Abstract
Sister chromatid cohesion is regulated by cohesin complexes and topoisomerase IIα. Although relevant studies have shed some light on the relationship between these two mechanisms of cohesion during mammalian mitosis, their interplay during mammalian meiosis remains unknown. In the present study, we have studied the dynamics of topoisomerase IIα in relation to that of the cohesin subunits RAD21 and REC8, the shugoshin-like 2 (Schizosaccharomyces pombe) (SGOL2) and the polo-like kinase 1-interacting checkpoint helicase (PICH), during both male mouse meiotic divisions. Our results strikingly show that topoisomerase IIα appears at stretched strands connecting the sister kinetochores of segregating early anaphase II chromatids, once the cohesin complexes have been removed from the centromeres. Moreover, the number and length of these topoisomerase IIα-connecting strands increase between lagging chromatids at anaphase II after the chemical inhibition of the enzymatic activity of topoisomerase IIα by etoposide. Our results also show that the etoposide-induced inhibition of topoisomerase IIα is not able to rescue the loss of centromere cohesion promoted by the absence of the shugoshin SGOL2 during anaphase I. Taking into account our results, we propose a two-step model for the sequential release of centromeric cohesion during male mammalian meiosis II. We suggest that the cohesin removal is a prerequisite for the posterior topoisomerase IIα-mediated resolution of persisting catenations between segregating chromatids during anaphase II.
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Acknowledgments
We thank Linda Wordeman, Bill Earnshaw, Jibak Lee and Erich A. Nigg for providing MCAK, INCENP, REC8 and PICH antibodies, respectively; Lorena Barreras for her technical assistance; and Kim Nasmyth for providing REC8-myc transgenic male mice. This work was supported by grants BFU2009-10987/BCM to A.V., BFU2009-08975 to J.L.B., SAF2011-25252 to A.M.P. and SAF2011-28842-C02-01 to J.A.S. from the Ministerio de Ciencia e Innovación and Ministerio de Economía y Competitividad (Spain) and grant FMM AP98712012 from the Fundación Mutua Madrileña to J.L.B.
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Fig. S1
Distribution of Topo IIα and SYCP3 in metaphase I and anaphase I chromosomes. Double immunolabelling of Topo IIα (green) and SYCP3 (red) in selected metaphase I bivalents (a, b) and an anaphase I chromosome (c) from spreaded spermatocytes. Topo IIα appears enriched at centromeres and also along the chromatid axes (arrows), while SYCP3 is accumulated at the inner centromere domain and also appears at the interchromatid domain, although the labelling interrupts at the chiasma sites (arrowheads). Scale bar, 2.5 μm (JPEG 458 kb)
Fig. S2
Relative distribution of Topo IIα and INCENP during chromosome congression to the metaphase II plate. Double immunolabelling of Topo IIα (green) and INCENP (red) on squashed prometaphase II (a, b) and metaphase II (c, d) spermatocytes. INCENP redistributes from a band traversing the inner centromere domain (a, b) to a single signal at the kinetochore region (c, d), while Topo IIα persists as a TOCOS, as indicated in the schematic illustrations. Scale bar, 0.5 μm (JPEG 300 kb)
Fig. S3
Distribution of PICH and Topo IIα in early anaphase. Double immunolabelling of PICH (green) and Topo IIα (red) and chromatin counterstaining with DAPI (blue) on Pam212 early anaphases. Elongated PICH strands are observed between Topo IIα signals at the centromeres. Scale bar, 3 μm (JPEG 1804 kb)
Fig. S4
Topo IIα distribution during spermatogonial mitosis. a–h Double immunolabelling of Topo IIα (green) and kinetochores (ACA; red) and chromatin counterstaining with DAPI (blue) in spermatogonial prophase and metaphase from squashed seminiferous tubules. i–l Immunolabelling of Topo IIα (green) and chromatin counterstaining with DAPI (blue) on a spreaded spermatogonial metaphase. Topo IIα appears enriched at the centromere regions and faintly represented at the chromatid axes. Two enlarged chromosomes are shown together with a schematic illustration (k, l). Scale bars, 3 μm in h and 5 μm in j (JPEG 1427 kb)
Fig. S5
Apoptosis after Topo IIα inhibition with ET during meiosis I and meiosis II. Double immunolabelling of Topo IIα (green) and kinetochores (ACA; red) and chromatin counterstaining with DAPI (blue) on squashed spermatocytes. Positive immunoreactivity can only be detected in live metaphase I (a) and metaphase II (k) spermatocytes. b–f Apoptotic metaphase I spermatocytes with hypercompacted chromosomes without apparent univalents (b, e) and with univalents (arrowheads in c, d and f). g, h Apoptotic anaphase I/telophase I spermatocytes with lagging chromosomes (g) and without lagging chromosomes (h). i, j Apoptotic metaphase I spermatocytes with fragmented chromosomes. l–o Apoptotic metaphase II spermatocytes with aligned chromosomes (l–n) and misaligned chromosomes (o). p–y Apoptotic anaphase II/telophase II spermatocytes without lagging chromosomes (p, r, u, w, x), with lagging chromosomes (arrowheads) and chromatin bridges (arrows) (q, s, t, v) and with fragmented chromosomes (y). Scale bar, 3 μm (JPEG 1572 kb)
Fig. S6
TUNEL assay after Topo IIα inhibition with ET. TUNEL assay (green) and chromatin counterstaining with DAPI (blue) on squashed spermatocytes after 24 h of ET treatment. Apoptotic cells correspond to those with hypercondensed chromosomes. The image is a projection of different focal planes throughout the cell volume. Scale bar, 5 μm (JPEG 478 kb)
Movie 1
Metaphase I spermatocyte shown in Fig. 1f–h. Double immunolabelling of Topo IIα (green) and kinetochores (red) and chromatin counterstaining with DAPI (blue) (MOV 469 kb)
Movie 2
Metaphase II spermatocyte shown in Fig. 3g–i. Double immunolabelling of Topo IIα (green) and kinetochores (red) and chromatin counterstaining with DAPI (blue) (MOV 259 kb)
Early anaphase II spermatocyte. Immunolabelling of Topo IIα (green) and chromatin counterstaining with DAPI (blue) (MOV 812 kb)
Anaphase I and telophase I spermatocytes after ET treatment shown in Fig. 6e–l. Double immunolabelling of Topo IIα (green) and kinetochores (red) and chromatin counterstaining with DAPI (blue) (MOV 2 mb)
Anaphase II spermatocyte after ET treatment shown in Fig. 7e–h. Double immunolabelling of Topo IIα (green) and kinetochores (red) and chromatin counterstaining with DAPI (blue) (MOV 1.03 mb)
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Gómez, R., Viera, A., Berenguer, I. et al. Cohesin removal precedes topoisomerase IIα-dependent decatenation at centromeres in male mammalian meiosis II. Chromosoma 123, 129–146 (2014). https://doi.org/10.1007/s00412-013-0434-9
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DOI: https://doi.org/10.1007/s00412-013-0434-9