Article |
Address correspondence to Christa Heyting, Molecular Genetics Group, Botanical Center, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, Netherlands. Tel.: 31-317-482150. Fax: 31-317-483146. E-mail: christa.heyting{at}wur.nl
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Abstract |
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Key Words: meiosis; axial element; rat; sister chromatid cohesion; synaptonemal complex
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Introduction |
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In meiosis, cohesins also function in chromosome segregation, but in a modified way. In the meiotic cell cycle, two nuclear divisions (meioses I and II) follow a single S phase. Sister chromatid cohesion can ensure proper chromosome segregation in both meiotic divisions, because it is released in two steps (Buonomo et al., 2000). At anaphase I, cohesion between sister chromatid arms is lost, which leads to disjunction of homologous chromosomes, and at anaphase II, centromeric cohesion is lost so that sister chromatids can separate (see discussion in Buonomo et al., 2000).
Cohesins not only function in chromosome segregation, but also in DNA repair by homologous recombination (Jessberger et al., 1996; Hirano, 2000; Jessberger, 2002). In the mitotic cycle, this role may include the assembly of recombinational repair complexes (Hartsuiker et al., 2001) and the direction of recombinational repair toward the sister chromatid rather than the homologous chromosome, if there is one (Grossenbacher-Grunder and Thuriaux, 1981). In somatic mammalian cells, recombination between sister chromatids is a prominent pathway of DNA repair (Johnson and Jasin, 2000). In meiosis, cohesins are also required for homologous recombination (Klein et al., 1999), but their role has been modified in such a way that recombination occurs preferentially between nonsister chromatids of homologous chromosomes rather than sister chromatids (Schwacha and Kleckner, 1997).
A third aspect of the altered role of cohesins in meiosis is their contribution to the assembly of synaptonemal complexes (SCs).* SCs are zipper-like protein structures that are assembled between homologous chromosomes (homologues) during meiotic prophase. They play an only partly understood role in adapting recombination and cohesion for meiosis (Roeder, 1995; Kleckner, 1996). SCs consist of two axial elements (AEs), which are connected by transverse filaments. Each AE supports the two sister chromatids of one homologue. Cohesins are required for the assembly of AEs and constitute part of AEs (Klein et al., 1999; Eijpe et al., 2000a; Pelttari et al., 2001).
Given these specific roles of cohesins in meiosis, it is not surprising that meiotic variants of cohesins exist. Meiotic cohesin Rec8 replaces Scc1 in all species analyzed thus far. Rec11 of Schizosaccharomyces pombe (Krawchuk et al., 1999) and mammalian STAG3 (Pezzi et al., 2000) are meiotic variants of Scc3, and SMC1ß is a mammalian meiotic variant of SMC1 (further denoted as SMC1) (Revenkova et al., 2001).
Previously, using Mabs 462 (anti-SMC3) and ß70 (anti-SMC1ß), we found that in rat, SMC1ß (Eijpe et al., 2000a) and SMC3 (Revenkova et al., 2001) colocalized with meiotic AE components SCP2 (Offenberg et al., 1998) and SCP3 (Lammers et al., 1994). This agreed with the colocalization of Smc3 with AE component Red1 in yeast (Klein et al., 1999). However, according to our first approximation, SCP2, SCP3, SMC1ß, and SMC3 appeared simultaneously in AEs in leptotene, after premeiotic S phase (Offenberg et al., 1998; Eijpe et al., 2000a; Revenkova et al., 2001). This was unexpected for SMC1ß and SMC3, because the cohesin complex as a whole is thought to bind to chromatin before S phase and to establish cohesion during S phase (Uhlmann and Nasmyth, 1998; Ciosk et al., 2000). Furthermore, SMC1ß and SMC3 had virtually disappeared from the chromosome arms at metaphase I (Revenkova et al., 2001), when arm cohesion is most needed for proper disjunction of homologues (Buonomo et al., 2000). In this study, we analyzed therefore in detail the presence and localization of cohesins in successive stages of meiosis of the male rat. We focused on REC8, which is a target of the cell cycleregulated protease that releases cohesion in yeast meioses I and II (Buonomo et al., 2000). Furthermore, we included SMC1 in the analysis and a new anti-SMC3 serum.
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Results |
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For labeling of SMC3, we used monoclonal (MSMC3) and polyclonal (R
SMC3) antibodies, which recognize the same band on Western blots, which is enriched in SCs (Fig. 2 B). In previous studies, we found that M
SMC3 specifically labeled the AEs and did not label somatic or premeiotic S-phase cells (Eijpe et al., 2000a; Revenkova et al., 2001). The new anti-SMC3 serum R
SMC3 predominantly produced a diffuse nuclear labeling in all cell types, including somatic and premeiotic S-phase cells (Fig. 3, GI); in Triton X-100treated sections, the diffuse labeling was largely lost from spermatocyte nuclei, whereas a weak labeling remained along the AEs (Fig. 3, JL) (also see next paragraph). To summarize, during premeiotic S phase, all three analyzed types of cohesin (SCC1/REC8, SMC1
/SMC1ß, and SMC3) are represented and are diffusely distributed through the nucleus.
Assembly of AEs
Before we analyzed the incorporation of proteins in AEs, we tested the antibodies on various types of preparations under various conditions. Anti-SMC1 and R
SMC3 antibodies could produce both a diffuse labeling throughout spermatocyte nuclei and labeling of the AEs; which labeling pattern prevailed depended on the conditions. Pretreatments that were likely to extract proteins and disrupt structures resulted in loss of the overall nuclear labeling but enhanced the labeling of AEs by anti-SMC1
(Eijpe et al., 2000a) and R
SMC3 (see above and Fig. 3, JL). Because isolated SCs are enriched in SMC1
and SMC3 (Eijpe et al., 2000a; Fig. 2 B), we think that these proteins make part of AEs and are rather inaccessible to anti-SMC1
or R
SMC3 but accessible to M
SMC3 within these structures. We therefore used M
SMC3 for detection of SMC3 within AEs.
We analyzed the incorporation of REC8, SMC1ß, SMC3, SCP2, and SCP3 in AEs using primarily dried down preparations (Fig. 5)
. REC8 formed short axial structures (REC8-AEs) before SMC1ß, SMC3, SCP2, and SCP3; the first short REC8-AEs appeared already during premeiotic S phase (Fig. 5, B, J, and K). After premeiotic S phase, the leptotene cells assembled increasingly longer REC8-AE fragments (Fig. 5, C, F, and N). SCP3 appeared in leptotene and localized along already formed REC8-AEs from its first appearance on; it formed dots along REC8-AEs (Fig. 5, B and C), which extended and fused until they lined REC8-AEs along their length (Fig. 5 D). At some sites in leptotene and zygotene nuclei, it appeared as if the chromatin surrounding the REC8-AEs also contained some SCP3 (Fig. 5 D, arrows). Using MSMC3 and Mab ß70 (anti-SMC1ß), we got corresponding results for SMC1ß and SMC3, as is shown for SMC3 in Fig. 5 (EH). R
SMC3 did not label AEs detectably in (pre)leptotene (unpublished data). Because we found previously that SCP2, SCP3, SMC1ß, and SMC3 appear along AEs simultaneously and colocalize with each other from their first appearance on (Offenberg et al., 1998; Schalk et al., 1998; Eijpe et al., 2000a; Revenkova et al., 2001), we conclude that these four proteins are all deposited along already existing REC8-AE fragments in leptotene spermatocytes.
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Bridges between desynapsed AEs
Fig. 6
shows REC8 and other AE components in later stages of meiosis. In pachytene, REC8 colocalized with other SC components (Fig. 6, A and B). However, in late diplotene, SCP2, SCP3, SMC1ß, and SMC3 started to accumulate in the centromeric and telomeric regions, whereas REC8 did not (Fig. 6, CE). Furthermore, in nuclei with almost complete desynapsis, some bivalents showed one or two bridges between AEs, which were labeled by anti-SCP3 (Fig. 6, G, J, and L). We found such bridges before by immunofluorescence labeling of SMC3 (using MSMC3), SCP2 (Schalk, 1999), or SMC1ß (Revenkova et al., 2001). These bridges do not contain REC8 however (Fig. 6, F and H). Cdk2, which marks the position of crossovers on the AEs until late pachytene/diplotene (Ashley et al., 2001), is still present at the position of part of the bridges (Fig. 6, K and L), which indicates that the bridges represent crossover sites. Thus, SMC1ß, SMC3, SCP2, and SCP3 fulfill functions at crossover sites that do not require the presence of REC8.
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Disappearance of cohesins and AE components from the centromeric regions at anaphase II
At the metaphase I to anaphase I transition, all SMC1ß, SMC3 (as detected by MSMC3), SCP2, and SCP3 had concentrated in the centromeric region (shown for SCP3 in Fig. 7
, AE; Revenkova et al., 2001). REC8 had disappeared from the distal regions and persisted in the proximal regions of the chromosome arms so that two groups of REC8-labeled dots remained, which flanked the centromeric regions (Fig. 7, C and D). In anaphase I, REC8 was confined in most bivalents to two spots, which flanked the kinetochores (Fig. 7, F and H), whereas SCP3 stayed accumulated in a broad area around the kinetochores (Fig. 7, F and G). This area became more compact and needle shaped in metaphase II (Fig. 7, L and M; see also the metaphase II/anaphase II nucleus in Fig. 7, I and J). We found this previously for SMC3, SMC1ß (Revenkova et al., 2001), and SCP2 (Schalk, 1999). At the metaphase II to anaphase II transition, some of these needle-shaped aggregates were still associated with REC8, whereas others were not. The colocalization of REC8 and SCP3 with kinetochores was also lost at the metaphase IIanaphase II transition (Fig. 7, I and J).
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Discussion |
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REC8 and AE assembly
Several investigations (Klein et al., 1999; Eijpe et al., 2000a; Pezzi et al., 2000; Pelttari et al., 2001; Revenkova et al., 2001) indicate that cohesins provide a basis for AEs. Our results support this idea and allow for distinguishing between the role of REC8 and other cohesins and AE components. We think that REC8 provides a basis for AE assembly because it forms AE-like structures before SMC1ß, SMC3, SCP2, and SCP3. It is not known what triggers the formation of REC8-AEs, but one possibility is phosphorylation of REC8, because the pattern of REC8 bands in preleptotene and midprophase (pachytene/diplotene) suggests that the degree of REC8 phosphorylation increases between these stages of meiosis (Fig. 2 D). Within REC8-AEs, REC8 molecules are initially not associated with SMC1 ( or ß) and SMC3. SMC1
disappears temporarily in late leptotene/early zygotene (Eijpe et al., 2000a), and SMC1ß appears in midleptotene. SMC3 is present throughout meiotic prophase but does not associate with AEs until late leptotene. Possibly, cohesin complexes are gradually rebuilt during meiotic prophase as the AEs are assembled, and this could be accompanied by phosphorylation of REC8 (Fig. 2 D). SMC1ß appears too late to contribute to the establishment of cohesion; possibly, it contributes to maintenance of cohesion, and/or it functions primarily in recombination.
AE components and meiotic sister chromatid cohesion at meiosis I
Of the analyzed AE components, only REC8 persisted along the chromosome arms until the metaphase I to anaphase I transition. In part of the metaphase I cells, we found some SMC1ß, SMC3 (using MSMC3), SCP2, and SCP3 along the chromosome arms. Because the abundance of these proteins along the arms was negatively correlated with the degree of chromosome condensation, we assume that these proteins detach from the arms as condensation proceeds and thus cannot contribute to cohesion until the metaphase I to anaphase I transition. Anti-SMC1
and R
SMC3 did not label metaphase I chromosomes. Thus, of the analyzed cohesins, only REC8 can ensure chromosome arm cohesion until metaphase I. It remains to be investigated whether proteins other than REC8 are required for arm cohesion maintenance, and if so, which. In anaphase I, REC8 disappeared from the chromosome arms, as expected for a cohesion protein. REC8 and the other analyzed proteins displayed an interesting pattern between anaphase I and II: whereas SMC1ß, SMC3, SCP2, and SCP3 occurred throughout the centromeric chromatin, REC8 occupied two spots, which flanked the kinetochores (Fig. 7, FH, and Fig. 9). This is consistent with REC8 being part of the physical link between centromeric regions of sister chromatids. The two REC8 spots furthermore reconcile two apparently contradictory observations on mammalian chromosomes. On the one hand, cohesion is specifically retained at the centromeres in mitotic metaphase chromosomes (for review see Rieder and Cole, 1999), and this is correlated with persistence of SCC1 in the centromeric region (Waizenegger et al., 2000). On the other hand, the central domain of the centromere undergoes microtubule-dependent elastic deformations during mitotic metaphase (Shelby et al., 1996), and it has been suggested that this could be due to transient local separations of sister chromatids (He et al., 2000). Two cohesion sites that flank the kinetochores can explain these observations. It should be noted, however, that we found two supposed cohesion sites per centromeric region in meiosis, whereas elastic deformation and transient separation of centromeric domains have been found in mitosis (Shelby et al., 1996; Waizenegger et al., 2000).
The role of SMC1ß, SMC3, SCP2, and SCP3 in the centromeric region in metaphase I to anaphase II remains to be investigated. It is possible that these proteins stabilize REC8-mediated arm cohesion until metaphase I and centromeric cohesion until anaphase II. After anaphase I, they might furthermore contribute to a change in the orientation of kinetochores. The change in the shape of the SCP3-labeled domain between anaphase I and metaphase II (Fig. 7, compare F and G with L and M) suggests a conformational change of the chromatin around the centromeres.
Role of REC8 in recombination
Cohesins, including REC8 (de Veaux et al., 1992; Klein et al., 1999), also function in homologous recombination. We proposed (van Heemst and Heyting, 2000) that after S phase, cohesins attract protein complexes that are involved in the early steps of homologous recombination. In mitotic G2, the cohesion proteins would then direct the homology search of broken DNA ends toward the corresponding segment of the sister chromatid. In meiosis, specific proteins, including AE components (Schwacha and Kleckner, 1997), would block the homology search on the sister chromatid and direct it toward the homologous chromosome. Here, one prediction of this model is confirmed, namely the association of recombination proteins (RAD51/DMC1 and possibly RAD50) with a cohesion protein, REC8 (Fig. 8, A and B). Interaction of Rad50 with Rad21 (homologous to Scc1) in the mitotic cycle of S. pombe has been proposed before (Hartsuiker et al., 2001).
One observation points to a different role in recombination of REC8 on the one hand and SMC1ß, SMC3, SCP2, and SCP3 on the other hand, namely the "bridges" between AEs in late diplotene. The persistence of Cdk2 at these bridges (Fig. 6 L) indicates that they mark sites of crossing over (Ashley et al., 2001). SMC1ß, SMC3, SCP2, and SCP3 are constituents of the bridges (Fig. 6, G, J, and L; Schalk et al., 1998; Revenkova et al., 2001), whereas REC8 is not. Bridges are found in only a small proportion of the bivalents (Schalk et al., 1998; Revenkova et al., 2001), and Cdk2 marks only some of the bridges. In male mouse meiosis, Cdk2 is lost from most crossover sites before desynapsis (Ashley et al., 2001). Possibly, Cdk2 monitors a late step in recombination at the DNA level, whereas the bridges represent an even later step, for instance, the start of the formation of recombinant chromatid axes. SMC1ß, SMC3, SCP2, and SCP3 might stabilize crossover intermediates until recombinant chromatid axes have been formed. Apparently, the latter step is usually, but not always, completed before desynapsis.
To summarize, the localization of REC8 differs from that of other analyzed cohesins in various stages of meiosis. Probably, REC8 provides a basis for AEs and RNs and ensures cohesion throughout meiosis. The role of SMC1, SMC1ß, and SMC3 is less clear, apart from an essential role of Smc3 in cohesion and recombination in yeast meiosis (Klein et al., 1999) and a likely role of SMC1ß and SMC3 in centromeric cohesion (Revenkova et al., 2001). SMC1
and diffusely distributed SMC3 might contribute to the establishment of cohesion, whereas SMC1ß and AE-associated SMC3, together with SCP2 and SCP3, possibly further support REC8-mediated cohesion, promote recombinational interactions with the homologous chromosome, stabilize crossover intermediates, and provide a basis for the formation of recombinant chromatid axes.
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Materials and methods |
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Immunocytochemistry
Paraffin sections (Heyting et al., 1983) and frozen sections (Eijpe et al., 2000a) of testes from Wistar rats were prepared as described. Rat spermatocytes were spread by the dry-down technique (Peters et al., 1997) or by agar filtration (Heyting and Dietrich, 1991). We performed immunofluorescence labeling according to Heyting and Dietrich (1991) and mounted the slides in Vectashield (Vector Laboratories) containing 2 µg DAPI per ml. For detection of BrdU, we treated preparations with 70% formamide, 1 mM EDTA in 2x SSC at 55°C for 2 min and washed them in 70, 90, 100% cold ethanol before incubating them with anti-BrdU antibodies. All monoclonal antibodies were diluted 1:1. Affinity-purified antibodies from rabbit serum 602 (RN), 610 (R
C), and 624 (R
SMC3) and chicken SN11 (Ch
N) were diluted 1:50. All secondary antibodies, conjugated to fluorescent dyes, were diluted according to the instructions of the suppliers. We used immunodepleted serum fractions as negative controls for R
N and R
C. Immunofluorescent preparations were analyzed as described by Revenkova et al. (2001).
Cell separation
We separated cells from rat testes by elutriation and density centrifugation in Percoll (Amersham Biosciences) (Heyting and Dietrich, 1991). For purification of preleptotene spermatocytes, we collected cells from a BrdU-labeled rat at 1,800 rpm and 1525 ml/min during elutriation, and then centrifuged these cells in 29% Percoll. The cell band with the highest density was enriched in preleptotene spermatocytes (10% Sertoli cells, 23.3% spermatogonia, 46% preleptotene [BrdU and REC8 positive; no SCP3], 15.6% leptotene [BrdU negative and REC8 positive; REC8-AEs with some SCP3; no synapsis], 4.1% zygotene, 1% spermatids). Pachytene and diplotene spermatocytes were purified from rat testis as described by Lammers et al. (1995).
Immunoprecipitation
We performed immunoprecipitations according to Goedecke et al. (1999), with some modifications. We incubated the cell lysates overnight at 4°C with affinity-purified primary antibodies (from rabbits), and then we added paramagnetic beads coupled to sheep antirabbit antibodies (Dynal A.S.) and incubated the mixture with the beads for another 4 h at 4°C. We performed all incubations and washes in the presence of the complete miniprotease inhibitor cocktail (Roche Diagnostics GmbH). After incubation, we collected the beads, washed them with 1% NP-40, 50 mM NaCl, 10 mM Tris-HCl (pH 8.0), eluted the bound proteins from the beads by boiling them in SDS-PAGE sample buffer (Lammers et al., 1994), and applied the eluted proteins to a 1-cm-wide slot of a polyacrylamide SDS gel. In parallel with the immunoprecipitates, we loaded one slot with total lysate of 5 x 105 purified spermatocytes and one slot with protein from 107 purified SCs. After electrophoresis, the gel was blotted onto nitrocellulose, and the resulting blot was stained with Ponceau S. The blot of each lane was cut into four strips, which were probed with various antibodies.
Other procedures
For BrdU incorporation, we injected rats intraperitoneally with 60 mg BrdU (catalog no. B3002; Sigma-Aldrich) per kilogram body weight at 3 and 2 h before sacrifice. SCs (Heyting et al., 1985) and nuclei from spermatocytes (Meistrich, 1975) or liver (Blobel and Potter, 1966) were isolated by described procedures. SDS-PAGE (Laemmli, 1970) and immunoblotting (Dunn, 1986) were performed as previously described. For immunoblot analysis of nuclei, we loaded 20 µg of protein per 2-cm-wide slot of a 718% linear gradient polyacrylamide SDS gel. After electrophoresis, we stained 0.5 cm of each lane with Coomassie blue and blotted the remainder onto nitrocellulose. For immunoblot analysis of whole cells, we loaded proteins from 106 cells per 2-cm slot of a 7.5% or 10% polyacrylamide gel. SCs were dephosphorylated according to Lammers et al. (1995); samples containing 4 x 107 isolated SCs were treated with AP, AP pretreated with 8-hydroxyquinoline-5-sulfonic acid (a specific inhibitor of AP; Simpson and Vallee, 1968), or AP buffer only. After treatment, each sample was dissolved in electrophoresis sample buffer, loaded onto a 1-cm-wide slot of a 7.5% SDS gel, electrophoresed, and blotted onto nitrocellulose. Immunoblots stained with Ponceau S were scanned using an Agfa Snapscan 1212 flatbed scanner before they were probed with antibodies. Binding of antibodies to the blots was detected by secondary antibodies conjugated to AP (Promega) and incubation in NBT/BCIP; the Ponceau S stain is lost during these steps. After incubation in NBT/BCIP, we scanned the blots again and processed the obtained images using the Corel Photopaint and CorelDraw software packages.
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Footnotes |
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* Abbreviations used in this paper: AE, axial element; AP, alkaline phosphatase; RN, recombination nodule; SC, synaptonemal complex.
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Acknowledgments |
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This work was supported by grant 901-01-097 of the Netherlands Organization for Scientific Research and grant QRLT-2000-00365 of the European Community to C. Heyting and a grant from the Human Frontier in Science Program to R. Jessberger.
Submitted: 13 December 2002
Accepted: 21 January 2003
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