Activation of nucleolar and extranucleolar RNA synthesis and changes in the ribosomal content of human embryos developing in vitro

RNA synthetic activity of human 2-16-cell embryos developing in vitro was studied by [3H]uridine light-microscope autoradiography. Parallelly cut thin sections were examined in the electron microscope. The first extranucleolar RNA synthesis was detected in 4-cell embryos, but nucleoli were never labelled until the 3rd cleavage (6-8-cell embryos). In 6-cell embryos the nucleolar labelling was mostly confined to a narrow peripheral zone. In later cleavage stages most of the blastomeres showed intensive labelling of nucleoli and extranucleolar chromatin. However, rather low levels of extranucleolar RNA synthesis and the absence of nucleolar activity were often seen even in blastomeres of fully compacted morulae. The activation of nucleolar RNA synthesis entailed a noticeable increase in the number of ribosomes (estimated by electron microscope morphometry) that followed a marked drop during the period between the 2-cell and 8-cell stages. The results indicate that the concentration of ribosomes in the preovulatory oocyte is a major factor of its developmental potential.     

Effects of alpha-amanitin on RNA synthesis by mouse embryos in culture

Investigations were conducted to test the effects of alpha-amanitin on RNA synthesis in preimplantation mouse embryos. Exposure of embryos in culture to 1-100 microgram/ml alpha-amanitin produced a dose- and time-dependence suppression of total RNA synthesis as measured by incorporation of [3H]uridine. Synthesis of polyadenylated RNA in blastocyst-stage embryos was abolished by alpha-amanitin-treatment at concentrations and exposure times that suppressed total RNA synthesis by less than 15%. DNA-dependent RNA polymerase activity was measured in lysates of embryos at several stages of preimplantation development. alpha-Amanitin suppressed total polymerase activity assayed under ionic conditions favorable to the detection of RNA polymerase II. Electrophoretic analyses revealed that preincubation of blastocysts in 100 microgram/ml alpha-amanitin reduced labelling of cytoplasmic 28S and 18S RNA by inhibition of both synthesis and maturation of nucleolar 45SrRNA-precursor. This action of alpha-amanitin on nucleolar RNA synthesis cannot be correlated with the minimal suppression of nucleolar RNA polymerase activity and suggests that the synthesis and processing of rRNA may be under control of nucleoplasmic gene products.     

Fibrillarin is essential for early development and required for accumulation of an intron-encoded small nucleolar RNA in the mouse

Fibrillarin, a protein component of C/D box small nucleolar ribonucleoproteins (snoRNPs), directs 2'-O-methylation of rRNA and is also involved in other aspects of rRNA processing. A gene trap screen in embryonic stem (ES) cells resulted in an insertion mutation in the fibrillarin gene. This insertion generated a fusion protein that contained the N-terminal 132 amino acids of fibrillarin fused to a beta-galactosidase-neomycin phosphotransferase reporter. As a result, the N-terminal GAR domain was present in the fusion protein but the methyltransferase-like domain was missing. The ES cell line with the targeted fibrillarin allele was transmitted through the mouse germ line, creating heterozygous animals. Western blot analyses showed a reduction in fibrillarin protein levels in the heterozygous knockout animals. Animals homozygous for the mutation were inviable, and massive apoptosis was observed in early Fibrillarin(-/-) embryos, showing that fibrillarin is essential for development. Fibrillarin(+/-) live-born mice displayed no obvious growth defect, but heterozygous intercrosses revealed a reduced ratio of +/- to +/+ mice, showing that some of the Fibrillarin heterozygous embryos die in utero. Analyses of tissue samples and cultured embryonic fibroblasts showed no discernible alteration in pre-rRNA processing or the level of the U3 snoRNA. However, the level of the intron-encoded box C/D snoRNA U76 was clearly reduced. This suggests a high requirement for snoRNA synthesis during an early stage in development.     

Small nucleolar RNAs and nucleolar proteins inXenopus anucleolate embryos

We investigated the presence and localization, in the cells of anucleolate mutant embryos of Xenopus laevis, of three representative small nucleolar RNAs (snoRNAs), U3, U15 and U17, and of two nucleolar proteins, nucleolin and fibrillarin. The levels of the three snoRNAs in the anucleolate mutant are the same as in normal embryos, in contrast to 5S RNA and ribosomal proteins. In situ hybridization showed that, in the absence of fully organized nucleoli, the three RNAs are diffusely distributed in the nucleus and partly associated with a number of small structures. Nucleolin and fibrillarin are also present in the anucleolate embryos as in normal embryos, although there is less nucleolin mRNA in the former. The two nucleolar proteins were localized by immunofluorescence microscopy. Fibrillarin, similar to its associated U3 and U15 snoRNAs, is diffusely distributed in the anucleolate nucleus and is partly associated with small structures, probably prenucleolar bodies and pseudonucleoli. Nucleolin also appears diffusely distributed in the nucleus with some spots of higher concentration, but with a different pattern with respect to fibrillarin. Received: 26 September 1996; in revised form: 14 February 1997 / Accepted: 24 February 1997     

Autoradiography of tritiated uridine in shoot apices ofChenopodium rubrum

The incorporation of uridine-5-³H into shoot apices ofChenopodium rubrum, plants was studied using autoradiography. The evaluation of the rate of incorporation into the nucleolus and the extranucleolar part of the nucleus as a function of the total radioactivity in the apex yields quantitative data on the distribution of labeling in these parts of the nucleus. Incubation of intact germinating plants in uridine-³H makes it possible to carry out chase experiments. Curves of uridine incorporation into the nucleolus and the extranucleolar part of the nucleus were obtained which demonstrated a non-linear course of incorporation. When incubating with uridine from 30 to 120 min the nucleolar/extranucleolar ratio of labelling was found to increase from 2 to 3. In chase experiments this ratio changed within three days from 3 to 1. Interpretation of these results in view of the function of RNA localized in different parts of the nucleus is discussed.     

Differentiation of 2-cell and 8-cell mouse embryos arrested by cytoskeletal inhibitors

Mouse embryos at the 2-cell stage were cultured in the presence of cytochalasin B (CB), cytochalasin D (CD), colchicine (COL) or colcemid (COM) for up to 72 h. Cleavage was arrested in the 2-cell and 8-cell embryos cultured in CB or CD but the blastomeres continued to differentiate, since chromosome replication occurred in the blastomeres at approximately the same time as control embryos underwent cleavage; an increase in the incorporation of [³H]uridine into RNA was also detected. Furthermore, the cleavage-arrested embryos acquired the necessary information to undergo morphogenesis; these embryos when explanted to fresh medium after 48 h culture in CB or CD underwent compaction within 15–60 min and started to cavitate to produce trophoblastic vesicles within 5–6 h at the same time as when the control embryos were undergoing compaction and beginning to form blastocoelic cavities. In contrast, the embryos arrested in the presence of COM or COL showed none of these differentiative, biochemical or morphogenetic changes. Hence, differentiation of blastomeres and morphogenesis is apparently coupled with nuclear divisions and the information does not reside within the blastomeres at the 2-cell or 8-cell stage. The trophoblastic vesicles produced after cleavage arrest subsequently gave rise to only trophoblast giant cells and no embryonic derivatives were detected.     

Nucleolar changes in bovine nucleotransferred embryos.

This study focused on nucleolar changes in bovine embryos reconstructed from enucleated mature oocytes fused with blastomeres of morulae or with cultured, serum unstarved bovine fetal skin fibroblasts (embryonic vs. somatic cloning). The nucleotransferred (NT) embryos were collected and fixed at time intervals of 1-2 h (early 1-cell stage), 10-15 h (late 1-cell stage), 22-24 h (2-cell stage), 37-38 h (4-cell stage), 40-41 h (early 8-cell stage), 47-48 h (late 8-cell stage), and 55 h (16-cell stage) after fusion. Immunocytochemistry by light and electron microscopy was used for structure-function characterization of nucleolar components. Antibodies against RNA, protein B23, protein C23, and fibrillarin were applied. In addition, DNA was localized by the terminal deoxynucleotidyl transferase (TdT) technique, and the functional organization of chromatin was determined with the nick-translation immunogold approach. The results show that fully reticulated (active) nucleoli observed in donor cells immediately before fusion as well as in the early 1-cell stage after fusion were progressively transformed into nucleolar bodies displaying decreasing numbers of vacuoles from the 2- to 4-cell stage in both types of reconstructed embryos. At the late 8-cell stage, morphological signs of resuming nucleolar activity were detected. Numerous new small vacuoles appeared, and chromatin blocks reassociated with the nucleolar body. During this period, nick-translation technique revealed numerous active DNA sites in the periphery of chromatin blocks associated with the nucleolar body. Fully reticulated nucleoli were again observed as early as the 16-cell stage of embryonic cloned embryos. In comparison, the embryos obtained by fetal cloning displayed a lower tendency to develop, mainly during the first cell cycle and during the period of presumed reactivation. Correlatively, the changes in nucleolar morphology (desegregation and rebuilding) were at least delayed in many somatic NT embryos in comparison with the embryonic NT group. It is concluded that complete reprogramming of rRNA gene expression is part of the general nuclear reprogramming necessary for development after NT.