126 NUCLEAR TRANSLOCATION OF NUCLEAR FACTOR KAPPA B AND ITS ROLE IN MOUSE PRE-IMPLANTATION DEVELOPMENT

Abstract

In mouse pre-implantation development, it has been reported that RelA, one of the subunits of nuclear factor kappa B (NF-ºB), is expressed in eggs and embryos from the Metaphase II oocyte to the blastocyst stage. However, the role of NF-ºB in the pre-implantation development has not yet been elucidated in detail. In this study, we examined (1) the activation of NF-ºB during mouse pre-implantation development and (2) the effect of a synthetic peptide inhibitor of NF-ºB, SN-50, which inhibits nuclear translocation of NF-ºB on the pre-implantation development. Fertilized one-cell embryos were collected 17 h post-hCG from the ampullae of oviducts of superovulated ICR mouse females that had been mated with the same strain of males and then were cultured in KSOM medium at 37^°C under 5% CO₂ in air for 4 d. To elucidate the timing of NF-ºB activation, we examined the localization of NF-ºB in the nucleus by an immunofluorescence approach using RelA antibody with a laser confocal microscope. RelA was distributed mainly in the cytoplasm of embryos from the one-cell stage through the blastocyst stages. The presence of RelA in the nucleus, evidence for NF-ºB activation, was observed in embryos from the one-cell to the compacted 8-cell stages. Moreover, we observed RelA punctate localization in nucleoplasm of embryos from the one-cell to the 4-cell stages, and nuclear dots were enriched conspicuously in the one-cell embryos and the late 2-cell embryos. These results suggest that NF-ºB is activated in embryos from the one-cell to the compacted 8-cell stages and that its activation seems to be particularly strong at the developmental stage when RelA appeared to be concentrated in nuclear dots, as it has been reported that NF-ºB and other transcription factors and co-activators form punctate structures called 'enhanceosom' in association with particular promoters in the nucleus. Next, we examined the effect of SN-50 on the pre-implantation development of mouse embryos. When embryos were treated with SN-50 at 20 ¼g/mL from the 2-cell stage, 63% (33 of 52) of the embryos developed to blastocysts, but 55% (18 of 33) of the blastocysts showed abnormal morphology, such as poor cavitation, and many degenerating cells extruded into the perivitelline space. The percentages of 2-cell embryos that formed morphologically normal blastocysts were significantly lower in the SN-50 treatment group (29%; 15 of 52) than in the untreated control group (76%; 35 of 46) and in the SN-50M (inactive analogue of SN-50, 20 ¼g/mL) treatment group (72%; 38 of 53). These experiments were done in 4 replicates, and the statistical analyses of the data were done by ANOVA and Fisher's PLSD test. Nuclear location of RelA was not observed in the embryos at the 4-cell stage when treated with SN-50 from the 2-cell stage, although observed in control and SN-50M-treated embryos. Furthermore, it was found that most of embryos (23 of 37) treated with SN-50 from the compacted 8-cell or morula stages developed normally to the blastocyst stage as control embryos (25 of 36). These results suggest that morphological aberration at the blastocyst stage is elicited by inhibiting NF-ºB activation.