Figure 6. STAG3 facilitates SPO11 function in meiotic DSB formation.

(A & B) B6N (wildtype), Spo11^+/−, Stag3^−/−, Stag3^−/− Spo11^+/− and Spo11^−/− zygotene (and zygotene-like) spermatocyte chromatin spreads immunolabeled with antibodies against the SC protein SYCP3 (red in panel A and green in panel B), DMC1 (green in panel A), DSB-associated phosphorylated histone γH2AFX (red in panel B); DNA is stained with DAPI (blue). Scale bars=10μm.

(C) Bar graph showing the distribution of zygotene and zygotene-like spermatocytes with and without DSBs in B6 (wildtype), and mutant Spo11^+/−, Stag3^−/− and Stag3^−/− Spo11^+/− spermatocytes.

(D) Dot-plots showing quantification of DMC1 foci in in zygotene and zygotene-like spermatocytes from different mouse strains. Error bars=SEM. P-values were calculated using Mann-Whitney U test with Tukey’s multiple testing corrections and are tabulated in Data S4.

(E) Blot of immunoprecipitated and radioactively labelled SPO11-oligonucleotide complexes. The left vertical line marks SPO11-specific signals, the right asterisk indicates nonspecific labelling of contaminants in 13dpp old testis from B6N (wildtype), Stag3^−/− and Spo11^+/− mice. Radioactive signals were quantified and corrected for background noise and normalized to wild type level. Blots were re-probed with anti-SPO11 antibody. Only one alternative isoform of SPO11 (β) protein was present in 13 dpp B6N (wildtype), Stag3^−/− and Spo11^+/− mice. The experiments were replicated twice. ±, plus/minus represents standard deviation. P-values were calculated using Welch's t-test.

See also Data S4.