Cbx3hypo/hypotestes had severe impairment of spermatogenesis. (a) HP1γ protein was found in nearly all cells of the wild-type testes. (b) In wild-type tubules, Sertoli cells stained strongly for HP1γ (black arrows) as did round spermatids (stage 2-6) (white arrows). Many round spermatid nuclei possessed an enriched focus of HP1γ staining that was characteristic of the single block of heterochromatin observed at this stage (see Additional file 5, Figures 5b). (c) HP1γ was largely excluded from meiotic metaphase chromosomes and was found surrounding the condensed chromosomes in the meiotic cytoplasm (black arrows). Mature sperm (white arrows) were negative for HP1γ. (d) Stage 10 spermatids, at around the time they elongated, were either positive (black arrows) or negative (white arrows) for HP1γ, indicating that it was during this stage that levels of HP1γ proteins decrease. Pachytene spermatocytes (yellow arrows) showed a few brightly stained spots that represent sites of constitutive heterochromatin (see Additional file 5, Figures 5a). (e) In Cbx3hypo/hypotestes, HP1γ staining was very weak and the tubules showed severely impaired spermatogenesis with greatly reduced numbers of cells and some tubules exhibiting a Sertoli cells-only phenotype (see upper right tubule). (f) In some tubules of Cbx3hypo/hypotestes, mature sperm could be observed (inset, black arrowhead). Bar = 100 μm.