Laurincik J, Bjerregaard B, Strejcek F, Rath D, Niemann H, Rosenkranz C, Ochs R L, Maddox-Hyttel P
Constantin the Philosopher University, Nitra, Slovak Republic.
Mol Reprod Dev. 2004 Jul;68(3):327-34. doi: 10.1002/mrd.20088.
The nucleolus formation was studied as an indirect marker of the ribosomal RNA (rRNA) genes activation in porcine embryos following oocyte maturation, fertilization, and culture in vitro. Nucleologenesis was assessed by transmission electron microscopy (TEM), light microscopical autoradiography following 20 min of 3H-uridine incubation, and immunocytochemical localization of key nucleolar proteins involved in rRNA transcription (upstream binding factor (UBF), topoisomerase I, and RNA polymerase I) and processing (fibrillarin, nucleophosmin, nucleolin) by confocal laser scanning microscopy. During the first four post-fertilization cell cycles, TEM revealed spherical nucleolus precursor bodies (NPBs), consisting of densely packed fibrils, as the most prominent intra-nuclear entities of the blastomeres. Fibrillo-granular nucleoli were observed in some blastomeres in a single embryo during the 5th cell cycle, i.e., the tentative 16-cell stage, where formation of fibrillar centres (FC), a dense fibrillar component, and a granular component on the surface of the NPBs was seen. In this embryo, autoradiographic labeling was detected over the nucleoplasm and in particular over the nucleoli. Fibrillarin was immunocytochemically localized in the presumptive NPBs of the pronuclei. This protein was again localized to the presumptive NPBs together with nucleolin from late during the 3rd cell cycle, i.e., the four-cell stage in some embryos. UBF, RNA polymerase I, and nucleophosmin were localized to the presumptive NPBs in a proportion of the embryos at the 4th cell cycle, i.e., the tentative eight-cell stage and onwards. Toposiomerase I was not localized to intra-nuclear entities even during the 5th post-fertilization cell cycle. Moreover, a considerable proportion of the blastomere nuclei apparently did not show localization of other nucleolar proteins. In conclusion, porcine embryos produced in vitro display a substantial delay in or even lack of the development of functional nucleoli.
研究核仁形成作为猪胚胎在卵母细胞成熟、受精和体外培养后核糖体RNA（rRNA）基因激活的间接标志物。通过透射电子显微镜（TEM）评估核仁发生，在3H-尿苷孵育20分钟后进行光镜放射自显影，并通过共聚焦激光扫描显微镜对参与rRNA转录（上游结合因子（UBF）、拓扑异构酶I和RNA聚合酶I）和加工（纤维蛋白、核磷蛋白、核仁素）的关键核仁蛋白进行免疫细胞化学定位。在受精后的前四个细胞周期中，TEM显示球形核仁前体（NPB），由紧密堆积的纤维组成，是卵裂球中最突出的核内实体。在第5个细胞周期，即暂定的16细胞期，在单个胚胎的一些卵裂球中观察到纤维颗粒状核仁，此时在NPB表面可见纤维中心（FC）、致密纤维成分和颗粒成分的形成。在这个胚胎中，在核质上检测到放射自显影标记，特别是在核仁上。纤维蛋白在原核的推定NPB中进行免疫细胞化学定位。从第3个细胞周期后期，即一些胚胎的四细胞期开始，这种蛋白质再次与核仁素一起定位于推定的NPB。在第4个细胞周期，即暂定的八细胞期及以后，UBF、RNA聚合酶I和核磷蛋白在一定比例的胚胎中定位于推定的NPB。即使在受精后的第5个细胞周期，拓扑异构酶I也不定位于核内实体。此外，相当比例的卵裂球细胞核显然没有显示其他核仁蛋白的定位。总之，体外产生的猪胚胎在功能性核仁的发育方面表现出显著延迟甚至缺乏。
