Nlrp2 , a Maternal Effect Gene Required for Early Embryonic Development in the Mouse Hui Peng 1 , Bohao Chang 1 , Chenglong Lu1, Jianmin Su 1 , Yongyan Wu1, Pin Lv1, Yongsheng Wang1, Jun Liu1, Bowei Zhang1, Fusheng Quan1, Zekun Guo 2 , Yong Zhang 1 * 1 Key Laboratory of Animal Reproductive Physiology and Embryo Technology, College of Veterinary Medicine, Northwest Agriculture and Forestry University, Yangling, Shanxi, People’s Republic of China, 2 Department of Biochemistry and Molecular Biology, College of Life Sciences, Northwest Agriculture and Forestry University, Yangling, Shanxi, People’s Republic of China Abstract Maternal effect genes encode proteins that are produced during oogenesis and play an essential role during early embryogenesis. Genetic ablation of such genes in oocytes can result in female subfertility or infertility. Here we report a newly identified maternal effect gene, Nlrp2, which plays a role in early embryogenesis in the mouse. Nlrp2 mRNAs and their proteins (,118 KDa) are expressed in oocytes and granulosa cells during folliculogenesis. The transcripts show a striking decline in early preimplantation embryos before zygotic genome activation, but the proteins remain present through to the blastocyst stage. Immunogold electron microscopy revealed that the NLRP2 protein is located in the cytoplasm, nucleus and close to nuclear pores in the oocytes, as well as in the surrounding granulosa cells. Using RNA interference, we knocked down Nlrp2 transcription specifically in mouse germinal vesicle oocytes. The knockdown oocytes could progress through the metaphase of meiosis I and emit the first polar body. However, the development of parthenogenetic embryos derived from Nlrp2 knockdown oocytes mainly blocked at the 2-cell stage. The maternal depletion of Nlrp2 in zygotes led to early embryonic arrest. In addition, overexpression of Nlrp2 in zygotes appears to lead to normal development, but increases blastomere apoptosis in blastocysts. These results provide the first evidence that Nlrp2 is a member of the mammalian maternal effect genes and required for early embryonic development in the mouse. Citation: Peng H, Chang B, Lu C, Su J, Wu Y, et al. (2012) Nlrp2, a Maternal Effect Gene Required for Early Embryonic Development in the Mouse. PLoS ONE 7(1): e30344. doi:10.1371/journal.pone.0030344 Editor: Hugh Clarke, McGill University, Canada Received October 23, 2011; Accepted December 14, 2011; Published January 25, 2012 Copyright: 2012 Peng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Key Project for Production of Transgenic Livestock, People9s Republic of China (No. 2008ZX08007-004, http:// www.most.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: zhangylab@gmail Introduction Maternal factors, which are encoded by maternal effect genes and accumulate during oogenesis, are critical to support the development of the preimplantation embryo [1]. Maternal effect genes were first described in mammals in 2000 [2,3], although some early pioneering investigations had been reported in Drosophila [4,5], Caenorhabditis elegans [6] and Xenopus [7]. Several maternal effect genes that function in oogenesis [8], meiotic maturation [9], preimplantation [8,10,11,12,13,14,15,16] and postimplantation development [17] have been identified in the mouse. However, knowledge of the maternal effect genes involved in female reproduction and early embryo development is still limited in mammals. NLRP (Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing Proteins) is a subfamily of the newly described CATERPILLER (CAspase-recruitment domain (CARD) Transcription Enhancer, R (purine)-binding, Pyrin, Lots of LEucine Repeats) family of proteins with a nucleotide-binding domain and a leucine-rich region [18,19]. The Nlrp gene family contains 20 members in the mouse. Recent studies have demonstrated that these genes play key roles in reproduction. For instance, the mouse Nlrp5 gene, known to encode for the Maternal Antigen That Embryos Require (MATER) [20], was one of the earliest maternal effect genes characterized at the molecular level in the mouse. Although showing fairly normal folliculogenesis, oocyte maturation and fertilization, Nlrp5 knockout female mice are infertile because of a block of embryogenesis at the 2-cell stage [3]. Recently a subcortical maternal complex (SCMC), constituting MATER, FLOPED, TLE6 and FILIA has been identified. This assembles during oocyte growth, [15,21]. It is essential for zygotes to progress beyond the first embryonic cell divisions. In vitro knockdown of Nlrp14 in mouse zygotes leads to developmental arrest between the 2-cell and the 8-cell stages in more than 50% of the embryos [22]. Besides Nlrp5 and Nlrp14, several other Nlrp genes also display specific or preferential oocyte expression patterns, the expression of which declines with oocyte aging [22,23]. Additionally, in studies on the oocyte-to-embryo transition in mouse, Nlrp2 (also known as Nalp2/Pan1/Pypaf2) was shown to be highly enriched in fully grown oocytes by reverse transcription polymerase chain reaction (RT–PCR) amplification [24]. Based on these limited previous studies, we hypothesized that Nlrp2 would be essential for early embryo development and that perturbing its function in the oocyte and zygote would disrupt early embryogenesis. To test this hypothesis, we employed an RNA interference (RNAi) approach to inhibit Nlrp2 function specifically in mouse germinal vesicle (GV) oocytes and zygotes. This is an efficient method for inhibiting the function of maternally and zygotically expressed genes in oocytes and zygotes [25,26,27,28]. Electropo- PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e30344