Figure 10. Developmental stages of Nlrp2 knockdown embryos. Percentage of embryos at different stages after being cultured for 3.5 days. doi:10.1371/journal.pone.0030344.g010 embryos (2-cell, 4-cell, 8-cell, morula and blastocysts) was performed according to previously described protocols [30]. Embryos were cultured in groups of 30–50 in microdrops (100 ml) of KSOMaa medium [68] supplemented with 4 mg/ml bovine serum albumin (KSOMaa-BSA) under liquid paraffin oil in a humidified atmosphere of 5% CO2/95% air at 37uC. Parthenogenetic Activation of Oocytes Metaphase II oocytes were subjected to parthenogenetic activation by exposure to activation medium (Calcium-free KSOMaa, 10 mM strontium chloride and 5 mg/ml cytochalasin B) for 6 h. The oocytes were washed in H-KSOM and cultured in KSOMaa-BSA. Pronuclear formation was scored and only activated embryos were used for quantitative RT–PCR, immu- noblotting and immunofluorescence. Collection of Spermatozoa Epididymides were removed from mature 12-week-old ICR male mice and punctured with hypodermic needles. The tissues were compressed to release spermatozoa into 1.5 ml polypropyl- ene centrifuge tubes; 500 ml of H-KSOM medium was added to each tube. Then the tube was centrifuged (3000 rpm, 3 min) to collect spermatozoa. Cell Culture RAW264.7 murine macrophage-like cells were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supple- mented with 10% fetal bovine serum (FBS, Gibco). Mouse D3 embryonic stem (D3 ES) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 20% FBS, 2 mM glutamine, 1% nonessential amino acids (Invitrogen, Carlsbad, CA, USA) and 0.1 mM b-mercaptoethanol. 1000 U/ml murine leukemia inhibiting factor (mLIF) was added to the culture medium to maintain ES cell pluripotency. Mouse F9 teratocarcinoma stem cells (F9 ES) were grown in DMEM supplemented with 10% FBS. Mouse D3 ES and F9 ES were Nlrp2 is Required for Early Embryonic Development plated on gelatinized multiwell tissue culture plates each containing 2 ml of medium. EMT6 mouse mammary carcinoma cells were cultured in Waymouth’s medium (Invitrogen) supple- mented with 15% FBS. All cell media were supplemented with 50 U/ml penicillin (Invitrogen) and 50 mg/ml streptomycin (Invitrogen). Cells were cultured at 37uC in a humidified 5% CO2/95% air incubator. RNA Isolation and Quantitative Real-Time RT–PCR Total RNA extracts from 4-week-old mouse tissues (ovary, uterus, testis, kidney, lung, heart, liver, brain, stomach, small intestine, muscle and spleen) and different cells (RAW264.7, D3 ES, F9 ES, EMT6, cumulus cells and spermatozoa) were purified with RNeasy Mini Kits (Qiagen, Valencia, CA, USA). cDNA synthesis was performed using PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa, Otsu, Japan). Pooled oocytes or embryos (10 per group) were lysed and first- strand cDNA directly was synthesized using SuperScriptH III CellsDirect cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s protocol. Lysis and reverse transcription were performed in the same tube. DNase I was added to eliminate genomic DNA prior to first strand synthesis. After synthesis, the first-strand cDNAs were amplified with specific primers by quantitative (q)RT–PCR. The primer pairs used for qRT–PCR are shown in Table 1. Each primer pair was confirmed by performing electrophoresis and melting temperature analysis of the PCR product to ensure its correct size and the absence of primer dimers. For negative controls, deionized water replaced cDNA in the real-time reaction tubes. The mRNA levels were quantified using SYBR Premix ExTaqTM II (TaKaRa) on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Inc., Carlsbad, CA, USA). Samples were denatured at 95uC, 1 min and then subjected to 40 cycles of amplification (95uC, 5 s; 60uC, 30 s). Each data point was the average of duplicate assays performed on three independently obtained samples and tran- script levels were calculated relative to the transcription of the housekeeping gene b-actin in every sample. Fold changes for each gene were calculated using the 22DDCT method [69]. In Situ Hybridization Ovaries from 3-week-old mice were fixed in 4% paraformalde- hyde in 0.1 M PBS for 1 h before dehydration and paraffin wax embedding and sectioning (6 mm). Hybridization was carried out with Nlrp2 ISH Detection Kits (Boster-Bio, MK-3516-m, Wuhan, China) according to the manufacturer’s instructions. The dig- oxigenin (DIG)-labeled oligonucleotide sequences used for in situ hybridization were 59–GTGACTCTCTTTATCCAAGCACTG- GGGCAGTGGTC–39 59–GGCCAGAAGTTCTGCTTCCAT- TTAGCTGTGTATCT–39 59–GGAGGGCCATGCAGCACA- ATTGCATGAGCAAATGG–39 for Nlrp2. After deparaffiniza- tion and rehydration, sections were permeabilized with proteinase K (Boster-Bio) for 30 s and washed three times in 0.1 M PBS. Then the sections were postfixed in 1% paraformaldehyde for 10 min, prehybridized for 4 h at 42uC and hybridized overnight at 42uC. After hybridization, the sections were rinsed in 26 SSC buffer for 10 min at 37uC, 0.56SSC for 15 min and twice in 0.26 SSC for 20 min at 37uC. Hybridized probes on sections were detected immunologically: sections were labeled with biotinylated anti-DIG antibody (Boster-Bio) and detected with Avidin, biotinylated peroxidase and DAB substrate solution (Boster-Bio) for 20–30 min at room temperature (RT). Control sections had the Nlrp2 probes omitted. Hybridization signals were examined using a Nikon Eclipse Ti-S microscope equipped with a 198 Nikon DS-Ri1 digital camera (Nikon, Tokyo, Japan). PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e30344