Figure 3. Cellular localization of NLRP2 protein. Confocal microscopic images of oocytes and preimplantation embryos. Each sample was counterstained with DAPI to visualize DNA (blue). The original magnification was 6200. doi:10.1371/journal.pone.0030344.g003 introduction of Nlrp2 siRNA (60 nM) (Fig. 6C). Moreover, analysis of NLRP2 protein levels by immunoblotting showed that this obviously decreased in oocytes 24 h after electroporation with Nlrp2 siRNA compared with control groups (Fig. 6D). Thus, the decline of Nlrp2 mRNA and protein in oocytes after electropora- tion with Nlrp2 siRNA did not affect oocyte maturation. Nlrp2 is Required for Early Embryonic Development Nlrp2 Knockdown in Oocytes Leads to Early Embryonic Arrest After Parthenogenetic Activation Metaphase II oocytes (after extrusion of the first polar body) derived from oocytes electroporated at the GV stage were subjected to parthenogenetic activation and then cultured in KSOMaa-BSA medium (see Materials and Methods). There was no significant difference in the parthenogenetic activation rate of the oocytes obtained from the EP control, control siRNA or Nlrp2 siRNA (20, 40 and 60 nM) groups (Fig. 7A). The embryonic developmental stages and their morphological appearance after being cultured for 3.5 days are presented in Figure 7B. The parthenogenetic embryos derived from the electroporation (EP) control and control siRNA reached the blastocyst stage at rates of 65% and 63%, respectively (Fig. 7C). However, the development of parthenogenetic embryos derived from Nlrp2 siRNA-electropo- rated oocytes appeared to be blocked mainly at the 2-cell stage (Fig. 7C). Nlrp2 Knockdown in Zygotes Causes Early Embryonic Arrest To further confirm the function of Nlrp2 in early embryonic development, the zygotes were electroporated with Nlrp2 siRNA. As shown in Figure 8A, the introduction of Nlrp2 siRNA resulted in the reduction of the targeted mRNA in time- and concentra- tion-dependent manners. Moreover, it also led to a decrease in the NLRP2 protein level compared with control groups (Fig. 8B). Morphological analysis suggested that embryos derived from Nlrp2 siRNA-electroporated zygotes appeared to be arrested, while the embryos derived from control groups reached the blastocyst stage (Fig. 9). The Nlrp2 knockdown embryos were mainly arrested between the 2- and 8-cell stages (Fig. 10). Overexpression of Nlrp2 in Zygotes Leads to Normal Development but Increased Apoptosis As described above, the knockdown of Nlrp2 with target-specific siRNA in oocytes or zygotes led to early developmental arrest. However, the effect of overexpression in zygotes is unknown. To determine this, zygotes were microinjected with pIRES2-AcGFP1- Nuc-Nlrp2 (pIRES2-Nlrp2), a bicistronic vector independently expressing NLRP2 and green fluorescent protein (GFP) (see Materials and Methods). Zygotes microinjected with pIRES2- Nlrp2 or pIRES2-AcGFP1-Nuc (pIRES2) reached the blastocyst stage and expressed GFP (Fig. 11A). The rates of blastocyst formation were similar between the two groups (Fig. 11B). As shown in Figure 11C, the Nlrp2 transcripts were reduced dramatically and were undetectable after the 2-cell stage in the control group. However, the expression of Nlrp2 persisted during preimplantation development in the group microinjected with pIRES2-Nlrp2. As expected, the level of NLRP2 protein in the pIRES2-Nlrp2-microinjected group gradually increased during preimplantation stages compared with the control group (Fig. 11D). To confirm whether accumulation of NLRP2 protein would influence cell fate and differentiation in blastocysts, we investigated the expression levels of OCT3/4 and CDX2, markers of pluripotency and trophectoderm differentiation [32,33,34,35], respectively. The final cell fate and differentiation was not affected with regard to the expression of these markers (Fig. 12A). We also examined whether accumulation of NLRP2 protein would lead to apoptosis in blastocysts. We found that the number of apoptotic blastomeres in the pIRES2-Nlrp2 microinjected group was significantly increased compared with the control group (Fig. 12B, C). PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e30344