were immediately downregulated and not detected after the 2-cell stage during preimplantation development (Fig. 1C). In addition, except for oocytes and cumulus cells, Nlrp2 mRNA was not detected in other cell lines (Fig. 1D), although human NLRP2 is expressed in various tumor lines [31]. NLRP2 Protein Levels Immunohistochemistry, immunofluorescence and immunoblot- ting were used to assess expression at the protein level. Immunohistochemistry of ovarian sections suggested that the NLRP2 protein was expressed in oocytes and granulosa cells at various follicular stages (Fig. 2A). After ovulation and fertilization, the protein (,118 KDa) was still present in cumulus–oocyte complexes (Fig. 2B) and remained present through the blastocyst stage (Fig. 2C) although Nlrp2 mRNA was not detected after the 2- cell stage (Fig. 1C). Cellular and Subcellular Localization of NLRP2 To determine the location of NLRP2 protein in oocytes and early embryos, we conducted confocal and immunogold electron microscopy analyses. Confocal microscopy demonstrated a predominant cytoplasmic location of the NLRP2 protein in oocytes and early embryos (Fig. 3). The distribution of NLRP2 protein in the subcellular organelles was monitored by immuno- gold staining and transmission electron microscopy. Immunogold particles were seen within the oocytes and the surrounding Nlrp2 is Required for Early Embryonic Development granulosa cells. They were mainly located in the cytoplasm, but some particles were present in the nucleus and close to nuclear pores in the oocytes (Fig. 4A), as well as the surrounding granulosa cells (Fig. 4B). Expression and Localization of Nlrp2 in Parthenogenetic Embryos The amount of Nlrp2 mRNA declined sharply after partheno- genetic activation of metaphase II oocytes (Fig. 5A). As the parthenogenetic embryos developed beyond the 2-cell stage, the transcripts virtually disappeared (Fig. 5A). However, the NLRP2 protein remained present at preimplantation stages (Fig. 5B). The protein was predominately localized in the blastomere cytoplasm (Fig. 5C). Thus, the expression profile of NLRP2 protein during embryogenesis, as determined by immunoblotting assay, was similar to that determined by confocal microscopic analysis. Nlrp2 Is Not Required for Oocyte Maturation Zona-weakened germinal vesicle (GV)-stage oocytes were electroporated with custom-made Nlrp2 siRNA mixture. As shown in Figure 6A, Nlrp2 siRNA had no obvious effect on oocyte maturation. However, the Nlrp2 gene was downregulated in a dose-dependent manner (Fig. 6B). The Nlrp2 knockdown effects were specific as the expressions of Nlrp4f, Nlrp5, Nlrp9c and Nlrp14, randomly selected members of the Nlrp gene family associated with reproduction and embryo development, were not altered by the Figure 2. Developmental expression of NLRP2 protein in mouse. (A) Immunohistochemical analysis of sequential sections from a 3-week-old mouse ovary using an anti-NLRP2 antibody. The original magnification was 640. (B) Immunofluorescent detection of NLRP2 in cumulus–oocyte complexes after permeabilization and incubation with an anti-NLRP2 antibody. The original magnification was 6100. (C) Immunoblots of lysates isolated from oocytes and preimplantation embryos. Molecular masses (KDa) are indicated on the left; b-actin was used as a control. doi:10.1371/journal.pone.0030344.g002 PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e30344