Telomere analysis.

<p>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. <i>Smc1β</i>^<i>-/-</i> versus <i>Rec8</i>^<i>-/-</i> etc.) are also <0.0001 according to Dunn’s multiple comparison test.</p

List of secondary antibodies used.

<p>List of secondary antibodies used.</p

Testis tubule analysis of wild-type and mutant mice.

<p>A. Immunofluorescence staining of testis sections of WT, SKO (single knockout) mice <i>Smc1β</i>^<i>-/-</i>, <i>Rec8</i>^<i>-/-</i>, <i>Rad21L</i>^<i>-/-</i>, and DKO (double knock out) mice: <i>Smc1β</i>^<i>-/-</i><i>Rec8-/-</i> and <i>Smc1β</i>^<i>-/-</i><i>Rad21L</i>^<i>-/-</i> mice, probed with anti-SYCP3 and anti-γH2AX; DNA was stained with DAPI (scale bar: 5 μm). Tubular stages are indicated by roman letters. B. Magnified images from Fig. 2A showing SYCP3-stained axes and γH2AX localization. The most advanced stages are shown, characterized by chromosome axes that are SYCP3-positive and γH2AX signals, which indicate the presence of DNA double-strand breaks und unsynapsed chromosomes. C. Immunofluorescence staining of testis sections of WT, SKO (single knockout) mice <i>Smc1β</i>^<i>-/-</i>, <i>Rec8</i>^<i>-/-</i>, <i>Rad21L</i>^<i>-/-</i>, and DKO (double-knock out) mice: <i>Smc1β</i>^<i>-/-</i><i>Rec8-/-</i> and <i>Smc1β</i>^<i>-/-</i><i>Rad21L</i>^<i>-/-</i> mice, probed with anti-SYCP1 and anti-SYCP3; DNA was stained with DAPI (scale bar: 5 μm). D. Magnified images from Fig. 2C showing SYCP1 (green)/SYCP3 (red)-stained axes indicated by yellow color.</p

Overview of cohesins in meiosis and meiotic cohesin mutant phenotypes.

<p>A. Graph illustrating the approximate occurrence of individual cohesin proteins throughout male meiosis, based on summarizing the current literature; for details see main text. B. Table showing some of the key phenotypes observed in individual mouse strains deficient for one or two cohesin proteins. The respective references are indicated. * T: arrest stage based on most advanced stage of tubular development; C: arrest stage based on appearance of chromosomes and associated proteins; in several instances, the named stages are approximations due to difficulties determining the corresponding normal stage; the mutant stages are often called “-like” stages. ** The <i>Stag3</i> mutants used by Hopkins et al, Ward et al., Fukuda et al., and Llano et al., were of distinct origin and express low levels of STAG3 and thus display a hypomorphic phenotype.</p

Super resolution telomere analysis.

<p>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).</p

Distinct Roles of Meiosis-Specific Cohesin Complexes in Mammalian Spermatogenesis

<div><p>Mammalian meiocytes feature four meiosis-specific cohesin proteins in addition to ubiquitous ones, but the roles of the individual cohesin complexes are incompletely understood. To decipher the functions of the two meiosis-specific kleisins, REC8 or RAD21L, together with the only meiosis-specific SMC protein SMC1β, we generated <i>Smc1</i>β^<i>-/-</i><i>Rec8</i>^<i>-/-</i> and <i>Smc1β</i>^<i>-/-</i><i>Rad21L</i>^<i>-/-</i> mouse mutants. Analysis of spermatocyte chromosomes revealed that besides SMC1β complexes, SMC1α/RAD21 and to a small extent SMC1α/REC8 contribute to chromosome axis length. Removal of SMC1β and RAD21L almost completely abolishes all chromosome axes. The sex chromosomes do not pair in single or double mutants, and autosomal synapsis is impaired in all mutants. Super resolution microscopy revealed synapsis-associated SYCP1 aberrantly deposited between sister chromatids and on single chromatids in <i>Smc1β</i>^<i>-/-</i><i>Rad21L</i>^<i>-/-</i> cells. All mutants show telomere length reduction and structural disruptions, while wild-type telomeres feature a circular TRF2 structure reminiscent of t-loops. There is no loss of centromeric cohesion in both double mutants at leptonema/early zygonema, indicating that, at least in the mutant backgrounds, an SMC1α/RAD21 complex provides centromeric cohesion at this early stage. Thus, in early prophase I the most prominent roles of the meiosis-specific cohesins are in axis-related features such as axis length, synapsis and telomere integrity rather than centromeric cohesion.</p></div

Localization of cohesin proteins.

<p>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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006389#pgen.1006389.s004" target="_blank">S4</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006389#pgen.1006389.s010" target="_blank">S10</a> 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.</p

DNA double strand break repair foci.

<p>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).</p

List of primary antibodies used.

<p>List of primary antibodies used.</p

Centromeric cohesion.

<p>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).</p