Fig 2: 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.

Fig 3: 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.

Fig 4: 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.

Fig 5: 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.