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a Laboratory of Reproductive Physiology, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B) is a transcription factor that controls the expression of a number of genes under cellular redox potential. It has recently been found that NF-B plays a pivotal role in morphogenesis and embryonic development, e.g., in formation of Drosophila malanogaster ventral structures and chicken limb buds. However, the role of NF-B in preimplantation development in mammals is not yet understood. In this study, we show that RelA, one of the subunits of NF-B, is expressed in mouse eggs and embryos from the metaphase II (MII) oocyte to the blastocyst stage. Therefore, it is thought that RelA is maternally expressed and that it continues to be expressed during preimplantation development. Immunofluorescence analysis showed that RelA protein was mainly distributed in the cytoplasm of embryos, whereas nuclear translocation of RelA, evidence for NF-B activation, was observed only at the early 1-cell stage. Finally we studied the effects of NF-B inhibitors, pyrrolidine dithiocarbamate and N-acetyl-L-cysteine, on the preimplantation development of mouse embryos. When these inhibitors were added to the culture medium from the early 1-cell stage, subsequent development through the 2-cell stage was inhibited. However, little, if any, influence on the progression through the 2-cell stage was observed when the inhibitors were added at the late 1-cell or the 2-cell stage. Taken together, the results suggest that the activation of NF-B at the early 1-cell stage is required for the development of mouse embryos beyond the 2-cell stage.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B) [16].
Regulation of NF-B activation is now well elucidated [17, 18]. NF-B, usually composed of p50 and RelA (p65) heterodimer, is maintained in an inactive state in the cytoplasm through binding to members of the IB family. This binding masks nuclear localization signals of NF-B, thereby preventing its nuclear translocation and transcriptional activation. When NF-B is activated by various inducers (such as cytokines, mitogens, and so on), phosphorylation, ubiquitin conjugation, and degradation of IB protein are serially induced, which causes dissociation of the NF-B/IB complex. Upon release from IB, the activated NF-B dimer migrates to the nucleus, binds to an NF-B enhancer located upstream of NF-B regulated genes, and up-regulates their transcription.
Involvement of NF-B or its homologue during development of several species has been reported. In developing Drosophila malanogaster embryos, Dorsal, one of the NF-B homologues, is translocated to the nucleus in response to a ventral signal after about 10 nuclear division cycles, which is responsible for the generation of all ventral structures [19]. In Xenopus embryogenesis, activation of NF-B is observed during insulin-induced oocyte maturation [20] and early development [21], but its role has not been elucidated. In chicken embryos, NF-B is activated in limb bud cells and then up-regulates expression of homeobox genes to promote proper embryonic development of the limb bud [22, 23]. In mouse embryonic development, NF-B-dependent expression of a LacZ transgene was observed in the nervous system, embryonic blood vessels, and the thymus of embryos at the midgestation stage of transgenic mouse development [24]. But activation of NF-B in the embryo during preimplantation has not yet been reported in detail. The present study was conducted to examine 1) the expression and activation of NF-B during mouse preimplantation development and 2) the effects of specific inhibitors of NF-B, pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC), on preimplantation development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ICR mouse females were superovulated with injections of 5 IU eCG followed 48 h later by 5 IU hCG. Unfertilized metaphase II (MII) oocytes were collected at 16 h post-hCG from the ampullae of oviducts by means of scratching the ampullae with a hypodermic needle. Fertilized 1-cell embryos were collected 16 h post-hCG from the ampullae of oviducts of superovulated females that had been mated with the same strain of males. The cumulus cells were removed by digestion with 0.1% hyaluronidase (Sigma Chemical Co., St. Louis, MO) for ~5 min. Embryos were cultured in KSOM medium [25] at 37°C under 5% CO2 in air. Then 2-cell-, 4-cell-, 8-cell-, morula-, and blastocyst-stage embryos were collected after, respectively, 22–26 h of culture (28–42 h post-hCG), 48–50 h of culture (64–66 h post-hCG), 60–65 h of culture (72–77 h post-hCG), 70–75 h of culture (86–91 h post-hCG), and 96–100 h of culture (112–116 h post-hCG). Synchronization of 1-cell-stage embryos was carried out as described by Ram and Schultz [26]. Briefly, immediately after collection of early 1-cell embryos, those with visible pronuclei were discarded; thereafter the appearance of the pronuclei was monitored by visual inspection every 30 min, allowing batches of synchronized embryos to be collected.
In an experiment to examine the effects of the NF-B inhibitors, NAC and PDTC, on the development of mouse embryos, 1-cell embryos collected at 16 h post-hCG and cultured in KSOM medium were transferred to the medium containing 1 µM PDTC (Wako Pure Chemicals, Osaka, Japan) or 1 mM NAC (Wako Pure Chemicals) at 16 h post-hCG (early 1-cell), 27 h post-hCG (late 1-cell), and 45 h post-hCG (2-cell) and then kept in culture. For control, embryos were cultured in KSOM medium with no inhibitors. Those eggs that had not formed pronuclei until 27 h post-hCG were regarded as unfertilized oocytes and removed from the culture. Development to the 2-cell, 4-cell, morula, and blastocyst stages was examined after 4 days of culture.
Immunoblot Analysis
Pools of 30 MII oocytes and embryos were solubilized in SDS sample buffer [27]; the resulting whole-embryo extracts were separated by SDS-PAGE on 10% gel, and proteins were transferred electrophoretically onto nitrocellulose membrane. The membranes were blocked in TBS (10 mM Tris, 150 mM NaCl, pH 7.4) containing 5% skim milk and then incubated in anti-RelA antibody (C-20 antibody raised against the COOH terminal peptide of RelA; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1000-fold with TBS containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature. After three washes in TBS-T, the membrane was incubated for 1 h at room temperature with anti-rabbit immunoglobulin antibody conjugated with peroxidase (Dako, Glastrup, Denmark) diluted 1000-fold with TBS-T, and the immunoreactive band was revealed using the enhanced chemiluminescence system (Amersham, Backinghamshire, UK). To compare the amounts of RelA at the various stages, we quantified the bands with ATTO densitograph software (ATTO, Tokyo, Japan); a range of oocytes was used to verify the proportionality between the intensity of the band and the amount of protein.
Immunofluorescence Staining
Embryos were fixed in 3.7% paraformaldehyde in PBS overnight at 4°C. The fixed embryos were permeabilized in 0.1% Triton X-100 in PBS for 15 min. They were then washed in PBS and placed in blocking solution (BS: 0.1% BSA, 0.01% Tween 20, and PBS) for 15 min. Embryos were then incubated for 1 h with C-20 antibody diluted 100-fold in BS. After a wash in BS, they were incubated with secondary antibody (anti-rabbit immunoglobulin antibody conjugated to biotin) diluted 500-fold in BS for 30 min, followed by an incubation in fluorescein isothiocyanate (FITC)-conjugated streptavidin diluted 100-fold in BS. They were washed again and mounted on coverslip. FITC fluorescence was visualized by excitation at 488 nm with the argon laser in a confocal laser microscope (LSM-410; Carl Zeiss, Oberkochen, Germany). To determine specificity of the primary antibody, C-20 antibody was pretreated with the blocking peptide (Santa Cruz Biotechnology) for 1 h at room temperature prior to incubation with oocytes/embryos. All the samples were processed and analyzed together under the same conditions and the same settings.
Quantitation of fluorescence was carried out with the confocal microscope and Zeiss image analysis system. Fluorescence intensity of nucleus and cytoplasm was measured in each of three different regions; Fn/c (fluorescence quantitated in the nucleus relative to that of cytoplasm) of each embryo was calculated by dividing the average nuclear value by the cytoplasmic value.
Statistical Analysis
Statistical analyses for treatment comparisons were carried out by ANOVA and Fisher's protected least-significance difference test. All percentage data were subjected to arcsine transformation before statistical analysis. Differences were considered significant at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We first determined the presence of RelA, a subunit of NF-B responsible for the strong transactivation of genes [28], in MII oocytes and embryos during preimplantation development by immunoblotting. A 65-kDa band was specifically labeled with C-20 antibody at all stages of development from MII oocytes to blastocysts (Fig. 1). However, the amount of RelA protein decreased after the 8-cell stage. Quantitative analysis showed that morulae and blastocysts contained approximately 60% and 99% less RelA than oocytes, respectively (data not shown). The presence of RelA protein in MII oocytes indicates that RelA is maternally expressed.
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Nuclear Translocation of RelA at Early 1-Cell Stage
Since it is amply documented that localization of NF-B in the nucleus is evidence for its activation [17, 18], we took an immunofluorescence approach using C-20 antibody with a laser confocal microscope. Immunofluorescence was observed mainly in the cytoplasm of embryos at the 1-cell to the morula stages (Fig. 2), but little fluorescence was observed in the blastocyst stage (data not shown). In each examination, pretreatment of C-20 antibody with the blocking peptide abolished the nuclear and cytoplasmic fluorescence (Fig. 2E), indicating staining specific for RelA protein. The presence of RelA protein in the nucleus was observed only at the 1-cell stage (Fig. 2), indicating that NF-B is stage-specifically activated. However, the fluorescence intensity in the nucleus varied among the 1-cell embryos observed (data not shown), suggesting that NF-B might be translocated to the nucleus during a limited time in the 1-cell stage. This observation allowed us to refine the timing for nuclear translocation of RelA. In this experiment, we synchronized the cell cycle of 1-cell embryos as described in Materials and Methods; embryos at ~30 min, 6 h, and 11 h post-pronuclear formation were fixed, and the immunofluorescence intensity in both the nucleus and cytoplasm of the synchronized embryos was examined. When embryos were examined immediately after pronuclear formation, obvious fluorescence was detected in the nucleus, though its intensity was slightly weaker than in the cytoplasm (Fig. 3A). However, fluorescence intensity in both the male and female pronuclei became weaker as the cell cycle proceeded; and 11 h after pronuclear formation, i.e., G2 of the first cell cycle, it was much weaker than in the cytoplasm (Fig. 3, B and C). These results indicate that NF-B is activated specifically at an early phase of the 1-cell stage.
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Effects of Potent Inhibitors of NF-B on Mouse Preimplantation Development
To ascertain whether NF-B activation is involved in preimplantation development, we examined the effect of specific inhibitors of NF-B, NAC, and PDTC on in vitro development of mouse embryos by adding them to the culture medium at the early 1-cell (16 h post-hCG), late 1-cell (27 h post-hCG), and 2-cell (45 h post-hCG) stages. When embryos were cultured in the presence of 1 µM PDTC from the early 1-cell stage, most embryos (91%) were arrested at the 2-cell stage and none developed beyond the 4-cell stage. However, 49% of embryos cultured in the presence of 1 µM PDTC from the late 1-cell stage developed to the 4-cell stage, and most developed up to the blastocyst stage. No influence was observed when PDTC was added from the 2-cell stage; most of the embryos cultured (
80%) were able to reach the blastocyst stage, as observed in the control (85%) (Fig. 4A). On the other hand, when 1 mM NAC was used from the early 1-cell stage, about half of the embryos examined were able to develop to the 4-cell stage but the proportion to blastocysts after 4-day culture was very low (16%). However, no such influence was observed on development to the 4-cell stage when embryos were cultured in medium containing 1 mM NAC from the late 1-cell or 2-cell stage (91% and 100% of embryos developed to the 4-cell stage, respectively, as observed in the control [96%]) (Fig. 4B). Effects of these inhibitors on the nuclear translocation of RelA were examined by immunofluorescence methods, but neither inhibitor showed any significant effects on the nuclear translocation of NF-B at the early 1-cell stage (data not shown). Therefore, they appeared to inhibit the activities of NF-B rather than its nuclear translocation (see Discussion). Since two different types of NF-B inhibitors had similar effects on the preimplantation development of mouse embryos, the developmental arrest observed may not be due to their cytotoxic action or other effects, such as an increase in intracellular GSH caused by NAC [29]. It is obvious from these results that treatment with NF-B inhibitors from the early 1-cell stage causes embryos to arrest at the 2-cell stage. This suggests involvement of NF-B activation at an early 1-cell stage in the transition of embryos from the 2-cell to the 4-cell stage.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B, is present in mouse MII oocytes and embryos during preimplantation development. The amount of RelA decreased greatly during development from the 8-cell to the blastocyst stages, and since RelA protein is present as early as the MII oocyte stage, it appeared that RelA protein in preimplantation embryos undergoes transcription from maternal message. In immunofluorescence experiments, nuclear translocation of RelA, evidence of activation of NF-B, was observed at the early 1-cell stage, when only maternally derived RelA exists. This activation of maternally derived NF-B explains why RelA knockout (RelA-/-) embryos develop normally until implantation [30], since RelA protein would be provided from their heterogeneous (RelA+/-) mother even if the genotype of embryos was RelA-/-. Similar importance for a maternally derived gene product was observed in E-cadherin knockout embryos, which compact normally with the aid of maternally provided E-cadherin [31, 32].
Inconsistent with our observations were results of Schmidt-Ullrich et al. [24], who reported that NF-B is not activated during preimplantation development as evidenced by experiments using transgenic mice harboring NF-B-dependent reporter gene constructs whose promoter region contains NF-B motif in immunoglobulin kappa light-chain enhancer or NF-B motif in p105 promoter. However, the authors did not scrutinize preimplantation embryos at various developmental stages. Moreover, some researchers have found that expression of reporter constructs driven by NF-B is selective, depending on which NF-B motif is used for the construct or how NF-B is activated. Baldwin et al. [33] found that in serum-stimulated Balb/c 3T3 cells, with mediation by NF-B activation, two reporter constructs containing an NF-B motif in class I major histocompatibility complex enhancer or NF-B motif in c-myc enhancer, respectively, were inducible, but one reporter construct containing an NF-B motif in immunoglobulin kappa enhancer was not. Kunsch et al. [34] also reported that there are numerous NF-B motifs with preferential binding affinities for individual NF-B-related proteins, such as RelA and p50, and therefore that the variations in the sequence and configuration of NF-B motifs will permit selective and specific actions of NF-B. Thus, transcriptional activation mediated by NF-B differs dramatically depending on the specific NF-B motifs present. To define whether NF-B is activated in mouse embryos during preimplantation development, it might therefore be necessary to examine the regulation of gene expression by NF-B in transgenic mice harboring NF-B-dependent reporter genes driven by a regulatory element that contains various types of NF-B motifs.
Experiments with NF-B inhibitors (NAC and PDTC) show that embryos at the early 1-cell stage are highly sensitive to these inhibitors, and treatment of embryos at the early 1-cell stage with these inhibitors greatly influenced the transition from the 2-cell to the 4-cell stage. On the other hand, 2-cell embryos are completely resistant to these inhibitors, and the inhibitors do not show any influence on their development to the blastocyst stage. This result suggests the possibility that the developmental arrest observed is due to their action as specific inhibitors of NF-B activity rather than their cytotoxic action. It has been believed that NAC and PDTC prevent the release of IB from NF-B by their antioxidant action so as to inhibit NF-B translocation to the nucleus [29, 35]. However, this consideration may not be correct; a recent study has shown that PDTC inhibits the DNA binding of NF-B to the target sequence without interfering with the nuclear translocation [36]. In the present study, immunofluorescence experiments revealed that even if embryos at an early 1-cell stage were treated with NAC and PDTC, totally unexpectedly, nuclear translocation of NF-B was not prevented. It seems possible that the developmental arrest of early 1-cell embryos with NF-B inhibitors may be attributed to inhibition of NF-B binding to DNA of the embryos. Therefore, it should be ascertained by gel shift assay whether NF-B binding to DNA in pronuclei of embryos at an early 1-cell stage is actually inhibited by these inhibitors; in the present study, this assay was impossible because not enough nuclear extract was obtainable from the mouse preimplantation embryos (data not shown). This is attributable in part to the small size and limited numbers of mouse embryos that can be obtained, which in general preclude most direct molecular approaches to identification of genes expressed and/or activated in the early embryo. With the recent development of polymerase chain reaction-mediated in vivo footprinting [37], which allows analysis of the protein-binding activity of the sequence of interest with minimal amounts of materials, the DNA-binding activity of NF-B in the embryos could be analyzed using this method if the genes driven by NF-B in the early 1-cell embryo were identified.
During preimplantation development, nuclear translocation of NF-B was limited at the early 1-cell stage. This suggests that NF-B may be activated during formation of 1-cell zygotes from MII-arrested oocytes through stimulation related to fertilization. Similar cell cycle-dependent activation of NF-B has been reported in Balb/c 3T3 cells during the G0-to-G1 transition [33]. When G0 cells were stimulated with serum growth factors, transient activation of NF-B was observed, but the activity was reduced during the first cell cycle and became undetectable in proliferating cells. Although MII-arrested oocytes are different from G0 cells, since they are regarded as quiescent cells, it is speculated that NF-B regulate certain genes involved in the transition of cells from quiescence to proliferation.
In conclusion, the results obtained in this study are to our knowledge the first evidence indicating activation of NF-B during preimplantation development. NF-B translocated to the nucleus at the early 1-cell stage might induce the expression of certain genes that are required for the transition of embryos from the 2-cell to the 4-cell stage. Understanding the role of NF-B in preimplantation development will help us reveal more about the molecular dynamics concerning the second cell cycle progression and ZGA of mouse preimplantation embryos.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Masayasu Yamada, Laboratory of Reproductive Physiology, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho Sakyo-ku, Kyoto 606-8502, Japan. FAX: 81 75 753 6329; yamada{at}jkans.jkans.kais.kyoto-u.ac.jp
3 Current address: Minase Research Institute, Ono Pharmaceutical Co., Ltd. Shimamoto, Mishima, Osaka, 618-8585, Japan.
Accepted: January 29, 1999.
Received: November 30, 1998.
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