Af2030

Whole testes were fixed with 4% paraformaldehyde, immobilized in paraffin and sectioned. After removal of paraffin, sections were stained at the following antibody concentrations: anti-LINE Orf1p (1:300), anti-IAP Gag (1:300), anti-MORC1 (1:100), anti-VASA (1:100, R&D Systems AF2030), stained with fluorescent secondary antibody and mounted with DAPI (4',6-diamidino-2-phenylindole). Slides were imaged by Confocal microscopy.

Pregnant mothers were injected intraperitoneally (i.p.) with 20 mg/kg 5-ethynyl-2-deoxyuridine (EdU) to facilitate in vivo analysis of gonadal cell proliferation. Flow cytometry was performed as previously described [40 (link), 77 (link), 78 (link)]. Dissociated gonadal cells were stained using antibodies specific for SOX9 and AMH allowing identification of Sertoli cells and SOX9 (Millipore AB5535, diluted 1/300) and AMH (Santa Cruz sc-6886, diluted 1/200) staining intensity in the Sertoli cell population. Cell cycle was measured in Eed^wt/wt, Eed^wt/hypo and Eed^hypo/hypo samples as previously described [77 (link), 78 (link)]. Germ cells were identified using an antibody specific for MVH (R&D Systems, AF2030 diluted 1/100; [77 (link), 78 (link)]).

Mouse embryos were fixed in 4% PFA in PBS at 4°C, embedded in paraffin, sectioned at 5μm, and immunofluorescence performed as described previously [39 (link)]. Primary antibodies used for this study were anti-HMGCS2 rabbit monoclonal (1:50; ab137043, Abcam), anti-SOX9 sheep polyclonal (1:100; [40 (link)]), anti-MVH goat polyclonal (1:200; AF2030, R&D systems), anti-AMH goat polyclonal (1:200; sc6886, Santa Cruz), anti-AMH goat polyclonal (1:50; AF1446 R&D systems), anti-SYCP3 mouse monoclonal (1:100; ab97672, Abcam), anti-FOXL2 rabbit polyclonal (1:300; [41 (link)]) and anti-CYP11A1 rabbit polyclonal [42 (link)]. Secondary antibodies used were donkey anti-rabbit Alexa 488, donkey anti-rabbit Alexa 568, donkey anti-goat Alexa 488, donkey anti-goat Alexa 546, donkey anti-mouse Alexa 488, and donkey anti-sheep Alexa 647 obtained from Invitrogen and used at 1:300. Images were taken with a Zeiss LSM 510 Meta confocal microscope at the Australian Cancer Research Foundation Dynamic Imaging Centre for Cancer Biology, University of Queensland and with a Zeiss LSM800 confocal microscope at the Biological Optical Microscopy Platform (BOMP) at the Department of Anatomy and Neuroscience, The University of Melbourne.

Western blotting was performed according to standard procedures. In brief, 20 mg of frozen tissue per sample was used. Proteins were isolated by mechanical destruction of the tissue in a TissueLyser at 50 HZ (Qiagen, Hilden, Germany). The samples were denatured for 5 minutes at 95°C. Samples were then run on a 10% SDS-page gel in Tris HCl buffer, pH 8.8, and then semi-dry-blotted onto a PVDF membrane (150 mA for 1 h). Blocking of unspecific binding was achieved by incubating the membrane in 5% skim milk powder diluted in TBS for 1 h. Primary antibodies against VASA (#AF2030, R&D Systems; 1 : 2000) and β-actin (Santa Cruz, SC-1616-R; 1 : 5000) were diluted in blocking buffer. Membranes were incubated in primary antibody solutions for 16 hours at 4°C and then washed three times with blocking solution supplemented with Tween 20. After incubation with the horseradish peroxidase-coupled secondary antibody (1 h at 20°C) the membrane was washed again. Detection of bound antibody was performed using the Amersham ECL Western Blotting Detection Reagents Kit (RPN2106). Signals were detected and documented using the ChemoCam Imager (INTAS, Göttingen, Germany).