Fig. 4. Association of macroH2A1.2 with the XY bivalent during male meiosis (green is macroH2A1.2 except in I and K, where it represents X chromosome paint; red is COR1 except in I and K, where it represents Y chromosome paint; centromeres, white; DAPI, blue; long arrow, X chromosome; short arrow, Y chromosome; arrowhead, PAR; open arrowhead, macroH2A1.2 PAR focus). (A) Early diplotene XY bivalent. (B) Same nucleus stained for macroH2A1.2. (C) Superimposition of (A) and (B). MacroH2A1.2 forms a sharp focus at the PARs, and, in contrast to M31 at this stage, is abundant at the centromeric heterochromatin of the X and Y chromosomes. (D) Diakinesis XY bivalent. (E) Same nucleus stained for macroH2A1.2. (F) Superimposition of (D) and (E). MacroH2A1.2 continues to associate with the PAR and the heterochromatin of the X and Y chromosome. (G) Metaphase I XY bivalent. (H) Same nucleus stained for macroH2A1.2, which no longer preferentially associates the PAR, but continues to associate with the X and Y centromeric heterochromatin. (I) Same nucleus stained with X and Y chromosome paints, which discriminates these chromosomes from one another. (J) Round spermatids stained for macroH2A1.2, which forms a sharp focus (open arrows) in half of the cells analysed (inset DAPI stained image). (K) Same spermatids stained with X and Y chromosome paints, which demonstrate that the sharp focus is present in Y-bearing, but not X-bearing, spermatids (notice that this focus is preserved after the FISH procedure and appears yellow in this image). (L) The region of heterochromatin from the lowest spermatid stained with CREST and macroH2A1.2. The heterochromatin is due to the clustering of centromeres, and the macroH2A1.2 is closely associated with a single (Y) centromere. Bars, 5 µm.