PMC

(a) Double labelling of SIX6OS1 (green) and SYCP3 (red) or (b) SYCP1 (red) in Rad21l^−/−, Rec8^−/−, Stag3^−/−, Syce3^−/− and Sycp1^−/− showing that loading of SIX6OS1 is dependent on synapsis. SIX6OS1 is detected in the synapsed LEs of meiotic cohesin mutants but is absent from unsynapsed AEs in Syce3^−/− and Sycp1^−/− spermatocytes. Scale bar, 10 μm.

(a) Double immunolabelling of SYCP3 (red) and SYCP1 (green) in wild-type pachytene, and Six6os1^−/− and Syce3^−/− pachytene-like arrested spermatocytes. Yellow letters indicate aligned/unaligned sex chromosomes in mutant spermatocytes. (b) Co-labelling of SYCP3 (red) and γ-H2AX (green) in Six6os1^−/− pachytene-like arrested spermatocytes, showing moderate staining in XY chromosomes. (c) Double immunolabelling of SYCP3 (red) and 53BP1 (green). 53BP1 signal is not observed in the XY chromosomes of mutant spermatocytes, in contrast to the strong labelling of the sex body in wild type. Scale bar, 10 μm.

(a) Double immunostaining of SIX6OS1 (green) and SYCP1, SYCE1, SYCE3, SYCE2 or TEX12 (red). Immunofluorescence signal levels were measured on synapsed chromosome axes. Left plots represent normalized signal intensity profiles of SIX6OS1 with each CE protein. Right plots show regression analysis of the correlation between each pair. (b) Plot of the mean correlation between SIX6OS1 and SYCP1, SYCE1, SYCE3, SYCE2 or TEX12 (n=38 axial elements (AEs), mean±s.d.). The best correlation value was obtained with SYCE1. Scale bar, 2.5 μm.

(a) Double labelling of spermatocyte spreads of wild-type pachytene, and Six6os1^−/− and Syce3^−/− arrested pachytene-like spermatocytes with SYCP3 (red) and SYCP1 (green). In Six6os1^−/− spermatocytes, SYCP1 does not localize to the unaligned-type (U-type) AEs but shows a very weak staining in spermatocytes with more aligned AEs (aligned-type, A-type). By direct comparison, in Syce3^−/− arrested spermatocytes SYCP1 localizes in a discontinuous pattern along AEs independent of whether or not they are closely aligned. (b) Double labelling of spreads of wild-type pachytene and Six6os1^−/− pachytene-like oocytes (aligned and unaligned) with SYCP3 and SYCP1. (c) Double labelling of spreads of wild-type pachytene and Six6os1^−/− pachytene-like spermatocytes of SYCP3 (red) and SYCE1, SYCE3, SYCE2 or TEX12 (green) (see also extended for staining in oocyte spreads). All proteins are completely absent from the AEs in Six6os1-deficient mice. Scale bar, 10 μm.

(a) Double immunolabelling of γ-H2AX (green) with SYCP3 (red) in wild-type and Six6os1^−/− spermatocytes (left panel). In wild-type pachytene, γ-H2AX labels intensely the chromatin of the unsynapsed sex bivalent. In Six6os1^−/− pachytene-like spermatocytes γ-H2AX labelling remains in the chromatin. Double immunolabelling of SYCP3 (red) and RAD51 (green) (central panel) or RPA (green) (right panel). Both RAD51 and RPA remain associated to the AEs in Six6os1^−/− pachytene-like spermatocytes, showing a higher number of foci than wild-type pachytene. (b) Immunostaining of spread preparations of wild-type pachytene and Six6os1^−/− pachytene-like oocytes for γ-H2AX (green), RAD51 (green) and RPA (green) together with SYCP3 (red). γ-H2AX labelling in Six6os1^−/− arrested oocytes is more restricted to the AEs than in spermatocytes. Plots under each image panel represent the quantification of intensity or number of foci from wild-type and pachytene-like arrested meiocytes. Welch's t-test analysis: *P<0.01; **P<0.001; ***P<0.0001. (See numeric data at ). Scale bar, 10 μm.

(a) Double immunolabelling of SYCP3 (red) with MLH1 (green). MLH1 foci are absent at the AEs/LEs of Six6os1^−/− meiocytes whereas at least one focus is present along each autosomal SC in wild-type pachytene meiocytes. (b) Immunostaining of SYCP3 (red) and ACA (green) in wild type and Six6os1^−/− spermatocytes. Okadaic acid-induced metaphase I plates of wild-type spermatocytes give rise to 20 bivalents each, with two opposed centromere signals (ACA) and positive staining for SYCP3, whereas Six6os1^−/− spermatocytes lead to 40 free univalents, each with an ACA signal and SYCP3 labelling the centromeric and interchromatid domain. Scale bar, 10 μm.

(a–c) HEK 293T cells were transfected or co-transfected with the indicated expression vectors. Protein complexes were immunoprecipitated overnight with either an anti-Flag, anti-EGFP or anti-Myc antibody, and were analysed by immunoblotting with the indicated antibody. (a) SIX6OS1 co-immunoprecipitates (co-IP) with SYCE1 (as well as in the reciprocal IP) but not with either SYCP1, SYCE3, SYCE2 or TEX12. (b) SIX6OS1-Flag co-immunoprecipitates with SIX6OS1-GFP, suggesting that it is able to form at least dimers. (c) SYCE1 co-immunoprecipitates with the SIX6OS1 N-terminal half (1–286) but not with the C-terminal half (287–574). Immunoprecipitation of SYCE1 and full length SIX6OS1 was used as positive control. (d) COS7 cells were transfected with Six6os1 alone (left panel) or in combination with Sycp1 and Syce1 (right panel). SIX6OS1 localization drastically changed in the presence of SYCE1 but not with SYCP1. (e) Sycp1, Syce3, Syce1 and Six6os1 were simultaneously co-transfected in COS7 cells and found to co-localize in the cytoplasm in the punctate pattern of SYCE1. The experiments were reproduced three times. Scale bars, 15 μm.

(a) Schematic representation of the wild-type locus (WT) and the genome editing strategy at the Six6os1 locus, showing the sgRNAs, the corresponding coding exons (light grey) and non-coding exons (open boxes). Thin (non-coding) and thick (coding sequences) lines under exons represent the expected transcript derived from WT and Six6os1 edited allele. ATG, initiation codon; TGA, stop codon. The nucleotide sequence of the 124 base pair deletion derived from PCR amplification of DNA from the Six6os1^{edited/edited} is indicated. Primers are represented by arrows. (b) PCR analysis of genomic DNA from three littermate progeny of Six6os1^+/− heterozygote crosses. The PCR amplification with primers F and R (indicated by arrows) revealed 413 and 289 bp fragments for wild-type and disrupted alleles, respectively. (+/+), (+/−) and (−/−) designate wild-type, heterozygous and homozygous knockout animals, respectively. (c) Double immunofluorescence of spermatocytes at pachytene stage obtained from Six6os1^+/+ and Six6os1^−/− mice using SYCP3 (red) and a goat polyclonal antibody against SIX6OS1 (green). Scale bar, 10 μm.

(a) Genetic ablation of Six6os1 leads to a reduction of the testis size (n=8 wild-type and knock out, Welch's t-test analysis: P<0.0001), and (b) a complete arrest of spermatogenesis in epithelial stage IV as shown in hematoxylin-eosin stained testis sections. Massive apoptosis of spermatocytes is indicated (asterisks). The spermatogenic arrest leads to empty epididymides and azoospermia. Scale bar in upper panels, 100 μm and in lower panels, 5 μm. (St) Seminiferous tubules. (Ep) Epididymides. (c) Tubule degeneration in juvenile mice (13 d.p.p. and 18 d.p.p.) lacking SIX6OS1 and spermatogenic arrest before pachytene studied by histology. At 13 d.p.p. spermatogenesis has reached late zygotene; at 18 d.p.p. it has reached late pachytene. Spermatocyte degeneration (apoptosis is indicated by asterisks) was first seen in 18 d.p.p. Six6os1^−/−. (d) Ovaries from Six6os1-deficient mice show atrophy with fibrosis and depletion of follicles. Comparative histological analysis of ovaries from Six6os1^−/− and wild-type mice at 6 days (6 d.p.p.), and 4 months (4 m) of age. Asterisks indicate corpora lutea. Scale bars represent 100 μm in 4 m, and 20 μm in 6 d.p.p.

(a) Relative transcription of Six6os1 and Rad21l (ref. ) mRNA by quantitative reverse transcription PCR (RT–qPCR) in mouse tissues. β-Actin transcription was used to normalize the expression (mean±s.d., three replicates). (b) Immunolabelling of in vivo electroporated SIX6OS1-GFP in mouse testis. SIX6OS1 was detected with anti-GFP (green) and endogenous SYCP3 was detected using mouse anti-SYCP3 (red). DNA was stained with DAPI (blue). During pachytene, SIX6OS1 colocalizes with SYCP3 along synapsed lateral elements (LEs) including the pseudoautosomic region (PAR) of the XY bivalent (spermatocytes). In diplotene and late diplotene, SIX6OS1 localizes at the still synapsed LEs. (c) Double immunolabelling of endogenous SIX6OS1 (green) and SYCP3 (red) in spermatocytes. DNA was stained with DAPI (blue). During pachynema, SIX6OS1 is located at the synapsed autosomal LEs and at the PAR of the sex XY bivalent. (d,e) Co-labelling of spermatocytes spread preparations with SIX6OS1 (green) and SYCP1, SYCE1, SYCE3, SYCE2 or TEX12 (red), showing that SIX6OS1 localizes to the synapsed LEs but best mirrors SYCE1 localization. (f) Immunoelectron microscopy of frozen mouse testis sections marked with goat anti-SIX6OS1 antibody. Left panel corresponds to an autosomal chromosome and right panel to the XY bivalent in which the PAR is shown. Gold particles 6 nm. Scale bar in b–e, 10 μm. PAR is indicated with an asterisk in b and c.