Nlrp2 is Required for Early Embryonic Development Figure 8. Nlrp2 mRNA and protein levels in Nlrp2 knockdown embryos. (A) The relative abundance of Nlrp2 transcripts in mouse embryos collected at 2 h, 11 h, 20 h, 29 h and 38 h after electropora- tion. Results have been normalized to the abundance in untreated zygotes and are expressed as the mean 6 SEM. (B) Immunoblots of mouse embryos at 4 h (1-cell), 28 h (2-cell) and 52 h (8-cell) after electroporation. doi:10.1371/journal.pone.0030344.g008 purchased from the Experimental Animal Center of The Fourth Military Medical University (Xi’an, China). They were maintained on a 14/10 h light/dark cycle with free access to food and water in the Laboratory Animal Facility of the College of Veterinary Medicine, Northwest A&F University. Chemicals All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, USA) unless stated otherwise. Sterile plastic ware was purchased from Nunclon (Roskilde, Denmark). Collection and Culture of Oocytes and Embryos Oocytes at the GV stage were obtained from the ovaries of 8– 10-week-old females injected with 10 IU of pregnant mare serum gonadotrophin (PMSG) to stimulate the growth of the follicles. Forty-eight hours after PMSG administration the ovaries were placed in Hepes-buffered KSOM medium (H-KSOM) [67] containing 0.1 mM 3-isobutyl-1-methyl-xanthine to inhibit re- sumption of meiosis and oocytes were released from the largest follicles by puncturing them with hypodermic needle. The oocytes were completely freed of attached cumulus cells by repeated mouth pipetting. Figure 9. Morphology of Nlrp2 knockdown embryos after being cultured for 3.5 days. Morphology (left) and DAPI staining (right). The original magnification was 6100. doi:10.1371/journal.pone.0030344.g009 Mature oocytes arrested in metaphase II were recovered from mice superovulated with intraperitoneal injections of PMSG and human chorionic gonadotrophin (hCG) 48 h apart. Metaphase II oocytes were collected from the oviduct ampullae at 16 h after the hCG injection. Cumulus masses were treated with hyaluronidase (1 mg/ml) to release ova. Metaphase II oocytes and cumulus cells were collected separately. Zygotes (1-cell embryos) were obtained from females induced to superovulate as described above and mated with males immedi- ately after hCG injection. Zygotes were collected from the oviducts 14 h after hCG injection. Cumulus cells surrounding zygotes were removed by brief exposure to hyaluronidase. Collection of PLoS ONE | www.plosone.org 9 January 2012 | Volume 7 | Issue 1 | e30344