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First published online June 23, 2005
doi: 10.1242/10.1242/jcs.02391

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DNA double-strand breaks and homology search: inferences from a species with incomplete pairing and synapsis

Adela Calvente^1,*, Alberto Viera^1,*, Jesús Page¹, M. Teresa Parra¹, Rocío Gómez¹, José A. Suja¹, Julio S. Rufas^1, and Juan L. Santos²

¹ Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
² Departamento de Genética, Facultad de Biología, Universidad Complutense de Madrid, 28040 Madrid, Spain

View larger version (119K): [in a new window]   Fig. 1. Meiotic -H2AX foci encompass cohesin-axis assembly. Early stages of male meiosis after double immunolabelling for the cohesin subunit SMC3 (green in first column) and the histone variant -H2AX (red in second column). Merged images (SMC3 and -H2AX) are presented in the forth column. The third column shows chromatin organisation (DAPI staining, blue). All images correspond to the accumulative superimposition of several focal planes. A progressive increase in the cohesin-axis maturation and the number of -H2AX foci can be detected. (A-D) Pre-leptotene. Short SMC3 stretches are detected in a discrete nuclear region (A). Few -H2AX foci (B) are located on these SMC3 threads (D). From early (E-H) up to late (I-L) leptotene, the maturation and linear organisation of the axes occur concomitantly with an increase of -H2AX foci. All -H2AX foci are located over the cohesin axes. (M-P) Zygotene spermatocyte characterised by the presence of parallel thick SMC3 threads located at a discrete region of the nucleus, denoting a bouquet-like organisation. Notice that in these stages no -H2AX staining is detected on the sex chromosome (X). Bar, 10 µm.

View larger version (93K): [in a new window]   Fig. 2. Discrete -H2AX foci are located over synapsed autosomal regions at pachytene. Three different pachytene spermatocytes after double immunolabelling of the cohesin subunit SMC3 (green) and the histone variant -H2AX (red). Merged images (SMC3 and -H2AX, fourth column) and DAPI stained nuclei (blue) are also shown. SMC3 staining indicates the absence of full synapsis (A,E,I). The number of -H2AX foci reveals the different pachytene substages. Early pachytene (A-D) with a large amount of -H2AX foci. The number of foci varies from seven to 12 in mid-pachytene (E-H), but foci are absent in late pachytene (I-L). Notice that the amount of synapsis is similar in all pachytene sub-stages. X indicates sex chromosome. Bar, 10 µm.

View larger version (101K): [in a new window]   Fig. 3. RAD51 foci appear over cohesin axes. Early meiotic stages of male meiosis after double immunololabelling of the cohesin subunit SMC3 (green in first column) and the recombinase RAD51 (red in second column). Merged images (SMC3 and -H2AX) are in the forth column and DAPI stained nuclei (blue) are observed in the third one. Several focal planes were merged to obtain each image. Early leptotene (A-D) is characterised by the presence of threads of cohesin axes in a polarised nuclear region (A). Notice that RAD51 foci (B) are located over SMC3 threads (D). Mid-leptotene (E-H). Progressive maturation of cohesin axes (E). The number of RAD51 foci rapidly increase (F) and remain associated to the cohesin axes (H). The characteristic bouquet-like arrangement can be identified in zygotene spermatocytes (I-L). Both SMC3 paired-axes (I) and RAD51 foci (J) are polarised in a discrete region of the nucleus. It is evident that the localisation of RAD51 foci is strictly restricted to paired cohesin axes (L). RAD51 foci are restricted to thick, synapsed, cohesin axes at early pachytene (M-P). Mid-pachytene (Q-T) shows a lower amount of RAD51 foci (R). Notice that, in all cases RAD51 signals are located over synapsed regions (D,H,L,P,T). X indicates sex chromosome. Bars, 10 µm.

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