(Abcam Ab52, Cambridge, UK; 1:1000), a rabbit anti-Becn1 polyclonal antibody (Santa Curz, SC11427, CA, USA; 1:200), a mouse anti-Atg5 monoclonal antibody (A kindly gift from Professor Hans-Uwe Simon, University of Bern, Switzerland; 1:500) or a rabbit anti-LC3 (Novus Biological, NB100-2220, CO, USA; 1:1000) polyclonal antibody in TBST, then washed three times (10 minutes each) with TBST. The membranes were then incubated for 1 hour with alkaline phosphatase (AP)-conjugated anti-mouse IgG (Promega, S372B, WI, USA; 1:1000). PCNA and beta-actin protein levels were evaluated by the detection of activity of alkaline phosphatase using a Lumi-Phos kit (Pierce Biotechnol- ogy, KJ1243353). Meiotic prophase I oocyte preparation Meiotic prophase I oocyte spreads were prepared using cytospinning. Briefly, ovaries were shredded, and the released cells were spun onto slides at 800 rpm for 2 minutes by cytospin (Ttettich, MIKRO22R, Tullingen, Germany). Then the slides were incubated with 1% paraformaldehyde solution at PH 7.4 (Sangon, Shanghai, China) for 5 minutes and incubated in 70% ethanol for 10 minutes. The tissues were blocked by BDT with 0.2% Triton-X (Sangon) for 15 minutes and incubated with mouse anti-PCNA monoclonal antibody (1:100) and rabbit anti-SCP3 (A kindly gift from Professor Peter Moens, Department of Biology, York University, Canada; 1:200) overnight at 37uC. These primary antibodies were detected by incubation at 37uC for 90 minutes with the following secondary antibodies: Alexa 555 donkey anti-mouse (Molecular Probes, A21432; 1:250) and Alexa 488 donkey anti-rabbit (Molecular Probes, A21206; 1:250). At last 10 ml of vectashield mounting medium (Vector Laboratories, H-1000, CA, USA) was applied per slide, and a cover slip was sealed in place. Culture of fetal mouse ovaries The ovaries from 18.5 dpc mice were cultured as described [42]. Briefly, the ovaries without attached mesonephroses were placed on Millicell-PC membrane inserts (Millipore, PIHT12R48, MA, USA) covered with only a thin film of culture medium, and on each membrane only one ovary was placed. The medium for organ culture contained DMEM-F12 (HyClone, SH40007-13, UT, USA)/a-MEM (HyClone, SH30256.01B) (1:1) supplemented with 0.23 mM pyruvic acid (Gibco, 11360-070, CA, USA), 3% (w/v) BSA (Sigma, A-7906, MO, USA) and insulin-transferrin- selenium mix (Gibco, 41400-045), and did not contain any antibiotics. The organ cultures were maintained at 37uC and 5% CO2 in modular incubation chambers (Thermo, 3111, MA, USA). The medium was changed every 48 hours after culture initiation with replacement of half of the complete medium. RNAi on cultured fetal mouse ovaries Ovary RNAi was initiated as described [38]. Cultured ovaries were transfected with 100 nM siRNAs (Invitrogen, CA, USA) using Metafectene (Biontex, T020-5.0, Munich, Germany) following supplier’s instructions. The medium was changed at the 48th hour after RNAi initiation with replacement of half of the complete medium without siRNAs. To determine the transfection efficiency of siRNAs, ovaries were transfected with100 nM Cyanine 3 (Cy3) labeled siRNAs targeting luciferase GL2 (Dharmacon, D-1001110-01-20, TX, USA). Ovaries were harvested after transfection for 96 hours, and cryostat sectioned at 10 mm or dispersed into single cells mechanically. Single ovarian cells were dropped onto microscope slides. Cy3-labeled cells in cryostat sections and on microscope PCNA Regulates Primordial Follicles Assembly slides were quantified using a BX61 Olympus fluorescence microscope. To determine if interferon-like response was invoked by siRNA transfection in cultured ovaries, real-time PCR was used to measure mRNA content of Oas1, a classical interferon target gene [43,44], in ovaries transfected with siRNAs targeting PCNA or luciferase GL2 mRNAs. The sequence of PCNA siRNAs is 59-GGCATTGCTA- GAAATTGAGAA-39 targeting 930–950 nt of PCNA mRNA [45,46], and the nontargeting control siRNAs sequence is 59- TTCTCCGAACGTGTCACG-39 which has no homology to any known mouse mRNAs. These siRNAs were chemically synthe- sized by Invitrogen. Proliferation and apoptosis analysis To assess proliferation, 18.5 dpc mouse ovaries were cultured in the medium with 100 mM bromodeoxyuridine (BrdU) (Sigma, B9285) added 24 hours before harvesting ovaries [47]. Only the cells undergoing DNA synthesis could incorporate BrdU. The ovaries were fixed and embedded in paraffin and sectioned at 5 mm. BrdU-labeled cells in the sections were detected immuno- histochemically using a mouse anti-BrdU monoclonal antibody (Molecular Probes, A21300; 1:100) and imaged using a BX61 Olympus fluorescence microscope. Apoptotic ovarian cells were detected in sections by the TUNEL assay according to the manufacturer’s specifications. Ovarian sections were imaged and TUNEL assay positive cells were counted manually using Image- Pro plus software. Isolation of oocytes and somatic cells, RNA isolation, RT-PCR, and real-time PCR Oocytes and ovarian somatic cells were isolated from the cultured ovaries using EDTA-Stab method reported by De Felici and McLaren and a method by O W-S and Baker TG, respectively [48]. The purity of the isolated germ cells and somatic cells was assayed by detecting the mRNA level of Cyp19, a specific somatic cell marker and Figa, a specific oocyte marker [49,50]. Ovaries and oocytes were collected in RNase-free tubes and total RNAs were extracted using Trizol reagents (Invitrogen, 15596018). cDNAs were synthesized using the prime ScriptTM 1st strand cDNA synthesis Kit (Takara, D6110A, Osaka, Japan) according to the manufacturer’s protocol. Briefly, ovaries were ground sufficiently using an RNase-free grinding rod in Trizol reagent, then methyl chloroform was added to the 1/5 volume of Trizol and mixed thoroughly. The mixture was centrifugated at 14,000 g (4uC) for 15 minutes. The supernatants were transferred to new RNA-free tubes and mixed with isopropyl ketone at a ratio of 1:1, then centrifuged at 14,000 g (4uC) for 10 minutes after incubation for 10 minutes. The supernatants were removed, 100 ml 75% ethanol (diluted in DEPC water) was added to the tubes and mixed gently. After centrifugation at 7,500 g (4uC) for 5 minutes, the supernatant was removed and the tubes were air- dried at room temperature. To synthesize cDNAs, 2 ml Oligo dT primer (50 mM), 2 ml dNTP mixture (10 mM) and 5 ml RNase-free H2O were added to each tube, and the tubes were heated at 65uC for 5 minutes, then snap-cooled on ice immediately. Four ml 5xPrimeScriptTM buffer, 0.5 ml RNase inhibitor (40 U/ml), 1 ml PrimeScriptTMRTase (200 U/ml) and 4.5 ml RNase-free H2O were added to each tube, and heated for 1 hour at 42uC for cDNA extension followed by inactivation of the enzymes at 70uC for 15 minutes. The mixture was then frozen at 230uC until used. Real- time quantification of mRNAs was done using a SYBR premix Ex TaqTM II kit (Takara, DRR081A) according to the manufacturer’s protocols. Briefly, real-time PCRs were performed in a total PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e16046