Nlrp2 is Required for Early Embryonic Development Figure 5. Developmental expression and localization of Nlrp2 in parthenogenetic embryos. (A) The relative abundance of Nlrp2 transcripts in mouse oocytes and parthenogenetic embryos. (B) Immunoblots of lysates isolated from parthenogenetic embryos. (C) Confocal microscopic images of parthenogenetic embryos. Each sample was counterstained with DAPI to visualize DNA (blue). The original magnification was 6200. doi:10.1371/journal.pone.0030344.g005 embryos, Nlrp2 mRNA was undetectable, but the protein persisted through to the blastocyst stage. The expression patterns of Nlrp2 mRNA and its protein in parthenogenetic embryos were identical to those in normally fertilized embryos, suggesting that Nlrp2 might be involved in two different developmental processes, but fulfill the same function. The NLRP2 protein was distributed predominantly in the cytoplasm both in normal fertilized embryos and parthenogenetic embryos as shown by confocal microscopy. Immunogold electron microscopy demonstrated that the protein was present in the cytoplasm and close to nuclear pore complexes. In addition, immunogold particles were detected within the nucleus. However, the NLRP2 sequence does not display a nuclear localization signal. It is likely that other proteins interacting with NLRP2 mediate its transport into the nucleus; indeed, this protein contains leucine- rich repeat domains, which are known to participate in protein– protein interactions [49,50,51]. The subcellular localization of NLRP2 protein differs from NLRP5 (MATER) in mouse [52] and bovine oocytes [53]. Such localization of NLRP2 in multiple organelles suggests its participation in different intracellular functions. Mouse embryos with mutations in maternal effect genes are characterized by developmental arrest during oogenesis or embryogenesis. Among the known maternal effect genes, only Formin-22/2 and basonuclin-deficient oocytes cannot undergo normal maturation [8,9]. Compared with Formin-2 and basonuclin-deficient oocytes, Nlrp2 knockdown GV-stage oocytes could reach metaphase II and emit the first polar body, suggesting that Nlrp2 is not required for the metaphase spindle positioning or migration. Moreover, Nlrp2 deficiency in oocytes does not perturb RNA polymerase I- or -II-mediated transcription, as the disruption of these processes leads to abnormal oocyte maturation [8,9,54]. However, the parthenogenetic embryos derived from Nlrp2 knockdown oocytes were mainly arrested at the 2-cell stage. Interestingly, the expression levels of Nlrp4f, Nlrp5, Nlrp9c and Nlrp14 were not affected in Nlrp2 knockdown oocytes, suggesting that the expression of other Nlrp genes was not able to compensate for the absence of Nlrp2. Furthermore, the predominant 2- and 8- PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e30344