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
Author for correspondence (e-mail: julio.s.rufas{at}uam.es)
Accepted 21 March 2005
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Summary |
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Key words: Double-strand breaks, Synapsis, Recombination, -H2AX, Meiosis, RAD51
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Introduction |
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In some model organisms, the successful pairing of homologous chromosomes depends on the meiotic recombination pathway initiated by programmed double-strand breaks (DSBs) catalysed by SPO11, a topoisomerase-II-like protein (Keeney et al., 1997; Baudat et al., 2000
; Romanienko and Camerini-Otero, 2000
; Grelon et al., 2001
; Peoples et al., 2002
). DSB formation requires the products of at least nine other genes that are involved in either stabilisation or recruitment mechanisms (Keeney, 2001
). DSBs also induce the phosphorylation of certain variants of the histone H2A, such as H2AX, H2Av (Redon et al., 2002
; Madigan et al., 2002
) and H2B (Fernández-Capetillo et al., 2004
). These modifications are associated with the recruitment of repair factors to damaged DNA in order to facilitate repair efficiency (Madigan et al., 2002
; Celeste et al., 2003
). DSB ends are degraded from their 5' end, which gives rise to single-stranded DNA that is thought to be used by recombinases to invade double-stranded DNA and form heteroduplex regions (Haber, 2002). Two major recombinases have been described: RAD51 and its meiosis-specific homolog DMC1, both of which are homologues of the bacterial RecA protein (Bishop et al., 1992
; Shinohara et al., 1992
; Bishop, 1994
). It is well established that RAD51 is involved in both mitotic and meiotic recombination (Shinohara et al., 1992
). By analogy with the functions of the RecA protein, RAD51 is also expected to be involved in homology search, (Ashley et al., 1995
; Rockmill et al., 1995
; Barlow et al., 1997
; Moens et al., 1997
) and recent evidence reinforces this suggestion (Franklin et al., 1999
; Moens et al., 2002
; Pawlowski et al., 2003
; Tsubouchi and Roeder, 2003
).
For decades, grasshoppers were considered a model organism for studying meiotic pairing and synapsis. Immunocytological studies in the species Locusta migratoria and Eyprepocnemis plorans on the location of the phosphorylated histone H2AX (-H2AX) which marks sites of DSBs in combination with the recombinase RAD51 and the cohesin subunit SMC3 have led us to suggest that, at least in the two species analysed, certain steps in the recombination pathway might be required for normal synapsis (Viera et al., 2004a
). This sequence of meiotic events also occurs in yeast (reviewed in Kleckner, 1996
; Roeder, 1997
), mouse and Arabidopsis thaliana (Grelon et al., 2001
; Mahadevaiah et al., 2001
), but not in Drosophila melanogaster and Caenorhabditis elegans (Derburg et al., 1998; Page and Hawley, 2001
).
To obtain a better understanding of the roles of DSBs and RAD51 in the processes of pairing and synapsis, we analysed here the sequence of the chromosomal localisation of -H2AX and RAD51 proteins in spermatocytes of the grasshopper Stethophyma grossum. This species displays singular meiotic features because there are three different synaptic situations within each spermatocyte: (1) Full synapsis in the three shortest bivalents of the complement, (2) partial synapsis restricted to centromeric ends in the remainder eight bivalents, and (3) the unsynapsed X chromosome (Jones, 1973
; Fletcher, 1977
; Wallace and Jones, 1978
; Jones and Wallace, 1980
). Therefore, this bizarre natural system provides the possibility of analysing, within the same nuclear environment, the relationship between recombination events and different patterns of pairing and synapsis.
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Materials and Methods |
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Feulgen-Rossenbeck staining
Testes were fixed in 3:1 ethanol:acetic acid and stored at 4°C until required. The fixed material was hydrated for 15 minutes in distilled water. Afterwards, the seminiferous tubules were immersed in 5N HCl at 20°C for 30-40 minutes, thoroughly washed in distilled water and stained with Schiff reagent (Merck) for at least 30 minutes. Two or three tubules were placed per slide and squashed into a drop of 50% acetic acid.
Antibodies
A polyclonal rabbit anti-SMC3 antibody (AB3914; Chemicon International) raised against a synthetic peptide of human SMC3 was used to detect the cohesin subunit SMC3 at a 1:30 dilution in PBS. It is important to note that only the lot number 220701985 of the antibody works in grasshoppers whereas the actual stock commercialised by Chemicon does not. A monoclonal mouse antibody (number 05-636; Upstate) raised against amino acids 134-142 of human histone -H2AX was used to detect the histone variant
-H2AX (Paull et al., 2000
) at a 1:500 dilution in PBS. This peptide sequence is identical in yeast and mouse (Redon et al., 2002
). A polyclonal rabbit anti-RAD51 antibody (Ab-1; PC130; Oncogene Research Products), generated against recombinant HsRAD51 protein, was used to detect the recombinase RAD51 at a 1:30 dilution in PBS. All antibodies used in the present study have previously been tested in grasshopper immunoblot assays (Viera et al., 2004a
). A monoclonal mouse anti-topoisomerase II
antibody (MAB4197; Chemicon International) against a major 170 kDa protein (that was identified as the
isoform of human topoisomerase II) was used to detected the topoisomerase II
protein at 1:5 dilution in PBS.
Immunofluorescence
Fixed spermatocytes were incubated overnight at 4°C with primary antibodies. Following three washes of 5 minutes in PBS, primary antibodies were revealed with the appropriate secondary antibodies conjugated with either FITC or Texas Red (Jackson ImmunoResearch Laboratories) at a 1:150 dilution in PBS, counterstained with 4',6-diamidino-2-phenylindole (DAPI) 10 µg/ml, thoroughly washed in PBS and finally mounted with Vectashield (Vector Laboratories). In double-immunolabelling experiments both primary antibodies were incubated simultaneously, except when they had been generated in the same host species. In this last case, slides were first incubated with the first primary antibody (anti-SMC3) for 1 hour at room temperature, rinsed three times for 5 minutes in PBS and incubated overnight at 4°C with an FITC-conjugated goat Fab' fragment anti-rabbit IgG (Jackson) at a 1:100 dilution in PBS. Afterwards, slides were rinsed at least six times for 5 minutes in PBS, incubated with the second primary antibody (anti-RAD51) for 1 hour at room temperature, rinsed three times for 5 minutes in PBS and then incubated with a Texas Red-conjugated goat anti-rabbit IgG (Jackson) at a 1:150 dilution. Finally, slides were counterstained with DAPI and mounted as previously described.
Observations were performed using an Olympus BX61 microscope equipped with a motorised Z-axis and epifluorescence optics. Stacks of images were captured with a DP70 Olympus digital camera using the AnalySIS software from Olympus and finally analysed and processed using the public domain ImageJ software (National Institutes of Health, USA; http://rsb.info.nih.gov/ij), VirtualDub (http://virtualdub.org/) and Adobe Photoshop 6.0 software.
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Results |
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DSBs appear at prophase I before synapsis
To establish accurately the sequence of prophase I stages we used an antibody against the SMC3 protein. This protein is a member of the structural maintenance of chromosomes (SMC) family of proteins, which is widespread in eukaryotes (Hirano, 2002). SMC3 is involved in sister chromatid cohesion during meiosis, and also seems to be essential for the organisation of an axial element (AE) structure in which chromatin loops are attached in mammal meiosis (Eijpe et al., 2000
; Pelttari et al., 2001
; James et al., 2002
). In grasshoppers, the AE development has been analysed indirectly at prophase I by an antibody against SMC3. Although it was not possible to assay the synaptonemal complex (SC) formation directly, it could be inferred from the identification of thick and thin SMC3 filaments, which correspond to synapsed and unsynapsed regions, respectively (Viera et al., 2004a
) (see also supplementary material Movie 1). On these grounds, we assume that in Stethophyma grossum the SMC3 immunolocalisation patterns reflect the different stages of synaptic development that can be related to the sequence of appearance of
-H2AX and RAD51 proteins throughout prophase I.
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In zygotene spermatocytes the SMC3 cohesin axis maturation increased and appeared as well-defined thin lines all over the nucleus. Interestingly, paired cohesin axes that form thick filaments (synapsed regions) were only concentrated over a determined nuclear region that resembled a `bouquet-like' arrangement (Fig. 1M). By contrast, the unsynapsed autosomal regions remained dispersed in the nucleus and are not associated in pairs (Fig. 1M). At zygotene, -H2AX was detected as ribbons (Fig. 1N) associated with the chromatin adjacent to autosomal regions that had undergone, or were undergoing, pairing and synapsis. At these regions, SMC3 axes displayed parallel trajectories to each other. However, the chromosomal regions in which pairing was never to be completed showed irregular SMC3 axis trajectories and no
-H2AX labelling (Fig, 1P). From mid-zygotene to pachytene
-H2AX signalling started to disappear (compare Fig. 1N and Fig. 2B).
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Surprisingly, pachytene spermatocytes maintained the marked nuclear polarisation of the cohesin-axis maturation and the synaptic state of homologues observed at zygotene (Fig. 2A,E,I). Therefore, throughout all pachytene sub-stages and within the same nucleus we found bivalents that achieved full synapsis, bivalents with incomplete synapsis (from one of their ends to almost half of their lengths) and the unsynapsed X chromosome. These results reinforced previous observations on the existence of partial synapsis in the spermatocytes of this species (Fletcher, 1977; Wallace and Jones, 1978
; Jones and Wallace, 1980
). At pachytene,
-H2AX labelling also appeared polarised and reduced to a few discrete foci (Fig. 2B,F,J), which were located over the autosomal synapsed regions (Fig. 2D,H,L and supplementary material Movie 3). The number of
-H2AX foci found at early pachytene (Fig. 2B) decreased by mid-pachytene (Fig. 2F) and was almost absent at late pachytene (Fig. 2J).
-H2AX was not observed until the formation of spermatids (data not shown). Spermatid labelling has previously been reported in mouse (Hamer et al., 2003
) and in two species of grasshoppers (Viera et al., 2004a
). Therefore, our results indicate that, in Stethophyma grossum spermatocytes, extensive DSBs formation occurs before pairing and is strictly restricted to those autosomal regions that undergo synapsis (compare with the prophase I sequence of these events in the standard grasshopper species Eyprepocnemis plorans, see supplementary material Figs S2 and S3).
-H2AX is absent from the single X chromosome
The single X chromosome usually occupied a peripheral zone of the nucleus during prophase I (X in Fig. 1C,G,K,O and Fig. 2C,G,K). No SMC3 signalling was detectable in the X chromosomes until late leptotene, when its cohesin axis became evident (compare Fig. 1A,C,E,G with I,K). Therefore, the pattern of conformation and maturation of the cohesin axis in the X chromosome was delayed compared with that in autosomes. Throughout prophase I, the X chromosome remained unsynapsed and without any signs of -H2AX labelling, even though it was preferentially embedded in the nuclear domain, which contained the autosomal synapsed regions encompassed with strong
-H2AX staining (Figs 1 and 2).
Unsynapsed chromosomal regions are devoid of RAD51 foci
RAD51 first appeared as few discrete foci on the incipient stretches of autosomal cohesin axes at early leptotene (Fig. 3A,B,D). During leptotene, the number of RAD51 foci increased dramatically but foci were restricted to the polarised nuclear domain in which the cohesin axis formation was advanced (Fig. 3E,F,H). The rest of the nucleus, showing cohesin axes in a pre-leptotene-like appearance, was devoid of RAD51 foci (Fig. 3H). At zygotene, RAD51 foci were polarised on a nuclear domain (Fig. 3J), being closely associated to the synapsed or almost synapsed autosomal regions that displayed the `bouquet-like' rearrangement (Fig. 3L). However, we did not detect RAD51 foci within the unsynapsed chromosomal regions that dispersedly occupied the rest of the nucleus (Fig. 3I-L). From early to mid-pachytene spermatocytes (Fig. 3M-T), the number of RAD51 foci decreased until they had disappeared completely in late pachytene nuclei. Notice that, whereas RAD51 labelling was never associated with the single X chromosome (X in Fig. 3C,G,K,O,S), the -H2AX labelling was.
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Discussion |
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In Stethophyma grossum spermatocytes only one (occasionally two, in M9) of all recombination events observed per bivalent lead to crossover-formation that manifested itself as a chiasma. Fully synapsed M9, S10 and S11 bivalents did not present chiasma localisation at metaphase I, whereas the longest bivalents (L1-M8), which presented incomplete pairing and synapsis, always displayed chiasma localisation. In some L1-M8 bivalents, chiasmata form very close to the centromere and in other ones they form at some distance from the centromere, but in no case does the centromere to chiasma distance exceed the length of the S11 chromosome (Perry and Jones, 1974; see also supplementary material Fig. S1). Moreover, it was observed that only a region comparable in length to the S11 bivalent is paired and synapsed in each of the longest bivalents (Wallace and Jones, 1978
; and this work). Thus, large bivalents would only produce a single crossover because they behave like shorts ones in respect of generating crossovers and the corresponding interference signalling along their length. Accordingly, the formation of a second chiasma in L1-M8 bivalents would be prevented because of a strict regulation of interference signals that can be transmitted either along chromosomal axes (Börner et al., 2004
) (reviewed by Bishop and Zickler, 2004
) or through the SC (reviewed by Sinohara et al., 2003). Notice that, in contrast to males, chiasmata are not localised in females of Stethophyma grossum (Perry and Jones, 1974
). The evolutionary reason for these differences between the sexes remains to be ascertained and further studies are necessary to investigate them.
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Acknowledgments |
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Footnotes |
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* These authors contributed equally to this work
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References |
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