Sycp1

Seminiferous tubules were collected from dissecting testes and washed with 1× PBS. Hypo extraction buffer was used to incubate within seminiferous tubules for 30 min, followed by disrupting within 0.1 M sucrose liquid to form a single-cell suspension. The cell suspension was mounted on slides treated with 1% PFA. Slides were air-dried in a humidified box for at least 6 h. After washing with 0.04% Photo-Flo (Equl, 1464510), the slides were stained for SYCP3, SYCP1 (1:200; Abcam), γH2AX (1:200; Millipore). Incubation with the specific antibody was conducted at RT for 30 min after antibody dilution buffer treatment. After washing three times in 1× Tris buffer, saline (TBS), blocking was conducted with 1× antibody dilution buffer at 4°C overnight. After washing three times with cold 1× TBS buffer, the corresponding secondary antibody and the fluorescent dye–conjugated TRITC were incubated with sections for 3 h at 37°C. All images were captured with a confocal laser scanning microscope (Leica TCS SP8).

Chromosome spreads were prepared by the “drying down technique” as previously described [49 ]. Briefly, ovaries were collected from E16.5 mice and incubated in warmed PBS until their use. Ovaries were then incubated in hypotonic extraction buffer [30 mM Tris, 50 mM sucrose, 17 mM trisodium citrate dihydrate, 5 mM EDTA, 0.5 mM DTT, and 0.5 mM phenylmethylsulphonyl fluoride (PMSF), pH 8.2] for 30 minutes, then teased apart in 100 mM sucrose. The ovarian single cell suspension was then pipetted onto slides wetted in 1% PFA with 0.2% Triton X-100 and allowed to settle overnight in a humid chamber at 37°C. The next day, slides were air-dried, incubated in 0.4% Photo-Flo (Kodak) for 2 minutes, air-dried again and then either stained or stored at -80°C. Spreads were stained by immunofluorescence as described above using primary antibodies against MLH1 (BD Pharmigen), SYCP1 (Abcam), CENP-A (Abcam), γH2AX (Millipore) or SYCP3 (Santa Cruz). To determine prophase I stage, SYCP3 configuration (as shown in S1 Fig) was used. To determine percent oocyte asynapsis, >20 CENP-A centromeric foci was used as a quantitative assessment. For both analyses, four animals yielding approximately 50 oocytes and 10–20 pachytene oocytes each were utilized. To quantify meiotic recombination, 25 or more pachytene oocytes per animal were scored for MLH1 foci on chromosome cores as visualized by SYCP3 staining.