC, differential interference contrast. B. Same as (A) except with anti-H3Cit2 + 8 + 17 antibodies. C. Same as (A) except with anti-H3Cit26 antibodies. D. Structure of Cl-amidine. The red square highlights the chlorine group that is replaced with hydrogen to generate H-amidine. MW., Molecular weight. E. Structure of H-amidine. F. Rate of development to the morula stage following Cl-amidine treatment. Pronuclear stage zygotes were cultured for ~68 hours in KSOM medium supplemented with 250 μM of Cl-amidine or H-amidine and observed by light microscopy. H-AM, H-amidine.