Nlrp2 is Required for Early Embryonic Development Figure 6. GV-stage oocyte maturation after electroporation with Nlrp2 siRNA. (A) Oocyte maturation rate following electroporation (EP) of GV-stage oocytes in the presence or absence of control and Nlrp2 siRNA. The numbers on top of each bar indicate the numbers of oocyte matured/ numbers of oocytes electroporated. (B) The relative abundance of Nlrp2 transcripts after electroporation with Nlrp2 siRNA. The data have been normalized to untreated oocytes. Statistical comparisons were made using ANOVA and LSD tests (* p,0.05). (C) Nlrp2, Nlrp4f, Nlrp5, Nlrp9c and Nlrp14 gene expression by qRT–PCR in mouse oocytes at 24 h after electroporation with Nlrp2 siRNA (60 nM). Results were normalized to control siRNA (100 nM) group. * p,0.05. (D) Immunoblots of mouse oocytes at 24 h after electroporation in the presence or absence of control and Nlrp2 siRNA. doi:10.1371/journal.pone.0030344.g006 cell stage arrest of embryos derived from Nlrp2 knockdown zygotes suggests that this maternal factor functions in early embryonic development. Previous studies also indicated that targeted invalidation/depletion of Nlrp5 and Nlrp14 does not reveal any gene compensation. In addition, microinjection with the pIRES2- Nlrp2 vector into zygotes did not affect subsequent cell fate and differentiation in terms of the expression and localization of OCT3/4 or CDX2. However, overexpression of Nlrp2 increased the incidence of apoptosis in blastocysts, implying that an excessive accumulation of NLRP2 protein might impair the viability of blastomeres or activate the pathway of cell apoptosis, because NLRP2 contains a pyridine domain at the amino terminal that is primarily involved in apoptosis [55,56,57]. There are several reproduction-related Nlrp genes with conserved functions in mammals and primates. Nlrp5 (NLRP5) transcripts are detected in the ovary in the mouse [3,20], bovine [53,58,59], pig [60], rhesus macaque monkeys [28,48] and humans [61]. Moreover it has been demonstrated that Nlrp5 plays key roles in early embryo development in the mouse [3] and rhesus macaque monkeys [28]. Nlrp14 is continuously expressing in germ cells [62] and in vitro knockdown experiments in zygotes led to developmental arrest in the mouse [22]. Moreover mutations in the testis-specific NLRP14 gene in men leads to spermatogenic failure [63], implying that these genes are conserved functionally in a wide range of mammalian reproductive systems. Germline mutations in NLRP2 result in a familial imprinting disorder (Beckwith–Wiedemann Syndrome) in humans [64], suggesting that this gene might have a similar function in the establishment and/or maintenance of genomic imprinting/methylation in the mouse. The Dnmt1o mutation causes disrupted genomic imprinting in early mouse embryogenesis [17]. Moreover, a gene disruption study determined that PGC7/Stella is indispensable for the maintenance of methylation involved in epigenetic reprogram- ming after fertilization [11,65,66]. Thus, the function of Nlrp2 might be similar to the maternal effect gene Dnmt1o and/or PGC7/ Stella during early preimplantation development. However, the PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e30344