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.
Introduction
During oogenesis, mammalian oocytes accumulate extensive amounts of mRNAs and proteins encoded by maternal effect genes. The selective knockout or knockdown of certain maternal effect genes is known to block preimplantation embryogenesis, suggesting that these maternal proteins play crucial roles in early embryo development (Christians et al. 2000, Leader et al. 2002, Burns et al. 2003, Payer et al. 2003, Wu et al. 2003, Ma et al. 2006).
Nlrp5/Mater is a member of the Nlrp family of genes, which is divided into immunization and reproduction clades (Tian et al. 2009) and represents one of the first maternal effect genes to be identified in mammals (Tong et al. 2000). Knockout of Nlrp5 results in developmental arrest in preimplantation embryos, and female Nlrp5-null mice are known to be infertile despite normal patterns of oogenesis, oocyte maturation and fertilization (Tong et al. 2000). Furthermore, the knockdown of Nlrp14, a gene associated with the Nlrp reproduction-clade, in mouse zygotes leads to early embryonic arrest between the 1-cell and 8-cell stages (Hamatani et al. 2004). While our previous study successfully verified that Nlrp2 is a maternal effect gene required for early embryonic development in the mouse (Peng et al. 2012), the precise molecular mechanism underlying the role of NLRP2 in regulating early embryo development still remains unclear.
NLRP proteins contain three conservative domains: an N-terminal pyrin domain (PYD), a central NACHT domain and a series of C-terminal leucine-rich repeats (LRRs); the PYD and LRR domains are known to play critical roles in protein–protein interactions (Kobe & Kajava 2001, Eibl et al. 2012). Furthermore, within the NLRP family of proteins, the immunization-related NLRP proteins, including NLRP1, NLRP3 and NLRP 12, have been shown to interact with certain proteins to form specific complexes, which play central roles in the mammalian immune response (Lukens et al. 2015, Arbore et al. 2016, Zhong et al. 2016). Moreover, NLRP5, a reproduction-related protein, interacts with FLOPED, FILIA and TLE6 and assembles a subcortical maternal complex (SCMC) located in the subcortex of mouse oocytes and early embryos (Li et al. 2008, Ohsugi et al. 2008). Genetic ablation of NLRP5, FLOPED or TLE6 leads to the developmental failure of preimplantation embryos (Tong et al. 2000, Li et al. 2008, Yu et al. 2014).
Yeast two-hybrid assays have also shown that human NLRP2/PYPAF2 protein exhibits moderate levels of interaction with FAF1 (Fas-associated protein factor) (Kinoshita et al. 2006). Another study, using a combination of immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), indicated that Faf1 products can be detected in both mouse oocytes and early embryos and that mutation of the Faf1 gene results in the death of Faf1GT/GT embryos (Adham et al. 2008). Taking our previous study of NLRP2 into account, we therefore hypothesized that the NLRP2 protein interacts with FAF1 to form a maternal complex that plays an essential role in the preimplantation development of mouse embryos.
In the present study, we used the mouse model to 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 characterization of NLRP2-binding proteins indicates a potential mechanism for NLRP2 in regulating early embryo development in the mouse.
Materials and methods
Animals
All experimental procedures were approved by the Animal Care Commission of the College of Animal Science, Fujian Agriculture and Forestry University. All mice were of the ICR strain and were purchased from the Experimental Animal Center of Fujian Medical University (Fuzhou, China). Mice were provided with water and mouse-chow ad libitum and were maintained on a 14/10 h light/darkness cycle in the Laboratory Animal Facility of the College of Animal Science, Fujian Agriculture and Forestry University.
Chemicals
All chemicals and reagents were purchased from Sigma-Aldrich unless stated otherwise.
Collection of oocytes and embryos
To induce super-ovulation, female mice (8–10 weeks of age) ICR were injected with 10 international units (IU) of pregnant mare serum gonadotrophin (PMSG) followed 48 h later by 10 IU of human chorionic gonadotrophin (hCG). Metaphase II oocytes and zygotes were then collected from the oviduct ampullae 16 h after the injection of hCG. Cumulus masses were removed from oocytes with hyaluronidase (1 mg/mL). Preimplantation embryos were recovered from the oviducts or uteri of super-ovulated female by flushing with HEPES-KSOM medium (Wang et al. 2008).
Synthesis of complementary DNA (cDNA) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)
Total RNA was extracted from a range of tissues from 4-week-old mice (ovary, testis, uterus, kidney, lung, stomach and small intestine) using an RNeasy Mini Kit (Qiagen). First-strand cDNA was synthesized using a PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa). Ten oocytes and embryos lysis, reverse transcription and qRT-PCR were performed using the Power SYBR Green Cells-to-Ct Kit (Thermo Fisher Scientific). The primer pairs used for qRT-PCR analysis were as follows: Faf1, 5′-AACCTGGGCTTGGGATCTGAC-3′ (forward), 5′-GTGCAATAACGCTGCCAAAGTG-3′ (reverse); β-actin, 5′-GAAGTGTGACGTTGACATCCG-3′ (forward), 5′-ACTTGCGGTGCACGATGGAGG-3′ (reverse). Relative expression levels were normalized using the levels of β-actin mRNA. Changes in Faf1 gene expression were calculated using established 2−ΔΔCT methodology (Livak & Schmittgen 2001).
Western blotting
Western blotting was carried out as described previously (Peng et al. 2014). In brief, protein samples from a range of mouse tissues, fifty oocytes and preimplantation embryos were homogenized and solubilized in RIPA lysis buffer (Beyotime; Jiangsu, PR China), separated by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were blocked with Tris-buffered saline (TBS pH 7.4)/0.1% Tween 20 (TBS/T) containing 5% non-fat milk and then incubated at 4°C overnight in TBS/T with 5% non-fat milk containing anti-FAF1 antibody (1:200, Santa Cruz). After several washes, membranes were incubated with horseradish peroxidase-linked secondary antibody (1:2000, Pierce) and washed. Finally, the enhanced chemiluminescence (ECL) Advanced Western Blotting Detection System (Pierce) was used to detect FAF1 protein on membranes. β-actin was used during Western blotting as a loading control (1:1000, Santa Cruz).
Co-immunoprecipitation
Co-immunoprecipitation of target proteins within ovarian lysates was performed with a Pierce Co-Immunoprecipitation Kit (26149, Pierce) in accordance with the manufacturer’s instructions. In brief, 1 mg of protein lysate was first pre-cleared by incubation with control agarose resin to minimize non-specific binding. Protein lysates were then loaded onto columns containing 1 µg of immobilized antibodies covalently coupled onto an amine-reactive resin and then incubated at 4°C overnight. Negative controls consisted of identical reactions with anti-IgG antibody. Resultant immunoprecipitates were subsequently recovered by centrifugation, washed and used for Western blot analysis. Western blotting with anti-FAF1 antibodies (1:200, Santa Cruz) or anti-NLRP2 antibodies (1:500, Abnova, Taipei) was performed when co-immunoprecipitating with anti-NLRP2 antibodies or anti-FAF1 antibodies respectively. Ovarian lysates were loaded as a positive control.
Immunofluorescence
Immunofluorescence was carried out as described previously (Peng et al. 2015a). In brief, thirty oocytes and embryos were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) and permeabilized with 0.2% Triton X-100. After incubation with 3% bovine serum albumin (BSA), oocytes and embryos were incubated with anti-NLRP2 (1:100, Abnova) and anti-FAF1 antibodies (1:100, Santa Cruz) at 4°C overnight, washed and incubated with Alexa Fluor 488-labeled and 555-labeled secondary antibodies (1:500, Beyotime). DAPI (Beyotime) was used to stain DNA. Fluorescence was finally detected using a Zeiss LSM 510 confocal microscope equipped with differential interference contrast optics (Carl Zeiss).
Microinjection of zygotes
Three hundred and twenty zygotes were microinjected with approximately 10 pL of either Nlrp2 siRNAs (2 µM, Santa Cruz) or Faf1 siRNAs (2 µM, Santa Cruz) in M2 medium as previously described (Peng et al. 2015b). Negative control siRNA (2 µM, Santa Cruz) was microinjected into one hundred and twenty zygotes to act as a control for the siRNA experiments. 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 for immunofluorescence.
Statistical analysis
All experiments were replicated at least three times and data are presented as means ± standard error of the mean (s.e.m.). Data were analyzed by one-way analysis of variance (ANOVA) and by the least significant difference (LSD) test using SPSS, version 13.0 software (IBM Corp.). P values <0.05 were considered statistically significant.
Results
Expression of Faf1 in mouse tissues and preimplantation embryos
Quantitative RT-PCR showed that Faf1 mRNAs were expressed at high levels in mouse ovary and testis and reduced in stomach (P < 0.05), to a much lesser extent in the uterus and lung (Fig. 1A; P < 0.05) and were undetectable in the kidney and intestines (Fig. 1A). The expression pattern of FAF1 protein, as determined by Western blotting, was similar to that determined by qRT-PCR assay (Fig. 1B), which showed that Faf1 transcripts were detectable in both mouse oocytes and preimplantation embryos (Fig. 1C). Transcripts of Faf1 were slightly increased in the zygote, obviously reduced at 2-cell stage and significantly increased at 8-cell stage (P < 0.05). Notably, a sharp reduction in Faf1 expression was detected from the 8-cell stage onwards (P < 0.05). The expression of FAF1 protein remained throughout the blastocyst stage (Fig. 1D).
Expression profiles of Faf1 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.
Citation: Reproduction 154, 3; 10.1530/REP-16-0629
Expression profiles of Faf1 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.
Citation: Reproduction 154, 3; 10.1530/REP-16-0629
Expression profiles of Faf1 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.
Citation: Reproduction 154, 3; 10.1530/REP-16-0629
Co-localization of NLRP2 and FAF1 in oocytes and preimplantation embryos
Previous studies have revealed moderate interaction between FAF1 protein and NLRP2 in humans (Kinoshita et al. 2006). If mouse NLRP2 interacts with FAF1 during development of the oocyte and preimplantation embryo, then it also follows that the two proteins will localize in an identical manner.
To investigate the specific localization of NLRP2 and FAF1, mouse oocytes and preimplantation embryos were permeabilized and stained with specific antibodies to NLRP2 and FAF1. Target proteins were then imaged by confocal microscopy using Alexa Fluor 488-labeled anti-NLRP2 secondary antibody (green) and Alexa Fluor 555-labeled anti-FAF1 secondary antibody (red). By merging the Alexa Fluor 488 and Alexa Fluor 555 signals, it was possible to demonstrate the co-localization of the two proteins in both the cytoplasm and nucleus during the development of oocytes and preimplantation embryos (Fig. 2).
Co-localization of NLRP2 and FAF1 proteins by confocal microscopy. Mouse oocytes, 2-cell embryos, 4-cell embryos, 8-cell embryos, morulas and blastocysts were fixed, permeabilized and stained with specific antibodies raised against NLRP2 (green) and FAF1 (red). Each sample was counterstained with DAPI to visualize DNA (blue). Original magnification was ×200.
Citation: Reproduction 154, 3; 10.1530/REP-16-0629
Co-localization of NLRP2 and FAF1 proteins by confocal microscopy. Mouse oocytes, 2-cell embryos, 4-cell embryos, 8-cell embryos, morulas and blastocysts were fixed, permeabilized and stained with specific antibodies raised against NLRP2 (green) and FAF1 (red). Each sample was counterstained with DAPI to visualize DNA (blue). Original magnification was ×200.
Citation: Reproduction 154, 3; 10.1530/REP-16-0629
Co-localization of NLRP2 and FAF1 proteins by confocal microscopy. Mouse oocytes, 2-cell embryos, 4-cell embryos, 8-cell embryos, morulas and blastocysts were fixed, permeabilized and stained with specific antibodies raised against NLRP2 (green) and FAF1 (red). Each sample was counterstained with DAPI to visualize DNA (blue). Original magnification was ×200.
Citation: Reproduction 154, 3; 10.1530/REP-16-0629
Interaction between NLRP2 and FAF1 proteins
To further determine whether FAF1 and NLRP2 proteins physically interact in the murine model, antibodies to NLRP2 and FAF1 were used in immunoprecipitation experiments with whole ovarian lysates. As shown in Fig. 3, FAF1 was immunoprecipitated by anti-NLRP2 antibodies but not anti-IgG antibody and was also detected by Western blotting using anti-FAF1 antibodies (Fig. 3A). A reciprocal experiment using antibodies raised against FAF1 to immunoprecipitate NLRP2 in whole ovarian lysates was also performed. NLRP2 was detected by Western blot using anti-NLRP2 antibodies (Fig. 3B).
Interaction of NLRP2 with FAF1. (A) Immunoprecipitation of FAF1 protein with antibodies to NLRP2. Mouse ovaries were immunoprecipitated with specific antibodies to NLRP2 (lane 1), anti-IgG (lane 2) or analyzed directly (lane 3). Proteins were resolved by SDS-PAGE and analyzed by Western blotting (WB). (B) Immunoprecipitation of NLRP2 protein with antibodies to FAF1. Mouse ovaries were immunoprecipitated with specific antibodies to FAF1 (lane 1), anti-IgG (lane 2) or analyzed directly (lane 3). Proteins were resolved by SDS-PAGE and analyzed by WB.
Citation: Reproduction 154, 3; 10.1530/REP-16-0629
Interaction of NLRP2 with FAF1. (A) Immunoprecipitation of FAF1 protein with antibodies to NLRP2. Mouse ovaries were immunoprecipitated with specific antibodies to NLRP2 (lane 1), anti-IgG (lane 2) or analyzed directly (lane 3). Proteins were resolved by SDS-PAGE and analyzed by Western blotting (WB). (B) Immunoprecipitation of NLRP2 protein with antibodies to FAF1. Mouse ovaries were immunoprecipitated with specific antibodies to FAF1 (lane 1), anti-IgG (lane 2) or analyzed directly (lane 3). Proteins were resolved by SDS-PAGE and analyzed by WB.
Citation: Reproduction 154, 3; 10.1530/REP-16-0629
Interaction of NLRP2 with FAF1. (A) Immunoprecipitation of FAF1 protein with antibodies to NLRP2. Mouse ovaries were immunoprecipitated with specific antibodies to NLRP2 (lane 1), anti-IgG (lane 2) or analyzed directly (lane 3). Proteins were resolved by SDS-PAGE and analyzed by Western blotting (WB). (B) Immunoprecipitation of NLRP2 protein with antibodies to FAF1. Mouse ovaries were immunoprecipitated with specific antibodies to FAF1 (lane 1), anti-IgG (lane 2) or analyzed directly (lane 3). Proteins were resolved by SDS-PAGE and analyzed by WB.
Citation: Reproduction 154, 3; 10.1530/REP-16-0629
Effect of Nlrp2 or Faf1 knockdown upon formation of the NLRP2–FAF1 complex
Faf1 gene mutation leads to early embryonic death (Adham et al. 2008). Zygotes that were microinjected with Faf1 siRNA predominantly arrested between the 1-cell and 8-cell embryonic stages (Fig. 4A), while a larger proportion of embryos derived from negative control siRNA reached the blastocyst stage (Fig. 4B; P < 0.05). Zygotes in which Nlrp2 had been knocked down showed a similar phenotype (Peng et al. 2012).
Nlrp2 or Faf1 knockdown in zygotes influences formation of the NLRP2–FAF1 complex. (A) Morphology of Faf1 knockdown embryos following culture for 3.5 days. Original magnification was ×100. (B) Blastocyst formation rate of zygotes obtained from the negative control siRNA (NC siRNA) and Faf1 siRNA groups following culture for 3.5 days. Bars with different superscripts are significantly different (P < 0.05). (C) Four-cell mouse embryos obtained from the Faf1 siRNA group, Nlrp2 siRNA group and NC siRNA group were fixed, permeabilized and stained with specific antibodies to NLRP2 (green) and FAF1 (red). Each sample was counterstained with DAPI to visualize DNA (blue) and original magnification was ×200. (D) Eight-cell mouse embryos derived from the Faf1 siRNA group, Nlrp2 siRNA group and NC siRNA group were fixed, permeabilized and stained with specific antibodies to NLRP2 (green) and FAF1 (red). Each sample was counterstained with DAPI to visualize DNA (blue) and original magnification was ×200.
Citation: Reproduction 154, 3; 10.1530/REP-16-0629
Nlrp2 or Faf1 knockdown in zygotes influences formation of the NLRP2–FAF1 complex. (A) Morphology of Faf1 knockdown embryos following culture for 3.5 days. Original magnification was ×100. (B) Blastocyst formation rate of zygotes obtained from the negative control siRNA (NC siRNA) and Faf1 siRNA groups following culture for 3.5 days. Bars with different superscripts are significantly different (P < 0.05). (C) Four-cell mouse embryos obtained from the Faf1 siRNA group, Nlrp2 siRNA group and NC siRNA group were fixed, permeabilized and stained with specific antibodies to NLRP2 (green) and FAF1 (red). Each sample was counterstained with DAPI to visualize DNA (blue) and original magnification was ×200. (D) Eight-cell mouse embryos derived from the Faf1 siRNA group, Nlrp2 siRNA group and NC siRNA group were fixed, permeabilized and stained with specific antibodies to NLRP2 (green) and FAF1 (red). Each sample was counterstained with DAPI to visualize DNA (blue) and original magnification was ×200.
Citation: Reproduction 154, 3; 10.1530/REP-16-0629
Nlrp2 or Faf1 knockdown in zygotes influences formation of the NLRP2–FAF1 complex. (A) Morphology of Faf1 knockdown embryos following culture for 3.5 days. Original magnification was ×100. (B) Blastocyst formation rate of zygotes obtained from the negative control siRNA (NC siRNA) and Faf1 siRNA groups following culture for 3.5 days. Bars with different superscripts are significantly different (P < 0.05). (C) Four-cell mouse embryos obtained from the Faf1 siRNA group, Nlrp2 siRNA group and NC siRNA group were fixed, permeabilized and stained with specific antibodies to NLRP2 (green) and FAF1 (red). Each sample was counterstained with DAPI to visualize DNA (blue) and original magnification was ×200. (D) Eight-cell mouse embryos derived from the Faf1 siRNA group, Nlrp2 siRNA group and NC siRNA group were fixed, permeabilized and stained with specific antibodies to NLRP2 (green) and FAF1 (red). Each sample was counterstained with DAPI to visualize DNA (blue) and original magnification was ×200.
Citation: Reproduction 154, 3; 10.1530/REP-16-0629
To further investigate the effect of Nlrp2 or Faf1 knockdown on the NLRP2–FAF1 complex, we collected 4-cell and 8-cell embryos derived from Nlrp2 or Faf1 knockdowns and carried out immunofluorescence analysis. The immunofluorescence of NLRP2 in 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 preimplantation embryos. To determine the location of NLRP2 and FAF1 proteins in oocytes and preimplantation embryos, confocal microscopic analyses were carried out. The results show that NLRP2 and FAF1 proteins were mainly localized to both the cytoplasm and nucleus during the development of oocytes and preimplantation embryos, implying that these two proteins may physically interact. This distribution of NLRP2 proteins is identical as determined by immunogold electron microscopic detection (Peng et al. 2012) and differs from that of NLRP5, which interacts with FLOPED, TLE6 and FILIA and formed a SCMC (Li et al. 2008, Ohsugi et al. 2008). This complex is mainly located in the subcortex of oocytes and preimplantation embryos and controls symmetric division of mouse zygotes by regulating F-actin dynamics (Yu et al. 2014). If NLRP2 interacts with FAF1 and formed a complex, the function of NLRP2–FAF1 complex will differ from that of SCMC, which provides a molecular marker of embryonic cell lineages (Ohsugi et al. 2008). However, NLRP2–FAF1 complex may participate in intracellular function of oocytes and preimplantation embryos.
Co-immunoprecipitation assays further confirmed that the NLRP2 protein interacted with the FAF1 protein, which is mainly composed of three protein-interaction domains, including a FAS-interacting domain (FID) at the N-terminus, and a death effector domain-interacting domain (DEDID) and multi-ubiquitin-related domains at the C-terminus (Menges et al. 2009). The PYD and LRR domains of the NLRP2 protein are also known to participate predominantly in protein–protein interactions. Previous research has indicated that the FID of FAF1 protein interacts with the PYD of several NLRP proteins (Kinoshita et al. 2006). It is also known that 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). Previous research has shown that the N-terminal UBA domain of FAF-1, but not UB1, interacts with NLRP12 PYD (Pinheiro et al. 2011). Consequently, it is logical to 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 knockdown in zygotes caused abnormal development of preimplantation embryo in the mouse (Adham et al. 2008, Peng et al. 2012). Meanwhile, zygotes derived from faf1 siRNA group arrested at early embryonic stages. Thus, both FAF1 and NLRP2 proteins are required for early embryonic development in the mouse. To further reveal the relationship between Nlrp2 or Faf1 knockdown and the formation of NLRP2-FAF1 complex, we collected 4-cell and 8-cell embryos obtained from Nlrp2 or Faf1 knockdowns and carried out immunofluorescence analysis. The results show that knockdown of the Nlrp2 or Faf1 gene in zygotes 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 development of cleavage-stage mouse embryos.
The degradation of maternal products is a critical developmental process during egg-to-embryo transition in mammals (Ramos et al. 2004, Mtango et al. 2014) and is partially controlled by the ubiquitin-proteasome pathway (Yu et al. 2015). FAF1 protein may serve as a scaffolding protein to assist the ubiquitin-mediated degradation of proteins (Song et al. 2005). Our present results show that the knockdown of Nlrp2 or Faf1 in zygotes influences formation of the NLRP2–FAF1 complex resulting in developmental arrest during early embryogenesis. The molecular mechanism underlying this effect is likely to involve interaction of the NLRP2–FAF1 complex with ubiquitinated proteins, thus playing a key role in ubiquitin-mediated protein degradation. Failure of the NLRP2–FAF1 complex to form in preimplantation embryos may result in the ubiquitin-proteasome pathway being unable to degrade maternal proteins in an effective manner and thus cause important development events to occur in an unregulated manner. However, the precise mechanism of NLRP2–FAF1 complex involved in preimplantation embryogenesis requires further elucidation.
In summary, the present study investigated the expression of FAF1 protein in mouse tissues and preimplantation embryos and revealed evidence of a key interaction between NLRP2 and FAF1 proteins in oocytes and early embryos. This observation creates a rationale for the developmental arrest observed in either Nlrp2 or Faf1 knockdown zygotes. Ensuing research should focus on the functional domains responsible for the interaction between NLRP2 and FAF1 and determine the specific functions of the NLRP2–FAF1 complex during early embryogenesis.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by grants from the National Natural Science Foundation of China (grant numbers: 31402079 and 31672415), FAFU Program for Distinguished Young Scholars (grant number: XJQ201509) and Foundation of Fujian Educational Committee (grant number: JK2014013).
Acknowledgments
The authors thank Prof. Xu Wu for helpful discussions and Dr Song Wang and Xiaojuan Chi for their generous technical assistance.
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