Fig. 4. Ezh2 and Eed co-localise at the X_i at the blastocyst stage. (A) Ezh2 distribution at the blastocyst stage of controls (fertilised or PG^+/+). In about 50% of blastocysts from fertilised oocytes, Ezh2 protein (green) is detected as a spot within the nucleus of TE cells (white arrowhead). The inset shows a higher magnification of a TE cell in a PG^+/+ blastocyst. The accumulation of Ezh2-Eed co-localises with a DNA dense region, presumably the inactivated X chromosome. As expected, PG^-/- blastocysts do not show Ezh2 staining above background (not shown). (B) Eed distribution in PG^+/+ blastocysts, which is similar to the staining for Ezh2 shown in A. The inset shows that Eed stays associated with one chromosome (DNA in red) during M-phase at the blastocyst stage, presumably the inactive X chromosome. (C) Eed staining in PG^-/- blastocysts shows none of the localisation of Eed seen in PG^+/+ blastocysts shown in B. (D) Ezh2 (red) and Eed (green) co-localise in trophectoderm cells at the blastocyst stage, presumably at the inactivated X chromosome. The image shows immunostaining of two TE cells from PG^+/+ blastocysts. (E) Eed co-localises with macro-H2A in trophectoderm cells at the blastocyst stage in PG^+/+ blastocysts. Macro-H2A is highly enriched on the X_i (Costanzi and Pehrson, 1998), indicating that Ezh2 and Eed are also associated with the X_i.