Fig 1.
Acetic orcein and silver staining of metaphase I spermatocytes (A, B). (C-N) γ-H2AX and RAD51 double immunolocalization in non-irradiated squashed spermatocytesof S.grossum. (A) The X chromosome is marked. Most bivalents show a single chiasma located near the pericentromeric regions. (B) Kinetochores of chromosomes are marked by asterisks. See text for the nomenclature applied to different bivalents. (C,G,H) γ-H2AX (green); (D,H,L) RAD51 (red); (E,I,M) DAPI; (F,J,N) merging of γ-H2AX and RAD51. (C-F) Leptotene. (G-J) Zygotene. (K-N) Pachytene. γ-H2AX and RAD51 signals are polarized in the same nuclear region. (C-N) images correspond to the sum of different focal planes. Bars represent 10 μm.
Fig 2.
Double immunolocalization of γ-H2AX (green) and RAD51 (red) in squashed spermatocytes of S. grossum irradiated with 5Gy dose of gamma rays.
The chromatin is counterstained with DAPI (blue). (A-D) Late leptotene. Immunolocalization of γ-H2AX and RAD51 one hour after irradiation. Whereas γ-H2AX labelling is distributed in the whole chromatin, RAD51 foci are concentrated in a nuclear region. (E-H) Zygotene. One day after treatment. The labelling of γ-H2AX covers the whole nucleus but that of RAD51 is polarized in a nuclear area. (I-L) Late zygotene. Three days after treatment. The distribution of γ-H2AX and RAD51 is maintained. All images are the sum of all focal planes of a nucleus. Bar represents 10 μm.
Fig 3.
Pairing and recombination in non-irradiated (A, C) and irradiated (B, D) S. grossum spermatocytes. (A, B) Polarized RAD21 axis labelling (green) in squashed late zygotene nuclei. (C-D) Double immunolocation of SMC3 (green) and RAD51 (red) in spread late zygotene nuclei. (A, B). (C, D) SMC3 paired cohesin axes are polarized despite the possible disturbances produced by the spreading procedure. RAD51 signals are almost absent in the unsynapsed chromosome regions of both non-irradiated (C) and irradiated (D) spermatocytes. A and B images are the sum of all the photographs taken at different focal planes and are at the same magnification. C and D are at the same magnification. Bars represent 10 μm
Fig 4.
Acetic orcein staining of squashed metaphase I chromosomes of S. grossum males 5–6 days post-irradiation.
Black arrowheads indicate chromosome fragments and secondary constrictions. (C-K) Centromere positions are indicated by black asterisks. Blue arrows mark the position of a chiasma between homologous chromosomes. (G-K) Red arrows indicate the chiasma joining non-homologous chromosomes. (A) Metaphase I. The single X chromosome, a bivalent with a single chiasma (II), a quadrivalent (IV) and a bichiasmatic long bivalent (II2Q) are indicated. (B) Metaphase I. A monochiasmatic bivalent (II) and a bichiasmatic bivalent (II2Q) are indicated. (A’, B’) Enlargements of the bichiasmatic bivalents of A and B respectively. (C, D) Bivalents with a single proximal chiasma. (E, F) Bichiasmatic bivalents at diakinesis (E) and metaphase I (F). Positions of chiasmata are indicated by blue arrows. (G, I, K) Quadrivalents. (H, J, L) Diagrams of quadrivalents shown in G, I and K. Homologous chromosomes are shown in red and green, respectively. A and B, A’ and B’ and C-K have the same magnification. Bars represent 10 μm
Fig 5.
Acetic orcein staining of squashed spermatocytes of S. grossum at anaphase I and second meiotic division, 8–12 days after irradiation.
(A, B) Anaphase I. (C) Telophase I. (D) Metaphase II. (E,F) Anaphase II. (G) Haploid spermatids. (H-J) Aberrant spermatids. Chromatin bridges (double white arrowheads), chromosomal fragments (black arrows), chromatin constrictions (black arrowheads), heteromorphic chromosomes (blue arrows) and lagging chromosomes (blue arrowhead) are indicated. Bars represent 10 μm.
Fig 6.
Projection of several focal planes of spermatocytes of S. grossum in which SMC3 has been located. Drawings of some bivalents have been superimposed in D, E and F. Notice that the homologous telomeric ends of long bivalents remain at the nuclear periphery. The separation between the unsynapsed ends is evident even in a Z projection (see red and green bivalents in D, E and F, and also S2 and S3 Videos). In E and F, short full synapsed bivalents are drawn in blue and purple. Bar represents 10 μm.
Fig 7.
Possible origin of quadrivalents.
Diagramatic representation of two long bivalents, partially synapsed, of S.grossum, depicted in green and red, respectively. Whereas chiasmata normally occur in the pericentromeric regions of homologous chromosomes, they could also take place between homologous subdistal regions of non-homologous chromosomes as a consequence of reciprocal translocations produced by exogenous DSBs and /or COs between homologous DNA sequences of non-homologous chromosomes (non-allelic homologous recombination) (blue arrow).