The number and developmental stage of embryos was then evaluated using light microscopy. Results showed that embryos arrested either at the 2 4 cell stage or at the 1 cell stage in the Cl amidine group, while 86. 1% of embryos in the H amidine group and 72. 3% of embryos in KSOM medium alone developed to the morula stage. We note here that the concentration enzyme inhibitor of Cl amidine used in our study is within the range of that used to functionally block PADI activity in somatic cells and that lower concentrations of Cl amidine did not affect embryonic development. Our finding that Cl amidine suppressed histone citrul lination in cleavage stage embryos suggested that the observed effects of Cl amidine on development were due to specific inhibition of PADI activity.

However, it is also pos sible that the inhibitor blocked development because of non specific toxic side effects. To address this possibility, we first examined embryo viability following Cl amidine and H amidine treatment using the vital dye propidium iodide. Results showed that nuclei from both Cl amidine and H amidine treated embryos were not stained with PI while nuclei from embryos that were treated with Cl amidine and extracted with 0. 1% Triton were strongly stained with PI. These results indicate that the plasma membrane of Cl amidine and H amidine treated embryos appeared func tional. To further confirm embryo viability, we next evalu ated the mitochondrial membrane potential of Cl amidine and H Amidine treated embryos using the JC 1 fluorescent dye, which accumulates in functional mitochondria as red staining aggregates.

Results showed that the mitochondrial membrane potential appeared to be similar between the Cl amidine and H amidine treatment groups, suggesting the Cl amidine does not affect mito chondrial health. Together, these findings suggest that PADI activity is required for progression of embryonic de velopment beyond the two to four cell stage. Treatment of embryos with C amidine suppresses histone H3 and H4 acetylation while having no apparent effect on the repressive H3K9 Di methyl modification As noted, histone acetylation is well correlated with acti vation of gene expression in somatic cells and is also believed to play an important role in modulating gene expression in preimplantation embryos.

In order to begin testing whether histone citrullination may play a role regulating gene activity in early embryos, Carfilzomib we tested whether suppression of histone citrullination with Cl amidine affected levels of histone acetylation on H3 and H4 tails. Results showed that Cl amidine treatment sig nificantly reduced levels of histone H4 acetylation. The fluorescent intensities for hyper acetylated H4 and H4K5 Acetyl in Cl amidine, TSA, and KSOM groups were presented in Figure 3B and 3F. Additionally, Cl amidine also dra matically reduced the level of acetylation on the H3 tail, namely H3K9 Acetyl and H3K18 Acetyl.