The experiments were repeated for four times and the total quantity of blastomeres examined at the 1-cell, 2-cell, and 4-cell stages were 12, 22, and 19, respectively. preimplantation development. These results not only demonstrate the conversion of 5mC to 5fC and 5caC in zygotes, but also indicate that both 5fC and 5caC are relatively stable and may be functional during preimplantation development. Together with previous studies, our study suggests that Tet-catalyzed conversion of 5mC to 5hmC/5fC/5caC followed by replication-dependent dilution accounts for paternal DNA demethylation during preimplantation development. dynamics and to understand their function, we developed rabbit polyclonal antibodies against 5fC and 5caC, respectively. Dot blot analysis demonstrates that both antibodies are highly specific with no obvious cross-reactivity for other cytosine modifications (Physique 1A). To explore their power in immunostaining, we co-stained 5mC with 5fC or 5caC using pronucleus stage 4-5 mouse zygotes and found that both 5fC and 5caC signals are enriched in the male pronucleus relative to female pronucleus (Physique 1B and ?and1C,1C, top panels). To test for antibody specificity, we performed competition assays, which demonstrate that this 5fC signal can only be competed by 5fC nucleoside, while the 5caC signal can only be competed by 5caC nucleoside. These results demonstrate that both antibodies are specific with little reactivity to other altered cytosine residues. Open in a separate windows Physique 1 Characterization of 5fC and 5caC antibody specificity. (A) The 5fC and 5caC antibodies recognize 5fC and 5caC-containing Povidone iodine oligo DNA in dot-blot assays. Different amounts of 38-mer DNA oligos where C are either C, 5mC, 5hmC, 5fC, and 5caC were spotted on membrane and were probed with 5hmC (Active Motif), 5fC, and 5caC antibodies, respectively. (B, C) Representative confocal microscopy images of zygotes co-stained with 5mC and 5fC (B) or 5caC (C) antibodies in the absence or presence of 2 M of competitive nucleosides indicated. Increase of 5fC and 5caC levels in the male pronucleus correlates with the decrease of 5mC level in zygotes Having exhibited the specificity of the 5fC and 5caC antibodies, we next analyzed the dynamics of 5fC and 5caC during pronuclear development in zygotes by immunostaining. While the intensities of 5fC signals in the male and female pronuclei are low until 6 h after fertilization, the transmission in the male pronucleus is usually significantly increased relative to the female pronucleus 8 h after insemination (Physique 2A, ?,2B).2B). Interestingly, the dynamics of 5fC increase correlate with the decrease of 5mC in the male pronucleus (Physique 2A, ?,2B).2B). Comparable staining using the 5caC antibody demonstrates that 5caC transmission is usually barely detected in both male and female genome until 6 h after fertilization, and then preferentially appears in the male pronucleus concurrent with decrease Povidone iodine of 5mC in the male pronucleus after 8 h post insemination (Physique 2C, ?,2D).2D). These results suggest that loss of 5mC Povidone iodine in the male pronucleus is usually Povidone iodine concurrent with the appearance of 5fC and 5caC. Together with previous findings that loss of 5mC is usually concurrent with the appearance of 5hmC 13, 14, 15, 16, and that Tet proteins are capable of iterative oxidation of 5mC to generate 5fC and 5caC 17, 18, the above results suggest that 5mC in the male pronucleus is usually converted to all three oxidation forms (5hmC, 5fC, and 5caC). In addition, these results also demonstrate that ES cell is not the only cell type where 5caC is usually detectable. Open in a separate window Physique 2 Loss of 5mC staining in the paternal pronucleus correlates with increase in 5fC and 5caC staining in zygotes. (A, C) Representative confocal microscopy images of mouse zygotes co-stained with 5mC (green), DXS1692E 5fC (A) or 5caC (C) (reddish) at different.