Hyttel P, Laurincik J, Zakhartchenko V, Stojkovic M, Wolf E, Müller M, Ochs R L, Brem G
Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark.
Cloning. 2001;3(2):69-82. doi: 10.1089/15204550152475572.
In the present study, immunofluorescence confocal laser scanning microscopy, autoradiography following (3)H-uridine incubation, and transmission electron microscopy were used to evaluate the nucleolar protein localization, transcriptional activity, and nucleolar ultrastructure during genomic reprogramming in bovine embryos reconstructed by nuclear transfer from in vitro-produced bovine morulae to activated cytoplasts. During the first cell cycle (one-cell embryos), no autoradiographic labelling was detected. Ultrastructurally, whorls consisting of densely packed fibrillar material were observed instead of nucleoli. During the second, third, and fourth cell cycle (two-, four-, and tentative eight-cell embryos), autoradiographically unlabelled nuclei contained vacuolated bodies consisting of densely packed fibrillar material. Also, during the fourth cell cycle, the first nucleoplasmic autoradiographic labelling was observed, but still without formation of fibrillo-granular nucleoli. During the fifth cell cycle (tentative 16-cell embryos), the nuclei displayed autoradiographic labelling over both nucleoplasm and presumptive nucleoli, and the formation of fibrillo-granular nucleoli was observed. In a certain proportion of blastomeres, however, abnormal patterns of nucleolar formation and apoptosis were noted. During the first two cell cycles, labelling of RNA polymerase I, fibrillarin, upstream binding factor (UBF), nucleolin (C23), and nucleophosmin (B23) was localized to nuclear entities. During the third cell cycle, labelling of topoisomerase I was observed in addition. During the fourth and fifth cell cycles, a substantial portion of the embryos presented blastomeres that lacked labelling of several of these nucleolar proteins. In conclusion, the nuclear transfer procedure was associated with remodelling of the nucleoli to an inactive form, followed by reformation of fibrillo-granular nucleoli during the fifth cell cycle. Moreover, a certain proportion of blastomeres failed to form functional nucleoli with respect to both ultrastructural organization and protein allocation.
在本研究中，利用免疫荧光共聚焦激光扫描显微镜、³H-尿苷孵育后的放射自显影术以及透射电子显微镜，评估了通过将体外产生的牛桑椹胚细胞核移植到激活的细胞质中构建的牛胚胎基因组重编程过程中的核仁蛋白定位、转录活性和核仁超微结构。在第一个细胞周期（单细胞胚胎），未检测到放射自显影标记。在超微结构上，观察到由紧密堆积的纤维状物质组成的漩涡，而非核仁。在第二个、第三个和第四个细胞周期（二细胞、四细胞和暂定的八细胞胚胎），放射自显影未标记的细胞核含有由紧密堆积的纤维状物质组成的空泡体。此外，在第四个细胞周期，观察到首次出现核质放射自显影标记，但仍未形成纤维颗粒状核仁。在第五个细胞周期（暂定的十六细胞胚胎），细胞核在核质和假定的核仁上均显示放射自显影标记，并且观察到纤维颗粒状核仁的形成。然而，在一定比例的卵裂球中，注意到核仁形成和凋亡的异常模式。在前两个细胞周期，RNA聚合酶I、纤维蛋白原、上游结合因子（UBF）、核仁素（C23）和核磷蛋白（B23）的标记定位于核实体。在第三个细胞周期，还观察到拓扑异构酶I的标记。在第四个和第五个细胞周期，相当一部分胚胎的卵裂球缺乏这些核仁蛋白中的几种的标记。总之，核移植过程与核仁重塑为无活性形式相关，随后在第五个细胞周期重新形成纤维颗粒状核仁。此外，在超微结构组织和蛋白质分配方面，一定比例的卵裂球未能形成功能性核仁。
