Fig. 7. Abnormal behaviour of chromosomes in east spermatocytes. Wild-type and east^hop-7 testes were labeled with antibodies to PH3 (green) and -tubulin (cyan). DNA was detected with TO-PRO-3 (red). (A,B) Primary spermatocytes in early prophase. The stage is inferred from the nucleation of astral microtubules (left panels). The central panels show the nuclei of the same cells in focal planes 3 µm below. Chromosomal DNA in wt is highly condensed and organized into three distinct clusters. DNA in the east spermatocytes is less condensed and does not show discernible clustering. Chromosomal DNA does not show any H3 phosphorylation (right panels, same focal planes as middle panels). The green label is due to a cross-reaction of the antimouse secondary with the rat primary antitubulin antibody. In C-E: top panels, PH3 labeling (green); middle panels, PH3+DNA labelings; bottom panels, PH3+DNA+-tubulin labelings. (C) As wt cells progress towards late prophase (left cell) and prometaphase (right cell), PH3 labeling can be detected, apparently coinciding with nuclear envelope breakdown (right cell). The organization of chromosomes into three clumps of DNA is maintained. (D) In east prometaphase, the number of DNA clumps can exceed four; here, two major and four minor clusters can be observed. (E) Two neighbouring spermatocytes of the same cyst in an east testis display different intensities of PH3 labeling (weak in the left, strong in right cell), suggesting that these cells are at the beginning of prometaphase. The chromosomes appear scattered and their arms become exposed and visible. By contrast, chromosome arms are not discernible in wt primary spermatocytes (C). (F,G) Primary spermatocytes during metaphase of wt (F) and east (G) testes. Left and right panels show focal planes of the same cells 2 µm apart. (F) In wt, chromosomes congregate to a single cluster in the middle of the spindle. (G) In east, some of the chromosomes (right panel) disperse away from the main DNA cluster (left panel). (H,J) Testes of wt (H) and east (J) pupae containing PH3-positive cells (arrows) that are shown in higher magnifications in (I) for wt and (K) for east. In (I,K): top left panels, PH3; top right, PH3+DNA; bottom left, tubulin; bottom right, PH3+DNA+tubulin. (J,K) Testes in east pupae contain small cells with PH3-positive nuclei (arrow), presumably spermatogonia. (I,H) In wt, small PH3-positive cells located near the tip of the testis are stem cells and spermatogonia that undergo mitosis. Note that the abnormal PH3-positive cells in east are not associated with mitotic spindles as in wt (I,K; compare bottom panels). Bar in (A) represents 10 µm and applies to all panels except (H,J). Bar in (H) equals 50 µm and also applies to (J).