Figure 6

Identification of H3T3ph in testicular cells. (a) Frozen sections of immature (7-day old) testis and kidney stained with aH3T3ph. H3T3ph is detected exclusively in mitotic spermatogonia and mitotic kidney cells (arrowheads). Scale bars, 10 μm. (b) Staining with aH3T3ph and aH3S10ph antibodies confirmed that both of these modifications are present in mitotically and meiotically dividing cells of the male germline. Scale bars, 20 μm. (c) Frozen sections of adult mouse testis depicting seminiferous tubules at different stages of the spermatogenic cycle (Latin numbers). Early round spermatids (eRS) and mature, elongated ones (mES) are shown on the left. Late round spermatids (lRS) are shown in the middle; and elongating spermatids (ES) are shown on the right. Enlarged images show details in each case. Note that in stages V–VIII tubules (left panel) no H3T3ph is detected. The arrowhead points to a chromocenter. Scale bars, 20 μm. (d) Competitive inhibition of aH3T3ph staining using the unmodified histone H3 peptide (H3T3K4R8) or the immunogenic (H3T3phK4R8) peptide to confirm specificity. Scale bars, 20 μm. The stick diagram indicates the modifications in residues 3–8 of histone H3, with the H3T3ph mark in red. (e) Double staining of stage V–VIII and VIII–X tubules with aH3T3ph and anti-HP1α antibodies. Insets correspond to groups of early round spermatids and late round/elongating spermatids, respectively. Scale bars, 20 μm. (f) Double staining with aH3T3ph/aH3S10ph and anti-Aurora B/H3S10ph antibodies during mitosis (spermatogonia), meiosis (spermatocytes) and interphase (spermatids). H3S10ph is present in the chromocenters of late round and elongating spermatids (left) and in the chromosomes of dividing cells, along with Aurora B (right). Scale bars, 20 μm. In all panels, DNA has been stained with TO-PRO 3. For details see text.