Ab13840

The pellet of ovarian cells was re-suspended
and incubated with polyclonal rabbit antimouse
MVH (ab13840, Abcam, Cambridge,
UK) and subsequently by goat anti-rabbit IgG
magnetic beads (130-048-602, MiltenyiBiotec
Inc., UK). The mixture of cells and magnetic
beads was placed on the magnetic bead separator,
while separation was performed following
the manufacturerʼs instructions and as described
in experiment 1. Isolated cells were cultured on
mitotically inactivated MEF in 4 -well plates at
37˚C in a 5% CO₂ atmosphere.

In brief, ovaries were fixed with 4% PFA at 4°C overnight, incubated in 30% sucrose (#S0389, Sigma-Aldrich) at 4°C for three days, frozen, sectioned, incubated with PBS containing 0.25% Triton X-100 for 15 min, and blocked with PBST containing 3% BSA for 30 min. Sections were incubated with a rabbit anti-VASA (DDX4) antibody (1∶200; #ab13840, Abcam, Cambridge, UK) for 1 h at room temperature, rinsed with PBS, and incubated with a goat anti-rabbit IgG secondary antibody labeled with Alex Fluor 594 (1∶300). Nuclei were stained with DAPI for 3 min. All sections were mounted with fluoroshield mounting medium (#ab104135, Abcam) on glass slides and observed using a confocal microscope (Leica TCS SP5, Wetzlar, Germany).

The isolated ovaries were fixed and permeabilized with 4% PFA and then prepared for paraffin section. After dewaxing and rehydration of paraffin sections, membrane permeation was performed after washing. Then the sections were blocked with blocking buffer (5% goat serum, 0.3% Triton™ X-100 in 0.1 M PBS) for 1 h at room temperature, followed by the incubation with primary antibody overnight at 4 °C. After washing with TBST, sections were incubated with the secondary antibody at 37 °C for 1 h. After washing again, DAPI was added to probe the nuclei and mounted with a mounting medium (H-1400, Vector Laboratories, Inc). Axio Scan.Z1 (Zeiss) and Zeiss LSM780 were used for widefield and confocal imaging, respectively. Images were captured and analyzed using software Zeiss Zen (v2.1) and Image J (v1.52i). The primary antibodies used for immunostaining included: anti-DDX4 (1:200, ab13840, Abcam), anti-AMH (1:400, sc-6886, Santa Cruz Biotechnology), anti-cleaved CASP3 (9664, CST), anti-Foxo3 (1:250, Santa Cruz Biotechnology), and anti-BDNF (1:100, Abcam, ab108319). The secondary antibodies included: Alexa Fluor 546 conjugated goat anti-mouse (Thermo Fisher Scientific, A-11030, 2 μg/ml) and Alexa Fluor 488 conjugated goat anti-rabbit (Thermo Fisher Scientific, A-11008, 2 μg/ml).

For most experiments (except as indicated below), OSCs were isolated from ovaries of young adult mice (2–3 months of age) by FACS using a C-terminal DDX4-specific antibody (ab13840, Abcam). The cells were analyzed immediately or established in culture without somatic feeder cells, as described^{16 (link), 17 (link), 46 (link), 93 (link)}. Purified mouse OSCs propagated under these conditions spontaneously differentiate into IVD-oocytes for up to 72 h after passage until confluence is regained, and the number of IVD-oocytes generated by a fixed number of OSCs seeded per well remains relatively constant over successive passages^{16 (link), 17 (link), 19 (link)}. Between passages 32–40, OSCs were transfected with the desired plasmids (pStra8-HSVtk or pStra8-Gfp, each containing a neomycin resistance gene) using Lipofectamine 2000 (Invitrogen) and then selected by G418 (Geneticin, Cellgro) over 2 weeks. Cells were then maintained in G418 for all experiments, and the number of IVD-oocytes generated and released into the medium after treatment with vehicle or GCV (2 μM) was then determined by direct visual counts under a microscope^{16 (link), 17 (link), 19 (link)}. In other experiments, GFP-positive cells in ovaries of pStra8-Gfp transgenic female mice were quantitated and then isolated by FACS for gene expression profiling.

Freshly collected tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for analysis using primary antibodies against Stra8 (rabbit polyclonal, ab49602; Abcam), Ddx4 (rabbit polyclonal ab13840, Abcam; goat polyclonal AF2030, R&D Systems), Sycp3 (rabbit polyclonal NB300-230, Novus Biologicals), Ser¹³⁹-phospho-H2afx (mouse monoclonal 05–636, Millipore) or GFP (chicken polyclonal ab13970, Abcam; rabbit polyclonal ab290, Abcam). For IF, detection was performed using donkey anti-chicken Alexa Fluor 488 (Jackson Immuno), donkey anti-goat Alexa Fluor 647 or donkey anti-rabbit Alexa Fluor 546 (Molecular Probes) as secondary antibody^{16 (link)}. For IHC, detection was performed using biotin-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) as secondary antibody for horseradish peroxidase-based DAB detection (Sigma-Aldrich). Images were captured using a Nikon E800/BioRad Radiance 2000 confocal microscope or a Nikon ECLIPSE TE2000-S microscope.

Ovarian tissues were homogenized in RIPA buffer (Thermo Scientific, Rockford, IL, USA) and centrifuged at 12,000 × g for 20 min. The protein content of samples was determined using a BCA protein assay kit (Bio-Rad Laboratories, Carlsbad, CA). Aliquots of each sample were separated via 12% SDS-PAGE and then transferred onto a nitrocellulose membrane, which was blocked with non-fat milk for 1 h. The membranes were probed overnight at 4°C with antibodies against the following proteins: HO-1 (ab68477; 1:2000; Abcam); NRF2 (ab92946; 1:3000; Abcam), TNF-α (SAB5700627; 1:1000; Sigma), IL-1β (AB1413-I; 1:4000; Sigma), IL-6 (SAB5700632; 1:2000; Sigma); MVH (ab13840; 1:3000; Abcam); OCT4 (ab184665; 1:2000; Abcam); Ki67 (ab16667; 1:4000; Abcam), PCNA (mAb2586; 1:3000; Cell Signaling Technology), ATM (ab199726; 1:2000; Abcam), RAD51 (ab176458; 1:3000; Abcam), and GAPDH (ab181602; 1:3000; Abcam). After washing with PBST buffer solution three times, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Beyotime Institute of Biotechnology) for 1 h at 25 °C, and the protein bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific Inc.). The blots were scanned and normalized to GAPDH for quantification.