REPRODUCTIONRESEARCH NLRP2 and FAF1 deficiency blocks early embryogenesis in the mouse Hui Peng1,*, Haijun Liu2,*, Fang Liu1, Yuyun Gao1, Jing Chen1, Jianchao Huo1, Jinglin Han1, Tianfang Xiao1 and Wenchang Zhang1 1College of Animal Science, Fujian Agriculture and Forestry University, Fujian, Fuzhou, People’s Republic of China and 2Tianjin Institute of Animal Science and Veterinary Medicine, Tianjin, People’s Republic of China Correspondence should be addressed to W Zhang; Email: [email protected] *(H Peng and H Liu contributed equally to this work) Abstract Nlrp2 is a maternal effect gene specifically expressed by mouse ovaries; deletion of this gene from zygotes is known to result in early embryonic arrest. In the present study, we identified FAF1 protein as a specific binding partner of the NLRP2 protein in both mouse oocytes and preimplantation embryos. In addition to early embryos, both Faf1 mRNA and protein were detected in multiple tissues. NLRP2 and FAF1 proteins were co-localized to both the cytoplasm and nucleus during the development of oocytes and preimplantation embryos. Co-immunoprecipitation assays were used to confirm the specific interaction between NLRP2 and FAF1 proteins. Knockdown of the Nlrp2 or Faf1 gene in zygotes interfered with the formation of a NLRP2–FAF1 complex and led to developmental arrest during early embryogenesis. We therefore conclude that NLRP2 interacts with FAF1 under normal physiological conditions and that this interaction is probably essential for the successful development of cleavage-stage mouse embryos. Our data therefore indicated a potential role for NLRP2 in regulating early embryo development in the mouse. Reproduction (2017) 154 245–251 Introduction (Peng et al. 2012), the precise molecular mechanism underlying the role of NLRP2 in regulating early embryo During oogenesis, mammalian oocytes accumulate development still remains unclear. extensive amounts of mRNAs and proteins encoded NLRP proteins contain three conservative domains: by maternal effect genes. The selective knockout or an N-terminal pyrin domain (PYD), a central NACHT knockdown of certain maternal effect genes is known domain and a series of C-terminal leucine-rich repeats to block preimplantation embryogenesis, suggesting that (LRRs); the PYD and LRR domains are known to play these maternal proteins play crucial roles in early embryo critical roles in protein–protein interactions (Kobe & development (Christians et al. 2000, Leader et al. 2002, Kajava 2001, Eibl et al. 2012). Furthermore, within Burns et al. 2003, Payer et al. 2003, Wu et al. 2003, the NLRP family of proteins, the immunization-related Ma et al. 2006). NLRP proteins, including NLRP1, NLRP3 and NLRP Nlrp5/Mater is a member of the Nlrp family of 12, have been shown to interact with certain proteins genes, which is divided into immunization and to form specific complexes, which play central roles reproduction clades (Tian et al. 2009) and represents in the mammalian immune response (Lukens et al. one of the first maternal effect genes to be identified in 2015, Arbore et al. 2016, Zhong et al. 2016). Moreover, mammals (Tong et al. 2000). Knockout of Nlrp5 results NLRP5, a reproduction-related protein, interacts with in developmental arrest in preimplantation embryos, FLOPED, FILIA and TLE6 and assembles a subcortical and female Nlrp5-null mice are known to be infertile maternal complex (SCMC) located in the subcortex despite normal patterns of oogenesis, oocyte maturation of mouse oocytes and early embryos (Li et al. 2008, and fertilization (Tong et al. 2000). Furthermore, the Ohsugi et al. 2008). Genetic ablation of NLRP5, knockdown of Nlrp14, a gene associated with the FLOPED or TLE6 leads to the developmental failure Nlrp reproduction-clade, in mouse zygotes leads to of preimplantation embryos (Tong et al. 2000, Li et al. early embryonic arrest between the 1-cell and 8-cell 2008, Yu et al. 2014). stages (Hamatani et al. 2004). While our previous study Yeast two-hybrid assays have also shown that successfully verified that Nlrp2 is a maternal effect gene human NLRP2/PYPAF2 protein exhibits moderate required for early embryonic development in the mouse levels of interaction with FAF1 (Fas-associated protein © 2017 Society for Reproduction and Fertility DOI: 10.1530/REP-16-0629 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/24/2021 08:57:36PM via free access 10.1530/REP-16-0629 246 H Peng, H Liu and others factor) (Kinoshita et al. 2006). Another study, using a First-strand cDNA was synthesized using a PrimeScript II 1st combination of immunohistochemistry and reverse Strand cDNA Synthesis Kit (TaKaRa). Ten oocytes and embryos transcriptase polymerase chain reaction (RT-PCR), lysis, reverse transcription and qRT-PCR were performed indicated that Faf1 products can be detected in both using the Power SYBR Green Cells-to-Ct Kit (Thermo Fisher mouse oocytes and early embryos and that mutation of Scientific). The primer pairs used for qRT-PCR analysis were the Faf1 gene results in the death of Faf1GT/GT embryos as follows: Faf1, 5′-AACCTGGGCTTGGGATCTGAC-3′ (Adham et al. 2008). Taking our previous study of NLRP2 (forward), 5′-GTGCAATAACGCTGCCAAAGTG-3′ (reverse); into account, we therefore hypothesized that the NLRP2 β-actin, 5′-GAAGTGTGACGTTGACATCCG-3′ (forward), protein interacts with FAF1 to form a maternal complex 5′-ACTTGCGGTGCACGATGGAGG-3′ (reverse). Relative expression levels were normalized using the levels of that plays an essential role in the preimplantation -actin mRNA. Changes in Faf1 gene expression were development of mouse embryos. β calculated using established 2−ΔΔCT methodology (Livak & In the present study, we used the mouse model to Schmittgen 2001). investigate the expression of FAF1 and demonstrated that NLRP2 protein interacts with FAF1 in both oocytes and cleavage-stage embryos. This novel step in the Western blotting characterization of NLRP2-binding proteins indicates Western blotting was carried out as described previously a potential mechanism for NLRP2 in regulating early (Peng et al. 2014). In brief, protein samples from a range of embryo development in the mouse. mouse tissues, fifty oocytes and preimplantation embryos were homogenized and solubilized in RIPA lysis buffer (Beyotime; Jiangsu, PR China), separated by sodium dodecyl Materials and methods sulfate polyacrylamide electrophoresis (SDS-PAGE) and then Animals transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked with Tris-buffered saline All experimental procedures were approved by the Animal (TBS pH 7.4)/0.1% Tween 20 (TBS/T) containing 5% non-fat Care Commission of the College of Animal Science, Fujian milk and then incubated at 4°C overnight in TBS/T with 5% Agriculture and Forestry University. All mice were of the ICR non-fat milk containing anti-FAF1 antibody (1:200, Santa strain and were purchased from the Experimental Animal Cruz). After several washes, membranes were incubated with Center of Fujian Medical University (Fuzhou, China). Mice horseradish peroxidase-linked secondary antibody (1:2000, were provided with water and mouse-chow ad libitum and Pierce) and washed. Finally, the enhanced chemiluminescence were maintained on a 14/10 h light/darkness cycle in the (ECL) Advanced Western Blotting Detection System (Pierce) Laboratory Animal Facility of the College of Animal Science, was used to detect FAF1 protein on membranes. β-actin was Fujian Agriculture and Forestry University. used during Western blotting as a loading control (1:1000, Santa Cruz). Chemicals All chemicals and reagents were purchased from Sigma- Co-immunoprecipitation Aldrich unless stated otherwise. Co-immunoprecipitation of target proteins within ovarian lysates was performed with a Pierce Co-Immunoprecipitation Collection of oocytes and embryos Kit (26149, Pierce) in accordance with the manufacturer’s instructions. In brief, 1 mg of protein lysate was first pre- To induce super-ovulation, female mice (8–10 weeks of age) cleared by incubation with control agarose resin to minimize ICR were injected with 10 international units (IU) of pregnant non-specific binding. Protein lysates were then loaded onto mare serum gonadotrophin (PMSG) followed 48 h later by columns containing 1 µg of immobilized antibodies covalently 10 IU of human chorionic gonadotrophin (hCG). Metaphase coupled onto an amine-reactive resin and then incubated II oocytes and zygotes were then collected from the oviduct at 4°C overnight. Negative controls consisted of identical ampullae 16 h after the injection of hCG. Cumulus masses reactions with anti-IgG antibody. Resultant immunoprecipitates were removed from oocytes with hyaluronidase (1 mg/mL). were subsequently recovered by centrifugation, washed Preimplantation embryos were recovered from the oviducts or and used for Western blot analysis. Western blotting with uteri of super-ovulated female by flushing with HEPES-KSOM anti-FAF1 antibodies (1:200, Santa Cruz) or anti-NLRP2 medium (Wang et al. 2008). antibodies (1:500, Abnova, Taipei) was performed when co-immunoprecipitating with anti-NLRP2 antibodies or anti- FAF1 antibodies respectively. Ovarian lysates were loaded as Synthesis of complementary DNA (cDNA) and a positive control. quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) Immunofluorescence Total RNA was extracted from a range of tissues from 4-week-old mice (ovary, testis, uterus, kidney, lung, stomach Immunofluorescence