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 was carried out as described previously and small intestine) using an RNeasy Mini Kit (Qiagen). (Peng et al. 2015a). In brief, thirty oocytes and embryos were

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Downloaded from Bioscientifica.com at 09/24/2021 08:57:36PM via free access NLRP2 interacts with FAF1 247 fixed with 4% paraformaldehyde in phosphate buffered Results saline (PBS) and permeabilized with 0.2% Triton X-100. After incubation with 3% bovine serum albumin (BSA), Expression of Faf1 in mouse tissues and oocytes and embryos were incubated with anti-NLRP2 preimplantation embryos (1:100, Abnova) and anti-FAF1 antibodies (1:100, Santa Quantitative RT-PCR showed that Faf1 mRNAs were Cruz) at 4°C overnight, washed and incubated with Alexa expressed at high levels in mouse ovary and testis and Fluor 488-labeled and 555-labeled secondary antibodies reduced in stomach (P < 0.05), to a much lesser extent (1:500, Beyotime). DAPI (Beyotime) was used to stain DNA. in the uterus and lung (Fig. 1A; P < 0.05) and were Fluorescence was finally detected using a Zeiss LSM 510 undetectable in the kidney and intestines (Fig. 1A). confocal microscope equipped with differential interference The expression pattern of FAF1 protein, as determined contrast optics (Carl Zeiss). by Western blotting, was similar to that determined by qRT-PCR assay (Fig. 1B), which showed that Faf1 Microinjection of zygotes transcripts were detectable in both mouse oocytes and preimplantation embryos (Fig. 1C). Transcripts of Faf1 Three hundred and twenty zygotes were microinjected with were slightly increased in the zygote, obviously reduced approximately 10 pL of either Nlrp2 siRNAs (2 µM, Santa Cruz) at 2-cell stage and significantly increased at 8-cell stage or Faf1 siRNAs (2 µM, Santa Cruz) in M2 medium as previously (P < 0.05). Notably, a sharp reduction in Faf1 expression described (Peng et al. 2015b). Negative control siRNA was detected from the 8-cell stage onwards (P < 0.05). (2 µM, Santa Cruz) was microinjected into one hundred and The expression of FAF1 protein remained throughout the twenty zygotes to act as a control for the siRNA experiments. blastocyst stage (Fig. 1D). Injected zygotes were cultured in KSOMaa-BSA medium in a humidified atmosphere of 5% CO2/95% air at 37°C. Some embryos were collected at the 4-cell stage and the 8-cell stage Co-localization of NLRP2 and FAF1 in oocytes and for immunofluorescence. preimplantation embryos Previous studies have revealed moderate interaction Statistical analysis between FAF1 protein and NLRP2 in humans (Kinoshita et al. 2006). If mouse NLRP2 interacts All experiments were replicated at least three times and data with FAF1 during development of the oocyte and are presented as means ± standard error of the mean (s.e.m.). preimplantation embryo, then it also follows that the Data were analyzed by one-way analysis of variance (ANOVA) two proteins will localize in an identical manner. and by the least significant difference (LSD) test using SPSS, To investigate the specific localization of NLRP2 and version 13.0 software (IBM Corp.). P values 0.05 were < FAF1, mouse oocytes and preimplantation embryos considered statistically significant. were permeabilized and stained with specific antibodies

Figure 1 Expression profiles ofFaf1 mRNAs and protein in murine tissues. (A) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) performed using total RNA extracted from the ovary (Ov), testis (Te), uterus (Ut), kidney (Ki), lung (Lu), stomach (St) and intestines (In) of 4-week-old mice. Data were normalized to expression levels in the ovary and expressed as means ± s.e.m. Bars with different superscripts are significantly different (P < 0.05). (B) Western blot of protein lysates isolated from mouse ovary (Ov), testis (Te), uterus (Ut), kidney (Ki), lung (Lu), stomach (St) and intestines (In). β-actin was used as a loading control. (C) The relative abundance of Faf1 mRNAs in mouse oocytes (Oo), 1-cell (1C) embryos, 2-cell (2C) embryos, 8-cell (8C) embryos and blastocysts (Bl). Data were normalized to expression levels in 8-cell embryos and expressed as means ± s.e.m. Bars with different superscripts are significantly different P( < 0.05). (D) Western blot of protein lysates isolated from oocytes (Oo), 1-cell (1C) embryos, 2-cell (2C) embryos, 8-cell (8C) embryos and blastocysts (Bl). β-Actin was used as a loading control. www.reproduction-online.org Reproduction (2017) 154 245–251

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Figure 3 Interaction of NLRP2 with FAF1. (A) Immunoprecipitation of Figure 2 Co-localization of NLRP2 and FAF1 proteins by confocal FAF1 protein with antibodies to NLRP2. Mouse ovaries were microscopy. Mouse oocytes, 2-cell embryos, 4-cell embryos, 8-cell immunoprecipitated with specific antibodies to NLRP2 (lane 1), embryos, morulas and blastocysts were fixed, permeabilized and anti-IgG (lane 2) or analyzed directly (lane 3). Proteins were resolved stained with specific antibodies raised against NLRP2 (green) and by SDS-PAGE and analyzed by Western blotting (WB). (B) FAF1 (red). Each sample was counterstained with DAPI to visualize Immunoprecipitation of NLRP2 protein with antibodies to FAF1. DNA (blue). Original magnification was 200. × Mouse ovaries were immunoprecipitated with specific antibodies to FAF1 (lane 1), anti-IgG (lane 2) or analyzed directly (lane 3). Proteins to NLRP2 and FAF1. Target proteins were then imaged were resolved by SDS-PAGE and analyzed by WB. by confocal microscopy using Alexa Fluor 488-labeled anti-NLRP2 secondary antibody (green) and Alexa Fluor antibodies raised against FAF1 to immunoprecipitate 555-labeled anti-FAF1 secondary antibody (red). By NLRP2 in whole ovarian lysates was also performed. merging the Alexa Fluor 488 and Alexa Fluor 555 signals, NLRP2 was detected by Western blot using anti-NLRP2 it was possible to demonstrate the co-localization of antibodies (Fig. 3B). the two proteins in both the cytoplasm and nucleus during the development of oocytes and preimplantation embryos (Fig. 2). Effect of Nlrp2 or Faf1 knockdown upon formation of the NLRP2–FAF1 complex Faf1 gene mutation leads to early embryonic death Interaction between NLRP2 and FAF1 proteins (Adham et al. 2008). Zygotes that were microinjected To further determine whether FAF1 and NLRP2 proteins with Faf1 siRNA predominantly arrested between the physically interact in the murine model, antibodies to 1-cell and 8-cell embryonic stages (Fig. 4A), while a NLRP2 and FAF1 were used in immunoprecipitation larger proportion of embryos derived from negative experiments with whole ovarian lysates. As shown control siRNA reached the blastocyst stage (Fig. 4B; in Fig. 3, FAF1 was immunoprecipitated by anti- P < 0.05). Zygotes in which Nlrp2 had been knocked NLRP2 antibodies but not anti-IgG antibody and was down showed a similar phenotype (Peng et al. 2012). also detected by Western blotting using anti-FAF1 To further investigate the effect of Nlrp2 or Faf1 antibodies (Fig. 3A). A reciprocal experiment using knockdown on the NLRP2–FAF1 complex, we collected

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Nlrp2 knockdown embryos was faint and difficult to detect, as was FAF1 immunofluorescence in Faf1- knockdown embryos (Fig. 4C and D). Furthermore, the immunofluorescence of NLRP2 in Faf1 knockdown embryos or that of FAF1 in Nlrp2 knockdown embryos was much weaker than that from embryos microinjected with negative control siRNA (Fig. 4C and D). Consequently, NLRP2–FAF1 complexes were rarely formed in Nlrp2 or Faf1 knockdown embryos, and it is for this reason that Nlrp2 and Faf1 knockdown embryos exhibited a similar phenotype and developmental fate.

Discussion Fas is a member of the family of tumor necrosis factor receptors and takes part in the process of apoptosis when activated by its ligand or by antibody crosslinking (Nagata 1994). A Fas-associated protein factor (FAF1), which interacts with the cytoplasmic domain of wild- type Fas, has been isolated by yeast two-hybrid screening methodology (Chu et al. 1995). Other experiments have shown that these two proteins also interact in a mammalian cell (Ryu & Kim 2001). The Faf1 gene is known to be located on chromosome 1 (1p32.3) in humans and chromosome 4 (4C7) in the mouse (NCBI information). Amino acid sequence analysis shows that human FAF1 protein exhibits 96% homology with the mouse FAF1 protein (data not shown), suggesting that the FAF1 protein may contain conserved domains and functions across different mammals. Nlrp2 is a maternal effect gene transcribed throughout oogenesis. However, levels of Nlrp2 mRNA are reduced rapidly upon activation of the zygotic genome in mice (Peng et al. 2012). In the present study, we detected the expression of Faf1 in a range of murine tissues, and in oocytes and embryos up to the blastocyst stage. Other experiments showed that NLRP2 and FAF1 proteins were present throughout oogenesis and the development of Figure 4 Nlrp2 or Faf1 knockdown in zygotes influences formation of preimplantation embryos. To determine the location of the NLRP2–FAF1 complex. (A) Morphology of Faf1 knockdown embryos following culture for 3.5 days. Original magnification was NLRP2 and FAF1 proteins in oocytes and preimplantation ×100. (B) Blastocyst formation rate of zygotes obtained from the embryos, confocal microscopic analyses were carried negative control siRNA (NC siRNA) and Faf1 siRNA groups following out. The results show that NLRP2 and FAF1 proteins were culture for 3.5 days. Bars with different superscripts are significantly mainly localized to both the cytoplasm and nucleus different (P < 0.05). (C) Four-cell mouse embryos obtained from the during the development of oocytes and preimplantation Faf1 siRNA group, Nlrp2 siRNA group and NC siRNA group were embryos, implying that these two proteins may fixed, permeabilized and stained with specific antibodies to NLRP2 physically interact. This distribution of NLRP2 proteins (green) and FAF1 (red). Each sample was counterstained with DAPI to is identical as determined by immunogold electron visualize DNA (blue) and original magnification was× 200. (D) Eight-cell mouse embryos derived from the Faf1 siRNA group, Nlrp2 microscopic detection (Peng et al. 2012) and differs siRNA group and NC siRNA group were fixed, permeabilized and from that of NLRP5, which interacts with FLOPED, stained with specific antibodies to NLRP2 (green) and FAF1 (red). TLE6 and FILIA and formed a SCMC (Li et al. 2008, Each sample was counterstained with DAPI to visualize DNA (blue) Ohsugi et al. 2008). This complex is mainly located in and original magnification was× 200. the subcortex of oocytes and preimplantation embryos and controls symmetric division of mouse zygotes by 4-cell and 8-cell embryos derived from Nlrp2 or Faf1 regulating F-actin dynamics (Yu et al. 2014). If NLRP2 knockdowns and carried out immunofluorescence interacts with FAF1 and formed a complex, the function analysis. The immunofluorescence of NLRP2 in of NLRP2–FAF1 complex will differ from that of SCMC, www.reproduction-online.org Reproduction (2017) 154 245–251

Downloaded from Bioscientifica.com at 09/24/2021 08:57:36PM via free access 250 H Peng, H Liu and others which provides a molecular marker of embryonic cell proteins in an effective manner and thus cause important lineages (Ohsugi et al. 2008). However, NLRP2–FAF1 development events to occur in an unregulated manner. complex may participate in intracellular function of However, the precise mechanism of NLRP2–FAF1 oocytes and preimplantation embryos. complex involved in preimplantation embryogenesis Co-immunoprecipitation assays further confirmed requires further elucidation. that the NLRP2 protein interacted with the FAF1 protein, In summary, the present study investigated the which is mainly composed of three protein-interaction expression of FAF1 protein in mouse tissues and domains, including a FAS-interacting domain (FID) preimplantation embryos and revealed evidence of a at the N-terminus, and a death effector domain- key interaction between NLRP2 and FAF1 proteins in interacting domain (DEDID) and multi-ubiquitin-related oocytes and early embryos. This observation creates a domains at the C-terminus (Menges et al. 2009). The rationale for the developmental arrest observed in either PYD and LRR domains of the NLRP2 protein are also Nlrp2 or Faf1 knockdown zygotes. Ensuing research known to participate predominantly in protein–protein should focus on the functional domains responsible for interactions. Previous research has indicated that the FID the interaction between NLRP2 and FAF1 and determine of FAF1 protein interacts with the PYD of several NLRP the specific functions of the NLRP2–FAF1 complex proteins (Kinoshita et al. 2006). It is also known that during early embryogenesis. the FID (residues 1–180) of FAF-1 protein contains an ubiquitin-associated domain (UBA, residues 1–57) and an ubiquitin-related domain (UB1, residues 99–180). Declaration of interest Previous research has shown that the N-terminal UBA The authors declare that there is no conflict of interest that domain of FAF-1, but not UB1, interacts with NLRP12 could be perceived as prejudicing the impartiality of the PYD (Pinheiro et al. 2011). Consequently, it is logical to research reported. hypothesize that the PYD of NLRP2 protein may interact with the FID or UBA of FAF1 protein. Previous study show that Faf1 gene mutation or Nlrp2 Funding knockdown in zygotes caused abnormal development of This work was supported by grants from the National Natural preimplantation embryo in the mouse (Adham et al. 2008, Science Foundation of China (grant numbers: 31402079 and Peng et al. 2012). Meanwhile, zygotes derived from faf1 31672415), FAFU Program for Distinguished Young Scholars siRNA group arrested at early embryonic stages. Thus, both (grant number: XJQ201509) and Foundation of Fujian FAF1 and NLRP2 proteins are required for early embryonic Educational Committee (grant number: JK2014013). development in the mouse. To further reveal the relation­ ship between Nlrp2 or Faf1 knockdown and the formation of NLRP2-FAF1 complex, we collected 4-cell and 8-cell Acknowledgments embryos obtained from Nlrp2 or Faf1 knockdowns and The authors thank Prof. Xu Wu for helpful discussions and carried out immunofluorescence analysis. The results Dr Song Wang and Xiaojuan Chi for their generous technical show that knockdown of the Nlrp2 or Faf1 gene in zygotes assistance. interfered with the formation of a NLRP2–FAF1 complex. Thus, it is logical to hypothesize that interaction between NLRP2 and FAF1 is probably essential for the successful References development of cleavage-stage mouse embryos. 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Seminars in P, Zhu MS et al. 2014 The subcortical maternal complex controls (grant number: XJQ201509) and Foundation of Fujian Immunology 6 3–8. (doi:10.1006/smim.1994.1002) symmetric division of mouse zygotes by regulating F-actin dynamics. Ohsugi M, Zheng P, Baibakov B, Li L & Dean J 2008 Maternally Nature Communication 5 4887. (doi:10.1038/ncomms5887) Educational Committee (grant number: JK2014013). derived FILIA-MATER complex localizes asymmetrically in cleavage- Yu C, Ji SY, Sha QQ, Sun QY & Fan HY 2015 CRL4-DCAF1 ubiquitin E3 stage mouse embryos. Development 135 259–269. (doi:10.1242/ ligase directs protein phosphatase 2A degradation to control oocyte dev.011445) meiotic maturation. Nature Communications 6 8017. 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