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Meiotic sex chromosome cohesion and autosomal synapsis are supported by Esco2

François McNicoll, Anne Kühnel, Uddipta Biswas, Kai Hempel, Gabriela Whelan, Gregor Eichele, Rolf Jessberger  Correspondence email
Published 12 February 2020. DOI: 10.26508/lsa.201900564

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Figures

  • Figure 1.
    Figure 1. Expression and localization of cohesion establishment factors during the first meiotic prophase.

    (A) Meiotic cohesin complexes are acetylated on their SMC3 subunit. The meiosis-specific subunit SMC1β was immunoprecipitated from mouse testis nuclear extracts and acetylation of SMC3 was verified by immunoblotting using an anti-acSMC3 antibody. The membrane was then incubated with anti-SMC1β, stripped, and re-incubated with an anti-SMC3 antibody. (B, C) ESCO2 is expressed at high levels in meiotic and postmeiotic cells. (B) FACS profile of testis cells stained with Hoechst 33342; S phase, 1N, 2N, and 4N cell populations are indicated. The purity of the populations in these FASCS sorts was between 86% and 95%. (C) Immunoblotting of protein extracts from sorted primary spermatocytes (4N, 3 × 105 cells) and spermatids (1N, 1 × 106 cells) using anti-ESCO2, anti-SMC1β, and anti-GAPDH antibodies. GAPDH was used as a loading control, showing that ESCO2 and SMC1β are both enriched in meiotic and postmeiotic cells. (D) AcSMC3 appears in synapsed regions of homologous chromosomes during zygonema. Immunofluorescence staining of spermatocyte chromosome spreads using anti-SYCP3 (red) and anti-acSMC3 (green) antibodies. One pair of homologous chromosomes in zygonema is shown. A white arrow indicates the region where the homologs are already synapsed, and a yellow arrow marks the region where synapsis remains to be completed. (E, F, G, H) Cohesin and cohesion establishment factors are enriched in synapsed axial element (AE) regions during pachynema. Immunofluorescence staining of pachytene chromosomes using anti-SYCP3 (red) and either (green) anti-SMC3, anti-acSMC3, anti-sororin, or anti-ESCO2. These structures were visualized by conventional fluorescence microscopy and entire sets of chromosomes from single cells are shown. Sex chromosomes are labeled as X and Y. (F) Asterisk in (F) indicates the pseudoautosomal region. (F, I, J) The nucleus shown in (F) was visualized using super-resolution structured illumination microscopy (SIM), and the detailed structure of one pair of synapsed autosomes and of the sex chromosomes is shown. SIM allows visualization of the two lateral elements of the synaptonemal complex, which are marked by white arrows. The AEs of sex chromosomes, which remain unsynapsed, also appear as close, parallel double-filaments, that is, AEs corresponding to the individual sister chromatids are visible (yellow arrows) when visualized by SIM during pachynema (see Fig 2). (K, L) An asterisk depicts the short region of homology between chromosomes X and Y (pseudoautosomal region) (K, L). (H, I, J) Same as (I, J), except that the cell shown in (H) was analyzed. Note the enrichment of both acSMC3 and ESCO2 on autosomes between the lateral elements of the synaptonemal complex and the virtual absence of both proteins from sex chromosome AEs. A stretch of visibly separate sister chromatids is indicated by yellow arrows. Scale bars = 5 μm.

  • Figure S1.
    Figure S1. Localization of acetylated SMC3 during meiotic prophase.

    (A) Immunofluorescence staining of spermatocyte chromosome spreads from wild-type mice using anti-SYCP3 (red) and anti-acSMC3 (green) showing different substages of the first meiotic prophase. X and Y mark the sex chromosomes. (A, B) Magnified examples from (A) showing the enrichment of acSMC3 in synapsed regions in zygonema and diplonema. Scale bars for all images = 5 μm.

  • Figure S2.
    Figure S2. Localization of sororin during meiotic prophase.

    (A) Immunofluorescence staining of spermatocyte chromosome spreads from wild-type mice using anti-SYCP3 (red) and anti-sororin (green) showing different substages of the first meiotic prophase. (A, B) Magnified examples from (A). One pair of homologous chromosomes in late zygonema is also shown. “X” and “Y” indicate the sex chromosomes. Scale bars for all images = 5 μm.

  • Figure S3.
    Figure S3. ESCO2 localizes to synapsed chromosomal axes during the first meiotic prophase.

    (A) Immunofluorescence staining of spermatocyte chromosome spreads from control (Esco2∆/+) mice using anti-SYCP3 (red) and antimouse ESCO2 (green) showing different substages of the first meiotic prophase. The same exposure time and conditions were used for all stages with anti-ESCO2, whereas the exposure time with anti-SYCP3 was varied to properly visualize the meiotic substage. (A, B) Magnified examples from (A). One pair of homologous chromosomes in late zygonema is also shown. “X” and “Y” indicate the sex chromosomes. Scale bars for all images = 5 μm.

  • Figure S4.
    Figure S4. Antimouse ESCO2 but not antihuman ESCO2 produces a specific immunofluorescence signal in mouse cell nuclei.

    (A) Immunofluorescence staining of MEFs derived from wild-type (Esco2+/+) or Esco2−/− mice using a rabbit polyclonal antibody raised against an epitope of mouse ESCO2 (red) and an antibody raised against proliferating cell nuclear antigen (PCNA) (green). Nucleic acids were stained with DAPI. Anti-ESCO2 stains the pericentric heterochromatin (PCH) regions in Esco2+/+ but not in Esco2−/− MEFs. Result previously published in Whelan et al (2011). (A, B) Same as in (A), except that a commercial antibody from Bethyl Laboratories (A301-689A) raised against human ESCO2 was used. The antibody yields a signal that does not localize to PCH regions in Esco2+/+ MEFs, and the signal does not significantly differ in Esco2−/− MEFs and, thus, is unspecific. Scale bars = 5 μm.

  • Figure 2.
    Figure 2. Esco2 deletion during meiosis causes a loss of sister chromatid cohesion along sex chromosome arms.

    (A) Germ cell–specific deletion of the Esco2 gene. Single-cell suspensions were prepared from testes of Esco2fl/∆ Vasa-cre mice, stained with PI, and sorted by FACS according to the genomic DNA content. DNA was extracted from sorted cells and analyzed by PCR. The PCR reaction yields a 231-bp product for the wild-type (wt) allele, a 347-bp product for the floxed (fl) allele, and a 170-bp product for the excised (∆) allele. DNA from the spleen of the same mice was used as control to verify whether excision is germ cell specific; DNA from the tail of wt mice was used to show the wt allele. See also Fig S5. (B, C, D, E) Esco2 deletion in spermatocytes causes sister chromatid cohesion defects along sex chromosome arms. (B) Immunofluorescence (IF) staining of spermatocyte chromosome spreads from control (Esco2+/∆) and Esco2fl/∆ Vasa-cre mice (Esco2∆/∆ spermatocytes) at different stages of meiotic prophase using an anti-SYCP3 antibody and visualized by conventional fluorescence microscopy. Representative images of each stage are shown. Sex chromosomes are labeled with X and Y in magnified images, and the pseudoautosomal region, where visible, is indicated by an asterisk. Examples of sister chromatid cohesion defects such as splits in axial elements are indicated by arrows. (C) Sex chromosome axial elements consist of two SYCP3-positive filaments, presumably one per sister chromatid. Super-resolution structured illumination microscopy was performed on pachytene chromosome spreads from Esco2+/∆ and Esco2∆/∆ spermatocytes after IF staining with an anti-SYCP3 antibody. The pseudoautosomal region is indicated by an asterisk, and visible individual strands (in the control) or examples of clear splits (in the Esco2∆/∆) are marked by yellow arrows. (D) Percentage of pachytene and diplotene spermatocytes showing no splits (-), splits on chromosome Y only (Y), on chromosome X only (X) or on both sex chromosomes (XY) when visualized by conventional fluorescence microscopy. >100 cells of each genotype where X and Y could be clearly identified were analyzed. (E) Esco2 deletion during meiosis does not affect centromeric and telomeric cohesion. IF staining of chromosome spreads from Esco2∆/∆ spermatocytes using different combinations of antibodies: top row, anti-SYCP3 (red) and anti-RAP1 (telomere-binding protein, green); bottom row, anti-SYCP3 (green) and anti-centromere (ACA, red), >80 cells were analyzed for ACA staining, including >40 cells in late pachynema. Scale bars for all images = 5 μm.

  • Figure S5.
    Figure S5. Excision of the Esco2fl allele during meiosis by Vasa-cre.

    (A) FACS profile of a single-cell suspension from Esco2fl/∆ Vasa-cre mouse testes stained with PI and sorted according to genomic DNA content using gates shown in red. 1N population contains exclusively spermatids (postmeiotic); 2N population contains mostly somatic cells; 4N population contains mostly primary spermatocytes (meiosis I). (A, B) Immunofluorescence staining confirming the purity of FACS-sorted 4N and 1N cell populations from (A) using anti-SYCP3 or antimouse Vasa homolog (MVH) antibodies (green). Nucleic acids were stained with DAPI (blue). MVH localizes to the chromatid body in spermatids. One nucleus from each picture was magnified and shown in a white box. Scale bars = 20 μm. (C) Lower magnification showing entire fields with larger numbers of cells of the sorted 4N and 1N populations, as well as of the unsorted testis cell suspension stained with DAPI. Scale bars = 20 μm. (A, B, C, D) Genotyping PCR using DNA (either 10 or 50 ng per reaction) extracted from the sorted populations shown in (A, B, C) or from testes of Esco2fl/∆ Vasa-cre mice. Tail DNA from a wild-type (wt) mouse was used as control. 1N* = DNA from 1N cells of control (Esco2fl/∆, Vasa-cre negative) mice was used as control. The PCR reaction yields a 231-bp product for the wt allele, a 347-bp product for the floxed allele, and a 170-bp product for the excised allele (Whelan et al, 2011). (D, E) Same as in (D), except that control (Esco2fl/∆, Vasa-cre negative) mice were used, and either 10 or 75 ng of DNA was used per reaction.

  • Figure S6.
    Figure S6. Excision of the Esco2fl allele during meiosis by Smc1β-iCre.

    (A) Genotyping PCR on DNA extracted from the tail, testis, or from FACS-sorted spermatocyte populations of adult mice of the indicated genotypes. (B) Genotyping PCR on the testis DNA from juvenile Esco2fl/fl Smc1β-iCre mice showing the appearance of the PCR product diagnostic for the excised allele. Excision appears earliest at day 7. (C) Staining of testis cyrosections from Esco2fl/∆ Smc1b-iCre mice with DAPI and anti-SYCP3 as indicated to determine the appearance of SYCP3-positive meiocytes and, thus, the kinetics of entry into meiosis in this mouse strain. To illustrate the variability, two representative images from the same animal for each time point are shown. Scale bars = 50 μm.

    Source data are available for this figure.

  • Figure S7.
    Figure S7. Block of spermiogenesis at the round-to-elongated spermatid stage in Esco2fl/∆ Vasa-cre mice and increased apoptosis.

    (A) Overview DAPI staining of testis cryosections from control (Esco2+/∆) and Esco2fl/∆ Vasa-cre mice, 6 wk of age. (B) Immunofluorescence staining of testis cryosections from control (Esco2+/∆) and Esco2fl/∆ Vasa-cre mice, 6 wk of age using anti-SYCP3 (green) and DAPI (magenta). Tubule stages are indicated by roman letters; PS, primary spermatocyte; RS, round spermatid; ES, elongating or elongated spermatid; MS, mature spermatids; Spg, spermatogonia. Note the reduced lumen and the relative abundance of round spermatids (less mature) and scarcity of elongating/elongated spermatids (more mature) in a representative testis tubule of Esco2fl/∆ Vasa-cre mice (n = more than 10 for each genotype). (C) Immunofluorescence staining of cryosections for apoptotic cells in tubules of 4-mo-old Esco2fl/∆ Vasa-cre mice. Anti-SYCP3, anti-cIPARP, and DAPI were used for staining. cIPARP (green) indicates apoptotic cells. (B) The tubular stage and individual cell types are indicated: Zsp, zygotene spermatocyte; MSp, metaphase spermatocyte; ES, elongated spermatid (* indicates aberrant shape); RS, round spermatid; PSp, pachytene spermatocyte; SpgB, spermatogonia (B). Scale bars = 20 μm for all images.

  • Figure S8.
    Figure S8. No up-regulation of Esco1 expression.

    (A, B) Quantitative real-time PCR for Esco1 mRNA expression was performed on RNA extracted from the indicated FACS-sorted populations of Cre-negative control mice and of Esco2fl/∆ Vasa-cre (A) or of Esco2fl/∆ Smc1β-iCre mice (B) (n = 4 for each). (A, B) The data were normalized against the controls; P-values comparing the test sample against the control were >0.04 for all samples except for 1N and 4N in (A) and 1N and 2N in (B).

  • Figure S9.
    Figure S9. Defects in the maintenance of sister chromatid cohesion along chromosome arms in the absence of Esco2 during meiotic prophase.

    (A) Super-resolution structured illumination microscopy showing the structure of sex chromosomes stained with anti-SYCP3 at different stages of control (Esco2fl/+ Vasa-cre) and Esco2fl/∆ Vasa-cre mice. (B) Immunofluorescence staining using anti-SYCP3 (red) and anti-SYCP1 (green) antibodies showing a spermatocyte at early diplonema (left), where most synaptonemal complexes are still present along the length of homologs, and a spermatocyte at later diplonema (right), where most synaptonemal complexes have been disassembled. “X” and “Y” indicate the sex chromosomes. Scale bars = 5 μm.

  • Figure S10.
    Figure S10. Generation of sex chromosome splits in spermatocytes of Esco2fl/∆ Smc1β-iCre mice.

    Immunofluorescence analysis of mid-pachynema spermatocyte chromosome spreads of Esco2fl/∆ Smc1β-iCre mice. Two examples of spermatocytes and three examples of magnified sex chromosomes and an autosome, stained with anti-SYCP3 are shown; some of the splits are indicated by yellow arrows. X and Y chromosomes are indicated as are autosomes (“A”), which are associated with the sex chromatin and show loss of synapsis. Scale bars = 10 μm in the top image and 2.5 μm in the lower images.

  • Figure S11.
    Figure S11. Analysis of splits on sex chromosomes of Esco2fl/∆ Vasa-cre and Esco2fl/∆ Smc1β-iCre mice.

    As indicated, for pachytene cells, the total length of the sex chromosomes are provided as are the total, additive length of all splits per X/Y chromosomes, the number of splits per X/Y chromosomes, and the length of splits relative to the chromosome length; n = 14 cells of each genotype.

  • Figure S12.
    Figure S12. Esco2fl/∆ Stra8-cre+ spermatocytes show split axes.

    Three examples of spermatocyte spreads of the Esco2fl/∆ Stra8-cre+ genotype (Esco2∆/∆) are shown next to a control (Esco2fl/∆ Stra8-Cre−). The spreads were stained with anti-SYCP3 and anti-γH2AX. Split axes of the sex chromosomes and of autosomes reaching into the sex chromatin are visible in mutant spermatocytes. Scale bars = 10 μm.

  • Figure 3.
    Figure 3. Cohesin enrichment on sex chromosomes and changes upon Esco2 deletion.

    (A, B, C, D, E, F, G, H, I, J) Immunofluorescence staining of chromosome spreads from control (Esco2+/∆; A, E and G, I and Esco2∆/∆; B, F and H, J) spermatocytes using different combinations of antibodies: (A, B) anti-SYCP3 (red) and anti-SMC3 (subunit present in all cohesin complexes, green), showing the enrichment of cohesin on sex chromosomes compared with autosomes in both Esco2+/∆ and Esco2∆/∆ spermatocytes. Magnified images on the right show the immunofluorescence signals on sex chromosomes obtained with both antibodies (colored) or with SYCP3 only (black and white). (A, B, C, D, E, F, G, H, I, J) Note the enrichment of cohesin at the centromere of the X chromosome in both Esco2+/∆ and Esco2∆/∆ spermatocytes (orange arrows) and its presence on either side of the splits in Esco2∆/∆ spermatocytes (yellow arrows); (C, D) anti-SYCP3 (red) and anti-RAD21 (cohesin subunit present in a subset of cohesin complexes, green), showing a similar staining pattern as total cohesin in (A, B); (E, F) anti-SYCP3 (red) and anti-RAD21L (meiosis-specific cohesin subunit, green), showing the enrichment of RAD21L on sex chromosomes compared with autosomes in Esco2+/∆ spermatocytes and the relatively low abundance of RAD21L in regions where sister chromatid cohesion is impaired in Esco2∆/∆ spermatocytes; (G, H, I, J) anti-SYCP3 (red) and anti-REC8 (meiosis-specific cohesin subunit, green), showing no particular enrichment of REC8 on sex chromosomes. (G, H, I, J) For (G, H) and (I, J), two independent anti-REC8 antibodies were used. (F) The blue arrow marks a synapsis-defective autosomal region with a sex chromosome-like morphology (F). “A” indicates autosomes, “X” and “Y” indicate the sex chromosomes; scale bars = 5 μm.

  • Figure S13.
    Figure S13. RAD21-based cohesin complexes are enriched on sex chromosome axial elements.

    Spermatocyte spreads of control (Esco2fl/+ Vasa-cre) and Esco2fl/∆ Vasa-cre mice, stained for RAD21 and SYCP3. RAD21 is enriched on the unsynapsed axial elements of sex chromosomes in wt and Esco2∆/∆ spermatocytes. Several examples are shown, representing distinct stages as indicated. (A) X, Y chromosomes and autosomes (A) are indicated. Scale bars = 10 μm.

  • Figure S14.
    Figure S14. RAD21L-based cohesin complexes are enriched on sex chromosome axial elements.

    Spermatocyte spreads of control (Esco2fl/+ Vasa-cre) and Esco2fl/∆ Vasa-cre mice, stained for RAD21L and SYCP3. RAD21L are enriched at the unsynapsed axial elements of sex chromosomes in wt and Esco2∆/∆ spermatocytes. Several examples are shown representing distinct stages as indicated; the sex chromosomes are also shown magnified. Yellow arrows point to split regions, which are devoid of RAD21L. Levels of RAD21L on autosomes do not significantly change upon Esco2 deletion. Two independent antibodies were used with the same results. X, Y chromosomes and autosomes (A) are indicated. Scale bars = 10 μm.

  • Figure S15.
    Figure S15. REC8-based cohesin complexes are present on pachytene autosomes and sex chromosomes in Esco2+/∆ and Esco2∆/∆ spermatocytes.

    (A) Immunofluorescence staining of spermatocyte chromosome spreads from control (Esco2+/∆) and Esco2fl/∆ Vasa-Cre mice using anti-SYCP3 (red) and guinea pig anti-REC8 (green) antibodies. The sex chromosomes are magnified and indicated by “X” and “Y.” REC8 is not enriched on sex chromosomes and does not accumulate at specific sex chromosome regions, but dots are seen at different locations in different spermatocytes. (A, B) Immunofluorescence staining of spermatocyte chromosome spreads similar to (A) from Esco2fl/∆ mice using anti-SYCP3 (blue) and guinea pig anti-REC8 (green) antibodies. Single color channels are also shown and partially asynapsed autosomes that are close to the sex body are indicated (white arrows) as are some of the asynapsed regions carrying splits (yellow arrows; one is shown magnified). REC8 is also not enriched on the asynapsed or synapsed regions of these autosomes. Scale bars = 10 μm.

  • Figure S16.
    Figure S16. REC8-based cohesin analyzed by z-stacking in Esco2∆/∆ spermatocytes.

    Immunofluorescence staining of spermatocyte chromosome spreads from control (Esco2+/∆) and Esco2fl/∆ Vasa-Cre mice using anti-SYCP3 (red) and rabbit pig anti-REC8 (green; independent of the antibody used in S14) antibodies. The sex chromosomes are shown magnified at the bottom and indicated by “X” and “Y.” Consecutive z-stacks are shown, each 0.2 μm apart. The sex chromosome split regions are indicated by a yellow arrow, the accumulation of REC8 signals on one sister chromatid within a split region is indicated by a light blue arrow. Scale bar = 5 μm.

  • Figure S17.
    Figure S17. The loss of sex chromosome cohesion in spermatocytes of Esco2fl/∆ Vasa-cre mice is due to a small dosage effect of ESCO2 during meiosis.

    (A) Immunofluorescence (IF) staining of spermatocyte chromosome spreads from control (Esco2fl/∆ Vasa-cre negative) and Esco2fl/∆ Vasa-cre mice using anti-SYCP3 (red) and rabbit antimouse ESCO2 (green) antibodies. Representative cells in diplonema are shown. (E) See below in (E) for quantification of the signal. (B) IF staining using anti-SYCP3 (green) and guinea pig antimouse ESCO2 (red) antibodies. (A) Note that unless otherwise stated, the rabbit antimouse ESCO2 antibody shown in (A) was used in this report. (C) Super-resolution structured illumination microscopy after IF staining of spermatocyte chromosome spreads from control (Esco2+/∆) and Esco2fl/∆ Vasa-cre mice using anti-SYCP3 (red) and anti-acSMC3 (green) antibodies. ESCO2 localizes between the lateral elements of the synaptonemal complex despite excision of the Esco2fl allele during meiosis (see Figs 2A and S5). (D) Immunoprecipitation (IP) from mouse testis nuclear extracts of control (Esco2+/∆) and Esco2fl/∆ Vasa-cre mice using an anti-SMC1β antibody followed by immunoblotting using anti-acSMC3 (SMC3 acetylated on K105/K106), anti-SMC3, and anti-SMC1β antibodies. A control IP was performed using an unspecific antibody (both were mouse monoclonal IgGs). Similar levels of acSMC3 were co-immunoprecipitated with SMC1β in the absence of Esco2. (A, E) The signal corresponding to ESCO2 in (A) was quantified in at least 40 spermatocytes of each genotype at diplonema. Only a slight but not statistically significant reduction in the mean signal intensity (22 versus 17 intensity units; reduction by 23%) was observed (P = 0.07). Scale bars = 5 μm.

  • Figure S18.
    Figure S18. Reduced ESCO2 and acSMC3 levels in Esco2fl/∆ Vasa-cre and Esco2fl/∆ Smc1β-iCre spermatids.

    (A) Graph showing the average ESCO2 and acSMC3 intensity per μm2 of control (Esco2fl/∆ Cre-negative) and Esco2fl/∆ Vasa-cre spermatids as measured using the ImageJ software. The mean intensity of ESCO2 is 65.74 (±30.04 SD, n = 53) and 56.34 (±26.55 SD, n = 51) for the two strains, respectively. The mean intensity of acSMC3 in Esco2fl/∆ Cre-negative and Esco2fl/∆ Vasa-cre spermatids is 131.2 (±83.05 SD, n = 60) and 57.41 (±33.45 SD, n = 35), respectively. The difference of ESCO2 intensity is barely significant (P = 0.095), whereas the intensity of acSMC3 is significant (P < 0.0001). (A, B) Same as in (A) but for the Esco2+/∆ Smc1β-iCre and Esco2fl/∆ Smc1β-iCre mice. The mean intensity of ESCO2 is 155.7 (±56.87 SD, n = 66) and 55.13 (±23.72 SD, n = 31), respectively. The mean intensity of acSMC3 is 493.5 (±285.8 SD, n = 55) and 61.60 (±31.03 SD, n = 28), respectively. The differences of ESCO2 and acSMC3 intensity between spermatids of the two strains are significant (P < 0.0001). Unpaired t tests were performed for all analyses.

  • Figure S19.
    Figure S19. Esco2 deletion during meiosis delays synaptonemal complex formation.

    (A) Left and middle panels, immunofluorescence (IF) staining of spermatocyte chromosome spreads from control (Esco2+/∆Vasa-Cre) and Esco2fl/∆ Vasa-cre (Esco2∆/∆) mice using anti-SYCP3 (green), anti-γH2AX (red), and anti-H1t (blue) antibodies. Right panel, percentage of prophase I cells counted in each stage. At least 200 cells per genotype were staged. H1t-positive cells include all cell types after mid-pachytene; H1t-negative cells all meiotic cells (SYCP3-positive) up to mid-pachytene. L, leptonema; Z, zygonema; P, pachynema; D, diplonema; RS, round spermatid. (A, B) IF staining of spermatocyte chromosome spreads using anti-SYCP3 (red) and anti-RNA pol II phospho S2 (elongating RNA polymerase, green) antibodies showing that synapsis-defective autosomal (A) regions are transcriptionally silenced during meiosis (similarly to sex chromosomes in both control (Esco2+/∆) and Esco2∆/∆ spermatocytes). (C) IF staining using anti-SYCP3 (green) and anti-centromere (ACA, red) antibodies. Centromeres of synapsis-defective autosomes are indicated by yellow arrows. The percentage of synapsis-defective autosomes where asynapsis was observed at the centromeric or non-centromeric ends is indicated at the left. Scale bars = 5 μm.

  • Figure 4.
    Figure 4. Sister chromatid cohesion supports synapsis during meiotic prophase.

    (A) Esco2 promotes synaptonemal complex formation. Immunofluorescence staining of chromosome spreads from Esco2∆/∆ spermatocytes using anti-SYCP3 (green), anti-γH2AX (marks unsynapsed chromatin, red), and anti-H1t (testis-specific histone variant demarcating the mid-pachynema onset, blue) antibodies. (A) Top row, example of cell with incomplete synapsis on one pair of autosomes (indicated by (A) in magnified image) around mid-pachynema, where spermatocyte nuclei are slightly H1t positive. (A) Bottom row, example of cell with one synapsis-defective autosome (γH2AX-positive, indicated by (A)) at early diplonema. Note the similar morphology of the synapsis-defective autosome axial element (AE) regions and of sex chromosome AEs; orange arrows indicate excrescences in proximity to the thickened centromeres. (B) Sister chromatid cohesion is weakened around mid-pachynema in synapsis-defective regions of Esco2∆/∆ spermatocytes and is partly lost in early diplonema. Super-resolution structured illumination microscopy showing the structure of synapsis-defective autosomes stained with anti-SYCP3 at different stages. Yellow arrows indicate unsynapsed regions of individual homologs and white arrows indicate synapsed regions. Note that sister AEs are not visible in unsynapsed regions during zygonema but can be clearly distinguished around mid-pachynema and further separate from each other in early diplonema. (C) Asynaptic autosomes acquire a sex chromosome-like morphology in Esco2∆/∆ spermatocytes. Immunofluorescence staining using different combinations of antibodies: top row, anti-SYCP3 (green) and anti-γH2AX (red), nucleic acids were stained with DAPI to show pericentric heterochromatin and centromeres of a synapsis-defective autosome pair are indicated by orange arrows; middle row, anti-SYCP3 (red) and anti-SYCP1 (transverse filament of the synaptonemal complex, green), where synapsed AE regions appear yellow in the merged image; bottom row, anti-SYCP3 (red) and anti-RAP1 (green). Scale bars for all images = 5 μm.

  • Figure S20.
    Figure S20. Synapsis defects in the absence of Esco2 are not due to a failure to repair DNA double-strand breaks characterized by DMC1.

    (A) Immunofluorescence staining of spermatocyte chromosome spreads from control (Esco2+/∆) and Esco2fl/∆ Vasa-cre mice using anti-SYCP3 (red) and anti-DMC1 (meiosis-specific recombination protein, green) antibodies. Left panel, typical examples of control cells in early pachynema (top), where most DMC1 foci still present are found on sex chromosomes, and in late pachynema (bottom), where all DMC1 foci have been processed. (A) Right panel, typical examples of cells from Esco2fl/∆ Vasa-cre mice with synapsis-defective autosomes (A) in early pachynema (top), where no unprocessed DMC1 foci are found in unsynapsed autosomal regions, and in late pachynema (bottom), where all DMC1 foci have been processed. The magnified insets show the presence of foci also within split regions. (B) Quantification of DMC1 foci on all chromosomes (total) and on sex chromosomes (XY) in early pachynema. >15 cells per genotype were counted. (A) X, Y chromosomes and autosomes (A) are indicated. Scale bars = 5 μm.

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Cohesin acetylase ESCO2 in spermatocytes
François McNicoll, Anne Kühnel, Uddipta Biswas, Kai Hempel, Gabriela Whelan, Gregor Eichele, Rolf Jessberger
Life Science Alliance Feb 2020, 3 (3) e201900564; DOI: 10.26508/lsa.201900564
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