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Files in this Data Supplement:
Fig. S1. Chiasma localization in Stethophyma grossum spermatocytes. (A) Feulgen stained first meiotic metaphase. The single sex chromosome (X) is displaced to one of the cell poles. Note that all of the bivalents with exception of the three shortest ones (M9, S10, S11) exhibit a proximal chiasma. (B) Chiasma positions (arrows) become more evident after centromere detection by means of immunostaining with a-DNA Topoisomerase-II, as shown in the enlarged bivalents.
Fig. S2. DSBs appearance in prophase I spermatocytes of the grasshopper Eyprepocnemis plorans. Different focal planes of three spermatocytes at leptotene (A,B,C), zygotene (D,E,F) and early pachytene (G,H,I). Immunolocalization of the cohesin subunit SMC3 (green) and the histone variant g-H2AX (red) are shown in the first two columns. Merged images appear in the third column. Early-mid leptotene is characterized by the presence of thin lines of cohesin axes in the whole nucleus (A). At this stage the first g-H2AX foci are observed (B). These are randomly distributed over cohesin axes (C). At zygotene (D), the beginning of synapsis is observed, and a similar cohesin axes morphogenesis is found among bivalents and. (E,F) Ribbons of g-H2AX locate over those autosomal regions that are undergoing synapsis in the bouquet area. At the zygotene/ pachytene transition (G,H,I), the synapsis is completed in all autosomal bivalents, with g-H2AX foci equally distributed over the paired autosomal cohesin axes. Bar represents 10 mm.
Fig. S3. The sequence of RAD51 foci distribution in prophase I spermatocytes of the grasshopper Eyprepocnemis plorans. Immunolocalization of the cohesin subunit SMC3 (green) and the recombinase RAD51 (red) of three spermatocytes at leptotene (A,B,C), zygotene (D,E,F) and early pachytene (G, H, I). Merged images are also shown in the third column. All images are the superimposition of different focal planes. Early–mid leptotene was devoid of RAD51 signalling (B). At zygotene the number of RAD51 foci has increased extraordinarily (E), they being distributed over the cohesin axes along the entire nucleus (F). At the zygotene/pachytene transition (G,H,I), a uniform pattern of RAD51 foci is observed over the length of paired cohesin axes. Bar represents 10 mm.
Fig. S4. g-H2AX appearance preceded RAD51 foci formation. Meiotic stages from preleptotene up to pachytene in Stethophyma grossum spermatocytes after double immunolocalisation of the recombinase RAD51 (green) and the histone variant g-H2AX (red). Spermatocytes were counterstained with DAPI (blue). Merged images of RAD51 and g-H2AX labelling are shown in the fourth column. Each image was obtained by the superimposition of several focal planes. At preleptotene (A-D) no RAD51 labelling was detected (A), however discrete initial g-H2AX foci were observed (B). Leptotene (E-H) presented a conspicuous g-H2AX staining and the formation of the initial RAD51 foci. Both proteins labelling appeared located in close proximity (H). From early (I-L) up to late zygotene (M-P) the amount of both RAD51 foci (I,L) and g-H2AX (J,N) decreased. RAD51 foci were located over the g-H2AX ribbons (L,P). This became more evident at late zygotene (P). At pachytene (Q-T) only a few number of discrete RAD51 and g-H2AX foci were detected. Some of them were associated but do not strictly colocalize (T). Bar in T represents 10 mm.
Movie 1. The pattern of synapsis in Stethophyma grossum spermatocytes. 3-D reconstruction of 60 focal planes throughout a single pachytene nucleus after SMC3 immunolocalization. The cohesin axes are revealed in green. Synapsed and unsynapsed chromosomal regions are denoted by thick and thin SMC3 filaments. In the selection that shows a magnification of the distal end of an autosomal bivalent (the position of telomeres is indicated; Tel), thicker and thin cohesin axes are marked by white and red arrows, respectively.
Movie 2. First g-H2AX foci appeared where cohesin axis morphogenesis is advanced.
3-D reconstruction of a Stethophyma grossum leptotene spermatocyte double immunolabelled against the cohesin subunit SMC3 (green) and the g-H2AX histone variant (red). Chromatin was counterstained with DAPI (blue), and the position of the X chromosome is indicated (X). Note the marked nuclear polarization of cohesin axes morphogenesis (top left) which is more advanced in the left nuclear domain. Initial double-strand breaks, revealed by g-H2AX foci (top right), also displayed nuclear polarization, appearing located over those cohesin axes presenting a more advanced morphogenesis (bottom right).
Movie 3. g-H2AX foci are only located over paired cohesin axes. 3-D reconstruction of a Stethophyma grossum pachytene spermatocyte after double immunolabelling against the cohesin subunit SMC3 (green) and the g-H2AX histone variant (red). Chromatin is counterstained with DAPI (blue), and the position of the X chromosome indicated. Discrete g-H2AX foci are restricted to those autosomal regions with paired cohesin axes. The single X chromosome, which remains unsynapsed, displays a single cohesin axis of similar thickness than that exhibit by autosomal regions that do not pair nor synapse.
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