Sycp3

Β-actin antibody (mouse, sc-47778; Santa Cruz); SYCP3 (mouse, sc-74569; Santa Cruz); γH2AX (rabbit, 9718; Cell Signaling Technology, Inc.); MVH (mouse, ab27591; Abcam); SOX9 antibody (rabbit, AB5535, Sigma-Aldrich); PLZF antibody (goat, AF2944, R&D Systems); SFRS2 polyclonal antibody (rabbit, 20,371–1-AP, Proteintech); SC35 antibody (mouse, S4045, Sigma-Aldrich); green-fluorescent Alexa Fluor® 488 conjugate of lectin PNA (L21409, Thermo). Horseradish peroxidase–conjugated secondary antibodies were purchased from Zhongshan Golden Bridge Biotechnology Co, LTD (Beijing). Alexa Fluor 488–conjugated antibody, 594–conjugated antibody and Alexa Fluor 647–conjugated antibody were purchased from Life Technologies.

β-actin antibody (mouse, sc-47778; Santa Cruz); SYCP3 (mouse, sc-74569; Santa Cruz); γH2AX (rabbit, 9718; Cell Signaling Technology, Inc.); MVH (mouse, ab27591; Abcam); SOX9 antibody (rabbit, AB5535, Sigma-Aldrich); PLZF antibody (goat, AF2944, R&D Systems); SFRS1 polyclonal antibody (rabbit, 12929-2-AP, Proteintech); c-Kit antibody (goat, AF1356, R&D Systems). Horseradish peroxidase-conjugated secondary antibodies were purchased from Zhongshan Golden Bridge Biotechnology Co, LTD (Beijing). Alexa Fluor 488-conjugated antibody, 594-conjugated antibody and Alexa Fluor 647-conjugated antibody were purchased from Life Technologies.

For western blotting, tissue or cell lysates were separated in SDS PAGE gel and transferred to nitrocellulose membrane, and was subsequently blocked with 5% skim milk at 4 °C overnight and then incubated with primary antibodies Sycp3 (Santa Cruz, sc-74,569), Stra8 (Abcam, ab49602), claudin11 (Abcam, ab175236), nectin2 (Abcam, ab135246), GATA2 (Abclonal, A0677), GFRα1 (Affinity, DF7309), β-catenin (Cell Signaling, 8480p), β-tubulin (Anbo, P07437) and others mentioned before at room temperature for 1 h. The membranes were incubated with HRP-conjugated goat anti rabbit or mouse IgG (Santa Cruz, sc-2004,sc-2005) and ECL, and finally exposed the film. For Co-IP, lysates of adult mice testes or SPCs were prepared using a standard protocol (Beyotime, P0013), and 0.5 mg total proteins diluted with TBST were incubated with mouse anti-ITGB1 antibody (BD, 610467) and rabbit anti-CDH1 antibody (Santa Cruz, sc-7870) respectively and rocked at 4 °C overnight. The pre-washed protein (A + G) sepharose beads (Beytime, P2012) were added afterwards and incubated at 4 °C for 4 h. The beads were then washed three times with TBST, pelleted and boiled in 1XSDS loading buffer, and finally analyzed by western blot.

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.