Axial element formation in absence of SMC1β/REC8 or SMC1β/RAD21L

An earlier report showed that the third kleisin RAD21 contributes little or not at all to the formation of AEs in spermatocytes, since there are extremely short SYCP3-positive AEs when the other two kleisins were absent, i.e. in Rad21L^-/-Rec8^-/- spermatocytes [10]. At least in this mutant background and for this central function, RAD21 cannot compensate for the loss of the two meiosis-specific kleisins. Thus, it appeared as if the majority of cohesin complexes in prophase I are based on either RAD21L or REC8. Here, we used single and double mutants lacking either REC8 or RAD21L only, or in combination with the SMC1^® deficiency to dissect the individual contributions of these cohesins to AE formation. Staining for SYCP3 was used to measure axis length at the most advanced spermatocyte stage in each "single-knockout" (SKO) and Smc1β^-/-Rec8^-/- and Smc1β^-/-Rad21L^-/- DKOs compared to wild type (wt) (Fig 3A and 3B).

The most advanced stage was assigned based on the appearance and extent of SYCP1 and γH2AX staining, where SYCP1 was present on some chromosomes and the previously diffuse γH2AX signal was reduced to one or two cloud-like structures (S1A and S1B Fig). We took into consideration that some short axes may represent fragments of the same chromosomes and therefore divided the total axes length of a cell by the normal number of chromosomes (21 including X and Y separately). In case of asynapsis of entire chromosomes the number of axes was increased, and we divided the total axes length by this increased number of axes, since each individual axes–whether synapsed or not–was added to the total axes length of a cell. Asynapsis of entire chromosomes was determined by counting the number of CENP-A signals for centromeres. This number was the same in all mutants at the leptotene stage (see below), and if increased at later stages the CENP-A signals on separate axes indicated asynapsis or loss of sister chromatid cohesion and thus an increased number of axes. The total axes length per cell (Fig 3B) was divided by the number of CENP-A positive chromosome axes.

In the most advanced spermatocytes of the Smc1β^-/-Rec8^-/- mutant, the axes are very short with an average axis length of 2.351 +/- 0.262 (SD) μm and thus shorter than the corresponding SKO or wt (wt: 6.56 +/- 0.253 μm; Smc1β^-/-: 3.27 +/- 0.451 μm; Rec8^-/-: 3.67 +/- 0.247 μm; Fig 3A and 3B). Thus, the removal of REC8 in addition to SMC1β further reduces axis length. Therefore, an SMC1α/REC8 complex should exist, unless one would propose a very distinct role of REC8 independent of any cohesin complex, which is very unlikely. The removal of this SMC1α/REC8 complex supposedly accounts for the additional length reduction in this mutant background. Based on the analysis of the Smc1β^-/-Rec8^-/- mutant, this SMC1α/REC8 complex contributes a moderate roughly 14% to axis length (i.e. the further reduction by 0.92 μm seen in the Smc1β^-/-Rec8^-/- mutant compared to the Smc1β^-/- mutant). Such numbers are obviously approximations only for a normal cell as they reflect the contributions in a mutant background. At any rate, this SMC1α/REC8 complex appears to be a minor complex. This is in agreement with the low efficiency or absence of co-precipitation of SMC1α and REC8 from wt or Smc1β^-/- testis extracts reported earlier [9, 13, 17].

Despite the moderate reduction is axis length in Rec8^-/- and Rad21L^-/- cells, the total axes length per cell is similar to wt (Fig 3B). This originates from the high levels of asynapsis in these mutants.

From the almost total reduction of axis length in STAG3 deficient spermatocytes [16] it is clear that cohesins determine the entire axis length. In the Smc1β^-/-Rec8^-/- mutant with only 2.35 μm of axis length left, these remaining axis–app. 36% of wt length only–must also be provided by some cohesin complex(es). Thus, the remaining app. 36% of axis length that still exists in Smc1β^-/-Rec8^-/- spermatocytes has to be supported either by SMC1α/RAD21 or SMC1α/RAD21L complexes, the only remaining complexes.

Due to non-homologous associations of AEs and to gaps in SYCP3-positive AEs, the measurement of axis length in Rad21L^-/- spermatocytes is very difficult, but an estimate that only takes non-associated, gap-less and clearly identifiable axes into account yields a length roughly comparable to that of the Rec8^-/- strain (Fig 3B). In Smc1β^-/-Rad21L^-/- spermatocyte spreads we observed very short SYCP3-stained axes, which often appeared as dots rather than as filaments; they measured 1.17 +/- 0.27 μm (Fig 3A and 3B).

Thus, in contrast to the Rad21L^-/- or Smc1β^-/- SKOs, the removal of SMC1β and RAD21L almost entirely abolishes formation of SYCP3-positive axes, with no obvious compensatory effect. This suggests that besides SMC1β complexes, an SMC1α/RAD21L complex contributes to axis formation. This further suggests that an SMC1α/RAD21 complex contributes little if any to axes length. In at least one report anti SMC1α precipitation co-precipitated RAD21L [13]. Since the combined loss of RAD21L and REC8 also causes almost complete loss of axes [10], this supports the above notion that an SMC1α/RAD21 complex does not significantly contribute to axis length and RAD21 cannot compensate for the loss of the two other kleisins, at least under conditions where other cohesins are absent. Similarly, since the Smc1β^-/-Rad21L^-/- and the Rec8^-/-Rad21L^-/- [10] DKOs essentially abolish axis formation, but the Smc1β^-/-, the Rec8^-/- and the Rad21L^-/- SKOs do not, the SMC1α/RAD21L and the SMC1β/REC8 complexes must very prominently if not almost entirely support axis formation, with the above mentioned minor contribution of SMC1α/REC8.

A potential role of RAD21 should therefore be mostly confined to other, specific functions such as supporting pachynema/diplonema events, perhaps formation of chiasmata, consistent with the reappearance of RAD21 seen in some studies at this stage.

It should be noted that all numbers provided here as percentage of contribution to axis length are based on comparison with the wt situation. In any mutant, compensatory mechanisms may arise that may affect these numbers such as increased expression or stability of the remaining cohesin complexes. Thus, conclusions are qualitative and only roughly quantitative. However, any compensatory effect, if it exists, may be very minor, since axes are reduced to almost dots in the Smc1β^-/-Rad21L^-/- spermatocytes, and a very similar observation has been made in a STAG3 deficient mutant–no rescue by other STAG proteins [16]. Therefore, the numbers suggest the relative importance of particular complexes to axis length not only in the specific mutant backgrounds, but very likely also with respect to wt cells. In any case, the analysis reveals the presence and functional capacities of certain cohesin complexes in spermatocytes.

Synapsis and DNA double-strand breaks

Synapsis is impaired in all mutants as co-staining for SYCP1 and SYCP3 showed (S1B and S1C Fig). However, it is not possible to precisely quantify the extent of synapsis for the individual mutants, since in absence of REC8 the SYCP1 deposits between sister chromatids yielding a “false” signal, and in the Smc1β^-/-Rad21L^-/- mutant very small axes or dots appear. Many (app. 70%) of these extremely small structures carry SYCP1, but the small size precludes quantification. Co-staining for HORMAD1, an asynapsis marker, and SYCP3 confirmed the extent of synapsis failure in all the mutants, since HORMAD1 is present in all cases (S1D Fig).

Detailed analyses of SYCP3- and SYCP1-stained axes of the wt and DKO spermatocytes by super-high resolution OMX microscopy (Fig 3C) showed the expected central localization of SYCP1 between the two SYCP3 axes in wt cells. In the Smc1β^-/-Rec8^-/- mutant, where no synapsis occurs and the SYCP3-stained axes therefore consist of two sister chromatids, the deposition of SYCP1 between the sister chromatids is clearly observed and is in agreement with previous reports that described this phenotype for the Rec8^-/- strain [19, 20]. In the Smc1β^-/-Rad21L^-/- mutant, SYCP1 is also deposited between sister chromatids, but in addition some individual SYCP1-positive sister chromatids were observed, typically 7 per cell (+/- 5.0; n = 40). This data indicates moderate loss of sister chromatid cohesion in this mutant and show that SYCP1 does neither require two sister chromatids in cohesion nor chromatids in SYCP3-mediated close proximity, nor two AEs, to associate along chromosomes. The deposition of SYCP1 at a single chromatid also suggests, that in mutant backgrounds SYCP1 deposition is not necessarily an indicator for synapsis. Therefore we prefer not to designate SYCP1 deposition on pairs of sister chromatids as "synapsis between sister chromatids".

In wt mouse spermatocytes, the sex chromosomes X and Y only pair at a short, centromere-distal region called the pseudo-autosomal region, PAR [24]. The largely unsynapsed sex chromosomes form a special chromatin domain, the sex body, which features silencer chromatin marks. This X/Y association is seen only in wt cells (Fig 3A, arrow), as is the characteristic sex body chromatin staining by γH2AX as one intense structure with the sex chromosomes embedded (S1A Fig, arrow).

Synapsis of homologs depends on programmed double-strand breaks (DSBs), which are repaired with progressing synapsis. Cohesin SMC1β is not required for generation of DSBs, but was shown to support their repair, which is delayed in absence of SMC1β [25]. DSBs can be visualized by staining of DSB repair proteins such as RAD51 or the meiosis-specific DMC1. In all mutants reported here, DMC1 foci and thus DSBs are produced (Fig 4).

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Fig 4. DNA double strand break repair foci.

Immunofluorescence staining of spermatocyte chromosome spreads of WT, SKO and DKO mice, probed with anti-SYCP3 (red) for AEs/LEs and anti-DMC1 (green) or anti RAD51 as indicated for DNA double-strand break repair foci. Upper panel: leptotene/early zygotene stage with many DMC1 foci; middle panel: late zygotene/early pachytene like stage with reduced number of DMC1 foci; lower panel: RAD51 foci on pachytene-stage like cells (scale bar: 5 μm).

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Quantification of DMC1 in the DKOs is very difficult as the axes are short or only dots exist where one cannot distinguish individual foci, particularly in the more advanced stages. Therefore we cannot provide exact numbers. The initial numbers of DMC1 foci, as much as recognizable, appeared to be very similar in the mutants and not unlike wt. The same was observed for RAD51 foci. In the more advanced stage of Smc1β^-/-Rec8^-/- cells the number of DMC1 foci is reduced to two or more foci per short axis. A similar reduction was observed in Rec8^-/- spermatocytes but not in Smc1β^-/- spermatocytes, where repair is delayed as previously reported [25]. One may speculate that in the Smc1β^-/-Rec8^-/- spermatocytes alternative repair pathways such as between sister chromatids supported by SYCP1 localized between sister chromatids are enhanced or that the foci are less stable. We also found several discrete DMC1 foci in Smc1β^-/-Rad21L^-/- spermatocytes, but due to the extremely short axes we cannot clearly distinguish foci. We observed a reduction of DMC1 signals in Smc1β^-/-Rad21L^-/- spermatocytes in the advanced stage, indicating that repair of DSBs happens or that the foci are not stable.

Centromeric cohesion

The analysis of centromeres of meiotic chromosomes reveals both, synapsis at centromeres and for centromeric sister chromatid cohesion. In wt pachynema spermatocytes 21 centromere signals indicate complete synapsis (except the sex chromosomes with their PAR-distal and thus non-synapsed centromeres) and complete centromeric sister chromatid cohesion. The appearance of 22 to 40 centromere signals indicates either loss of synapsis, or loss of centromeric cohesion in presence of full synapsis, or partial loss of both cohesion and synapsis. More than 40 centromere signals indicate loss of some or all (80 signals) centromeric cohesion and synapsis. In early zygotene cells, where no synapsis exists and thus this analysis of cohesion is less perturbed by synapsis, more than 40 centromere signals (from 40 AEs, i.e. 2 x 19 autosomes plus X and Y) indicate loss of centromeric cohesion. Since the DKOs develop to an early/mid zygonema-like chromosomal stage, we analyzed all cells at the leptotene/early zygotene stage to be able to test for centromeric cohesion independent of synapsis, i.e. we expect 40 centromere signals in wt cells.

Previously it has been shown that depletion of REC8 causes synapsis failure. However, REC8 is not required for the establishment of centromeric sister chromatid cohesion in meiocytes as in the most advanced Rec8^-/- spermatocytes never more that 40 centromeres were observed [19]. We also observed in average 37.76 (+/- 1.787, n = 42) centromeres in Rec8^-/- spermatocytes by staining with anti-centromeric antibodies (ACA) (S2 Fig), which recognize centromeric and pericentromeric heterochromatin. Staining for CENP-A, an inner centromere component showed 40.5 (+/- 2.84; n = 34) signals in leptonema Rec8^-/- spermatocytes (Fig 5A and 5B).

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Fig 5. Centromeric cohesion.

A. Immunofluorescence staining of spermatocyte chromosome spreads of leptotene stage of WT, SKO and DKO mice, probed with anti-SYCP3 for AEs/LEs and anti-CENP-A for the inner kinetochore (scale bar: 5 μm). CENP-A signals near the ends of chromosomes mark the centromeres and were counted. B. Graph showing the average number of CENP-A signals of WT, SKO leptotene/early zygotene spermatocytes, indicative of centromeric cohesion. (N = 14, Smc1β^-/-; N = 34, Rec8^-/-; N = 59, Rad21L^-/-; N = 21, Smc1β^-/- Rec8^-/-; N = 22, Smc1β^-/- Rad21L^-/-); red bars indicate SD. There was no significant difference between any wt or mutant strain (all p vales >0.04; Dunn’s multiple comparison test).

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This indicates proper centromeric cohesion despite absence of REC8. In Smc1β^-/-Rec8^-/- spermatocytes we observed an average of 36.12 (+/- 5.930; n = 33) ACA signals, and 41.4 (+/- 4.5; n = 21) CENP-A signals (Fig 5A and 5B; S2 Fig), again suggesting maintenance of centromeric cohesion, although the very slight increase in CENP-A signals may hint at a minor trend towards weakening of centromeric cohesion. The situation is similar in the other mutants: Rad21L^-/- spermatocyte spreads showed 30.44 (+/- 4.330; n = 52) ACA and 39.8 (+/- 6.4; n = 59) CENP-A signals on average in accordance with a previous report that used ACA [21]. The low number of distinguishable ACA signals may be caused by telomere fusions (see below), which can bring the relatively broad centromeric heterochromatin in very close proximity. CENP-A, which provides a more specific signal just at the inner kinetochore, would not be as much affected. Smc1β^-/-Rad21L^-/- spermatocytes showed 39.09 (+/- 5.39; n = 46) ACA signals and 41.0 (+/- 4.6; n = 22) CENP-A signals. Statistically significant differences were not found for CENP-A signals, and only some differences between certain pairs of mutants were statistically different for the ACA signals with a p-value >0.05. Overall there appears to be slightly more variation in centromere numbers in the DKOs, but this suggests very limited if any weakening of centromeric cohesion.

This data indicates that none of the three meiotic cohesins analyzed here are required for early prophase cohesion. Thus, an SMC1α complex, i.e. SMC1α/RAD21, must provide most if not all centromeric cohesion at this very early stage of male meiosis. This is consistent with the notion that SMC1β is only expressed after entry into meiosis [7], and is in agreement with the only partial loss of centromeric cohesion in okadaic acid-induced metaphase I Smc1β^-/- spermatocytes when these were derived from zygonema cells. When the cells originated from early/mid pachynema, complete loss of centromeric cohesion was observed [17]. This suggests that SMC1β complexes are loaded onto meiotic chromosomes during prophase I, or at least become cohesive then, and successively take over the duty of maintaining centromeric cohesion from the SMC1α/RAD21 complex, which progressively vanishes after entry into meiosis.

How can one reconcile this with our analysis of Smc1β^-/-Spo11^-/- and Smc1β^-/- spermatocytes, which showed some centromeric cohesion deficiency in absence of SMC1β [25]? In that study, spermatocytes of the most advanced stages were analyzed, i.e. late zygonema for Smc1β^-/-Spo11^-/- or early/mid pachynema for Smc1β^-/-. At this stage, partially synapsed axes carrying three separate centromeres were observed in Smc1β^-/- cells, which clearly indicate both, synapsis failure combined with loss of cohesion at the centromeres in at least 8% of the cases. If centromeric synapsis fails, the one strong signal derived from 4 very closely juxtaposed centromeres falls apart into two signals; 3 or 4 signals then indicate loss of cohesion. In Smc1β^-/-Spo11^-/- chromosomes, which do not synapse, one or two ACA signals were observed and indicated loss of cohesion in about a third of the cells. We suggest that while at leptonema the SMC1α/RAD21 complex still provides most centromeric cohesion, it is partly replaced by SMC1β complexes when synapsis starts to happen, i.e. in zygonema. This interpretation would fit to the above notion of loading of SMC1β complexes onto centromeres after entry into meiotic prophase I.

Presence of cohesin proteins on mutant spermatocyte chromosomes

In the absence of SMC1β, SMC1α and SMC3 localize to AEs of early prophase I cells [17] suggesting that the SMC1〈/SMC3 heterodimer forms complexes with RAD21L, REC8 or RAD21 or either of them in vivo. Evidence from immuno precipitation experiments differs somewhat between distinct reports [9, 13, 15, 17], but neither of the three complexes can be surely excluded at this time.

We analyzed chromosomal localization of the remaining cohesin proteins in Smc1β^-/-Rec8^-/- and Smc1β^-/-Rad21L^-/- spermatocytes in comparison to wt and the SKOs (Fig 6; for single channel images see S3 to S9 Figs).

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Fig 6. Localization of cohesin proteins.

Immunofluorescence staining of spermatocyte chromosome spreads of WT, SKO and DKO mice probed with anti-SYCP3, anti-SMC3, anti-SMC1β, anti-SMC1α, anti-RAD21L, anti-RAD21, anti-STAG3 or anti-REC8 (scale bar: 5 μm). All cohesins are labeled green and show either rather uniformly along the chromosomes or in a dot-like pattern. The single channel images provided in S4–S10 Figs may allow a more precise observation of staining patterns. Please note that while the comparison of mutants and WT stained with one particular antibody is possible, the interpretation of comparisons of staining patterns of different antibodies had to be made with much caution, since because antibody affinities and other features differ.

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The one cohesin present in all known cohesin complexes, SMC3, localizes to the chromosomes in all mutants, although at much reduced amounts in the two DKOs. The pattern also becomes more dotty, much less uniform in the DKOs and also in Rad21L^-/- than in wt cells. Whether the occasional signals located off the axes are true chromatin signals cannot be ascertained, although such signals rarely appear outside the nuclei; still at least some can be unspecific signals derived from sticky chromatin. The presence of SMC3 in the DKOs indicates that there are SMC1α complexes, either with RAD21 and/or with either RAD21L or REC8. This is consistent with the notion above on the role of SMC1α complexes in cohesion and axis length. SMC1β is present in REC8 and RAD21L deficient cells, indicative of SMC1β in association with either one or two of the other kleisins that are present in these mutants. SMC1α is present along the chromosome axes in all SKOs, as well as in the Smc1β^-/-Rec8^-/- chromosome spreads. A somewhat weaker signal was observed in the Rad21L^-/- spreads. This may indicate the disappearance of an SMC1αRAD21L complex. The Smc1β^-/-Rad21L^-/- preparations show the weakest SMC1α signal, if any. In most cells no specific SMC1α signal was observed. Since the removal of SMC1β in addition to RAD21L should, in principle, not affect SMC1α that is not associated with RAD21L, the nearly complete absence of SMC1α is not immediately intuitive. We think that the absence of a discernible signal may be due to the extremely short, dot-like axes, which would not allow SMC1α to efficiently associate with them. Thus, the SMC3 and SMC1α patterns are largely consistent with each other, although different antibodies generate different intensities, which therefore can hardly be directly compared.

Similarly, RAD21L is present in all spreads except those of Rad21L^-/- and Smc1β^-/-Rad21L^-/- mice. Widespread signals for REC8 are seen in the Rad21L^-/- spreads, much less is present in Smc1β^-/- and Smc1β^-/-Rad21L^-/- samples. In agreement with the above notion of a minor contribution of SMC1α/REC8 complex to axis formation, this suggests that the majority of REC8 complexes are SMC1β based. RAD21 is more chromatin-spread and axes-associated in wt, and there is still some RAD21 on Smc1β^-/- and Rad21L^-/- spermatocyte chromosomes, but less on Rec8^-/- chromosomes. Very little is seen on either of the DKO spreads, likely because these cells do not enter pachynema, the stage when RAD21 would reappear. Together this is in accordance with the notion that SMC1α/RAD21 complexes exist on spermatocyte prophase I chromosomes. Consistent with the finding of normal numbers of centromere signals in all mutants, we found that those other two kleisins that are present in a given kleisin mutant localize to the centromeres.

The only meiosis-specific SA-type cohesin STAG3/SA3 localizes all along the wt and Smc1β^-/- chromosomes indicative of SMC1α/STAG3 complexes as reported before [17]. STAG3 also associates along Rec8^-/- chromosomes and is present at least in a dotty pattern on Rad21L^-/- chromosomes. However, Smc1β^-/-Rec8^-/- chromosome spreads show STAG3 signals, which suggest that a complex of SMC1α/STAG3 provides the signals seen on Smc1β^-/- and on DKO chromosomes. The STAG3 signals seen on Smc1β^-/-Rad21L^-/- miniature axes are consistent with a SMC1α/STAG3/REC8 complex, which should not be eliminated in this DKO. In immuno precipitation experiments, STAG3 co-precipitated with either of the SMC1 variants, and precipitated with RAD21, RAD21L and REC8 although not in all experiments reported [9, 13, 17, 21].

While obviously the absence of one cohesin does not preclude others to associate with chromosomes, one potential caveat of the analysis is that chromosome association or cohesin expression of one cohesin could be increased if another one is missing. However, the strong phenotypes observed in each mutant clearly indicate that no full, probably not even relevant, compensation by other cohesins exist. Changes in expression levels of other cohesins in a particular single cohesin mutant were not observed so far [9, 13, 17, 21].

Telomere deficiencies

Earlier we reported telomere deficiencies in Smc1β^-/- spermatocytes [26]. These deficiencies included shortened telomeres, SCs without telomeres, telomeres that have apparently been broken off SCs, and telomere fusions. To test the contribution of individual kleisins to telomere integrity, we stained chromosome spreads of wt and all mutants by FISH for telomeric sequences (telo-FISH), by anti-RAP1 for this telomere-specific protein, and by anti-SUN1 for association of telomeres with the SUN/KASH complex (reviewed in [27]), which anchors telomeres at the nuclear membrane in early prophase I (Fig 7A–7D; S11 Fig).

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Fig 7. Telomere analysis.

A. Staining of spermatocyte spreads by Telo FISH (red) to assess telomere length and anti-SYCP3 for AEs/LEs in WT, SKO and DKO mice (scale bar: 5 μm). Magnified images of individual chromosomes are shown below. Telomeres are seen as red dots at the ends of chromosomes or on disrupted chromosome structures. WT chromosomes show FISH signals of about equal intensities at both ends. Examples for aberrant telomeres are indicated as follows: SCs lacking telomeres—white arrow head; isolated telomeres/broken off SCs, yellow arrow; telomere fusions or close associations—white arrow. B. Immunofluorescence staining of spermatocyte chromosome spreads of WT, SKO and DKO mice, probed with anti-SYCP3 (red) for the AEs/LEs and anti-RAP1 (green) to stain telomeres (scale bar: 5 μm). Aberrant telomeres are marked as above. C. Immunofluorescence staining of spermatocyte chromosome spreads of WT, SKO and DKO mice, probed with anti-SYCP3 (green) for the AEs/LEs and anti-SUN1 (red) to stain telomere attachments (scale bar: 5 μm). D. Graph showing the average telomere intensity, maximum intensity and area of WT, SKO and DKO spermatocyte spreads as measured using the ImageJ software. The boxes show the median value, the upper 75th and the lower 25th percentile, along with maximal and minimal values. The p values for all comparisons of a mutant to wt are <0.0001. The p values for all other paired comparisons (e.g. Smc1β^-/- versus Rec8^-/- etc.) are also <0.0001 according to Dunn’s multiple comparison test.

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Because of the very short or dot-like axes of the two DKOs it is not possible to quantify individual telomeres or telomere-like structures, but a qualitative description can be provided. In wt cells, 19 autosomes in full synapsis generate 38 telomere signals, the X and Y chromosomes yield 3 telomere signals, since the synapsed PAR telomere appears as one, together 41 signals. The loss of PAR synapsis in all mutants increases this number to 42. A fully unsynapsed autosome shows 4 signals, if there is additional loss of cohesion, 8 signals emerge. Telomere aberrations such as telo-less axes or solitary telomere fragments perturb these numbers.

In all mutants, telo-FISH shows aberrant telomeres with telomeric DNA that seems to have ruptured off the axes, with axes that lack telomere signals, and with telomeric ends tightly associated, perhaps fused, or clustered (Fig 7A). These phenotypes are most prominently seen in SMC1β and RAD21L deficient mutants. Rec8^-/- spermatocytes show the fewest telomeric aberrations, although often 3- to 4 chromosomes display a low intensity FISH signal at one end. FISH signals are directly proportional to telomere length (see below and, for example [26]). RAP1 staining (Fig 7B) confirms that many telomeres are deficient in the DKOs, since many axes lack RAP1 signals. These aberrations were also confirmed by staining for TRF2 (see below; Fig 8 and S12 Fig).

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Fig 8. Super resolution telomere analysis.

SIM analysis of wild-type and mutant spermatocyte telomeres in chromosome spreads, stained with anti TRF2 and anti SYCP3. Sex chromosomes in wild-type are marked by a blue arrow. High magnification excerpts labeled with a grey asterisks are additional examples from separate images (scale bar: 5 μm).

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Similarly, the SKO and DKO show reduced number of SUN1 spots (Fig 7C), indicative of failure to associate with the nuclear periphery. We counted the number of SUN1 telomere signals in all the mutants of the most advanced stage. We observed in average 45.32 (+/- 3.787, n = 26) SUN1 signals in wt zygotene/pachytene spermatocytes. In Rec8^-/-, Smc1β^-/- and Rad21L^-/- spermatocytes we observed 64.08 (+/- 5.787, n = 25), 43.3 (+/- 2.787, n = 26) and 42.3 (+/- 6.787; n = 26) SUN1 signals, respectively. In Smc1β^-/-Rec8^-/- and Smc1β^-/-Rad21L^-/- spermatocytes we observed in average only 44.3 (+/- 5.987, n = 23) and 39.3 (+/- 10.587, n = 34) signals (S11 Fig). Differences were statistically significant with a p-value <0.05 for the comparisons of wt versus Rec8^-/-, Smc1β^-/- versus Rec8^-/-, Rec8^-/- versus Smc1β^-/- Rec8^-/-, Rec8^-/- versus Rad21L^-/-, and Rec8^-/- versus Smc1β^-/-Rad21L^-/-. Thus, the only statistically significant difference was observed with the REC8 deficiency. Detailed interpretation of these numbers is difficult as many processes contribute in different ways. Increased SUN1 numbers may result from unsynapsed chromosomes that each form SUN1 foci. Telomere fragments may also form SUN1 foci as seen mostly in Rad21L^-/- cells. Decreased SUN1 foci most likely reflect the loss of telomere ends seen in all mutants, not compensated for by unsynapsed chromosomes. Why, for example, there are fewer SUN1 foci in the Smc1β^-/-Rec8^-/- cells than in the Rec8^-/- spermatocytes, which show comparable levels of asynapsis, can only be speculated about: SMC1β may be much more required to preserve telomeric DNA and its structure than REC8, consistent with the many aberrations seen in the Smc1β^-/- spermatocytes [26]. Overall, this data reflects the expected telomere and telomere attachment deficiencies.

Measurements of the intensity of the FISH signal (Fig 7D) showed that in all mutants there is a shift towards lower intensity, which indicates shorter telomeres. Telomere intensities peak in the wt at 35000 to 40000 units. The Smc1β^-/- spermatocytes display a peak around 20000 units and thus feature shorter telomeres as reported before [26]. In Rec8^-/- spermatocytes the median intensity is at app. 7500 units and thus telomeres are even shorter than in Smc1β^-/- spermatocytes. Unexpectedly, Smc1β^-/-Rec8^-/- spermatocytes show more intense telomere signals, indicating that other modes of telomere protection become effective if these two cohesins are absent. The median intensity in Smc1β^-/-Rec8^-/- spermatocytes is similar to that in Smc1β^-/- spermatocytes, and the difference is not statistically significant. Both however show higher intensity and thus longer telomeres than in Rec8^-/- spermatocytes. This indicates SMC1β is a main contributor to telomere length and it does so without REC8, i.e. in a different complex. The effect of REC8 deficiency can thus only be brought about by an SMC1α/REC8 complex. Rad21L^-/- and Smc1β^-/-Rad21L^-/- spermatocytes show very short telomeres, with average intensities peaking at around 10.000, and there is no statistically relevant difference between these two strains. This suggests that RAD21L is mainly associated with SMC1β in this function. The variation in length is particularly extensive in the Rad21L^-/- and Smc1β^-/-Rad21L^-/- spermatocytes. Together this suggests that an SMC1β/RAD21L complex and an SMC1α/REC8 complex are mainly responsible for proper telomere length. There is no additive effect of removing RAD21L in addition to SMC1β. This supports the notion that an SMC1α complex featuring RAD21L does not significantly contribute to telomere length.

To reveal ultrastructural features of telomeres we performed super-resolution imaging (SIM) on anti TRF2-stained telomeres of wild-type and mutant spermatocytes (Fig 8, S12 Fig).

The analysis confirmed the presence of telomere aberrations on mutant chromosomes as described above (Fig 8). In addition, we observed loop-like structures on many wild-type telomeres, but rarely on mutant telomeres (Fig 8, S12 Fig). Plotting a 3D-image from signal intensities to analyze contour plots shows a circle of 4 telomere spots in many wt instances (S13 Fig). Quantification of these loop-like structures showed that 64% of the wt, but less than 10% of the mutant chromosomes carry such structures (S14 Fig). Multiple telomere signals were seen in a third of wt samples, but in half or more of the mutants. The mutants often (36 to 48%) also showed only one telomere signal per chromosome, i.e. one end lacked a signal, which happened only in 4% of wt cases. Stretches of telomere signals were observed only in mutants. We assume that almost all wt telomeres feature these loop-like structures, since depending on the specific plane the telomeres were looked at, one may not be able to see all of them as distinct circles, and some may be lost upon chromosome spreading. The non-paired ends of sex chromosomes of wt often also show circles. This suggests that cohesins, particularly SMC1β complexes, support formation of a more closed conformation at the very end of telomeres, which may represent a protective structure. These loops are reminiscent of TRF2-positive t-loops reported from somatic cells [28] and of telomere complexes reported recently for spermatocytes [29].

Conclusions

In conclusion, the different cohesin complexes that exist in mammalian spermatocytes contribute distinctly to different structures and processes in these cells. S1 Table summarizes the most important observations. Some of our conclusions assume that there is no role of kleisins independently of a cohesin complex, i.e. independently of either SMC1α or SMC1β. Formally this can hardly be excluded, but there is no evidence for this. We think the assumption that kleisins work only within cohesin complexes is very reasonable. So far all known functions of kleisins are consistent with their association with cohesins, and thus the interpretations provided above and below are the most straightforward.

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Table 1. List of primary antibodies used.

https://doi.org/10.1371/journal.pgen.1006389.t001

Several complexes contribute to axes formation and define their length, but to different extent. As determined in mutant backgrounds, SMC1β complexes determine about half of axes length, SMC1α complexes provide the other half with an SMC1α/RAD21L complex supporting axes length most prominently with roughly one-third, the SMC1α/REC8 complex only contributes a minor fraction. A significant contribution by RAD21 complexes is unlikely. The additive effect of distinct cohesin complexes to axes length suggests that the amount of cohesin available to be loaded onto meiotic chromosomes determines axes length, perhaps more so than the particular type of cohesin, i.e. whether it is an SMC1α or SMC1β cohesin complex. Whether individual complexes prefer to associate with certain sequences or DNA structures along chromosomes is not known but not unlikely given the association of cohesin in mitotic cells with binding sites for transcriptional regulators. Synapsis is supported by all complexes, although to a different extent. It also remains unclear by which mechanism(s)–directly or indirectly–synapsis is promoted by cohesins beyond formation of an axis-loop-structure. Sex chromosome pairing at the short PAR is particularly vulnerable to loss of any cohesin, since all mutants fail in X/Y pairing. The previously observed dependence of X/Y pairing on cohesin dosage supports this notion [30]. Centromeric cohesion at the leptonema/early zygonema stage does not depend significantly on the meiosis-specific cohesins and thus relies on cohesion established during premeiotic S phase by SMC1α cohesin. Together with earlier publications it becomes clear that with progression of meiosis, cohesion increasingly depends on meiosis-specific cohesins, since SMC1α vanishes and SMC1β becomes prominent. Telomeres suffer from any absence of meiosis-specific cohesins, but the most from absence of RAD21L or REC8 with SMC1β. The mode of telomere protection, however, remains to be elucidated, but the TRF2 patterns revealed here by SIM hint at loop-like protective structures at spermatocyte telomeres. T-loops were initially described in 1999 [31], and TRF2-dependent t-loops recently demonstrated for somatic cells were indeed suggested to protect telomeres from non-homologous end-joining and ATM-triggered DNA damage signaling [28].

Mice

Smc1β^–/–mice have been previously described [32, 33]. In Smc1β^–/–mice, exon 10 was targeted representing 40% of the hinge domain. Generally, mice were bred and maintained in the animal facility of the Medical Faculty, Technische Universität Dresden (Dresden, Germany) according to institutional guidelines. All experiments were performed with approval by the State of Saxony. Rad21L^-/- and Rec8^-/- mice were generated as described previously [19, 34]. All mice were in the C57BL/6 genetic background. Number of mice used for the experiments: N = 5, Smc1β^-/-; N = 4, Rec8^-/-; N = 4, Rad21L^-/-; N = 4, Smc1β^-/- Rec8^-/-; N = 4, Smc1β^-/- Rad21L^-/-.

Single cell suspension and chromosome spreads

Surface-spread chromosomes were prepared by detergent spreading adapted from Wojtasz et al. [35]. Testis was taken from the sacrificed mice and tunica albuginea was removed. Tubules were digested in 1 ml of 1 μg/ml of collagenase type I—PBS buffer for 10’ at 32°C with slight agitation. Tubules were the centrifuged to pellet the cells and excess collagenase was removed. Pellet was then resuspended in 500μl of 0.025% trypsin and incubated for 5’ at 32°C. Then 200 μl of media with FCS was added to the Single cell suspension. Cells were then filtered through 40 μm to remove the cell debris and centrifuged. Pellet was then resuspended in 300 μl of PBS. Now single cell suspension was used for the chromosome spreads. 1.5 μl of single cell suspension were dropped on 7 μl of 0.25% of NP40. Cells were allowed to lyse for 2 mins and then fixed by adding 24 μl of S fix (1% paraformaldehyde, 10 mM sodium borate buffer pH 9.2). Samples were incubated for 1 hour at room temperature in a humid chamber. Slides were dried under a hood and washed two times for one minute with 0.4% Agepon (AgfaPhoto) and another three times for one minute with water. Slides were used immediately or kept at -20°C until IF staining.

Testis cryosection

Testis were removed from sacrificed mice and placed in 2% (v/v) of formaldehyde/PBS for 40’ at RT for fixation before incubation in 30% sucrose/PBS overnight. Subsequently, testes were mounted in O.C.T (Sakura Finetek Europe), shock-frozen on dry ice and stored at -80°C. 8μm thick sections were made from the frozen testis, placed on the slides and dried for at least 30 min at RT. Then slides were treated with ice cold methanol for 10’ and 1’ with ice cold acetone. After completely drying, the slides were kept at –80°C or used immediately for the staining. The tubular stages were defined primarily based on cell associations and DAPI staining (centromeric and pericentric heterochromatin clustering) as described in [36].

Immunofluroscence staining

Chromosome spreads and sections were treated in the same way. Slides were blocked with either blocking buffer (2% BSA, 0.1% Triton X in PBS) or 10% goat serum for at-least 1hr at RT before the primary antibody treatment. Slides were incubated with primary antibodies for at-least 3 hrs. at 37°C. Then slides were washed with blocking buffer and incubated with secondary antibodies for at-least 1hr. After the secondary antibody treatment slides were washed with blocking buffer and mounted with Vectashield containing 1μg/ml of DAPI. Statistics was performed using the 1-way Anova test, the Dunn’s test, the Whitney-Mann test or the Wilcoxon test as indicated.

Immuno-Telo FISH staining

Telo-FISH of the G-strand was performed using the Telomere PNA FISH/Cy3 kit (Dako). The hybridization were done for 3 h at RT after denaturation at 80°C for 5 min. Cells from WT, SKO and DKO mice were always hybridized at the same time and compared with each other. Telomere intensity were obtained with equal exposure between all the genotypes and the relative length of telomeres was estimated by measuring the fluorescence intensity using ImageJ.

Microscopy and image analysis

Fluorescence was visualized with Zeiss Axiophot fluorescence microscope and analysis of images was performed using ImageJ version 1.43u. Image analysis of SIM images was done using the 3D surface plot plugin in of ImageJ. Grid size and smoothing was kept as 256 and 10.0 values, respectively, for all images.

Antibodies

The following antibodies (Tables 1 and 2) were used in this study:

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Table 2. List of secondary antibodies used.

https://doi.org/10.1371/journal.pgen.1006389.t002