The relationship of ribosomal RNA synthesis to the formation of segregated nucleoli and nucleolus-like bodies

The relationship of ribosomal RNA (rRNA) synthesis to nucleolar ultrastructure was studied in partial nucleolar mutants of Xenopus laevis. These mutations are the result of a partial deletion of rRNA genes and therefore alow studies on nucleolar structure and function without using drugs that inhibit rRNA synthesis. Ultrastructural studies demonstrated that normal embryos have reticulated nucleoli that are composed of a loose meshwork of granules and fibrils and a typical nucleolonema. In contrast, partial nucleolar mutants in which rRNA synthesis is reduced to less than 50% of the normal rate have compact nucleoli and nucleolus-like bodies. The compace nucleoli contain granules and fibrils, but they are segregated into distinct regions, and a nucleolonema is never seen. Since other species of RNA are synthesized normally by partial nucleolar mutants, these results demonstrate that nucleolar segragation is related specifically to a reduction in rRNA synthesis. The nucleolus-like bodies are composed mainly of fibrils,and the number of such bodies are composed mainly of fibrils, and the number of such bodies present in the different nucleolar mutants is inversely related to the relative rate of rRNA synthesis. Although the partial nucleolar organizers produce segregated nucleoli in these mutants, they organize morphologically normal, but smaller, nucleoli in heterozygous embryos. Alternative explanations to account for these results are discussed.     

Nucleolar structure and synthetic activity during meiotic prophase and spermiogenesis in the rat

The ultrastructure of nucleoli was examined in developing rat spermatocytes and spermatids, with the help of serial sections. In addition, the radioautographic reaction of nucleoli as examined in rats sacrificed 1 hr after intratesticular injection of 3H(5')-uridine and taken as an index of the rate of synthesis of ribosomal RNA (rRNA). Primary spermatocytes from preleptotene to zygotene have small nucleoli typically composed of fibrillar centers, a fibrillar component, and a granular component, within which are narrow interstitial spaces. During early and mid-pachytene, nucleoli enlarge to about nine times their initial size, with the fibrillar and granular components forming an extensive network of cords--a nucleolonema--within which are wide interstitial spaces. Meanwhile, there appear structures identical to the granular component but distinct from nucleoli; they are referred to as extranucleolar granular elements. Finally, from late pachytene to the first maturation division, nucleoli undergo condensation, as shown by contraction of fibrillar centers into small clumps, while fibrillar and granular components condense and segregate from each other, with a gradual decrease in interstitial spaces. In secondary spermatocytes, nucleoli are compact and rather small, while in young spermatids they are also compact and even smaller. Nucleoli disappear in elongating spermatids. In 3H-uridine radioautographs, nucleolar label is weak in young primary spermatocytes, increases progressively during early pachytene, is strong by the end of mid pachytene, but gradually decreases during late pachytene up to the first maturation division. In secondary spermatocytes and spermatids, there is no significant nucleolar label. In conclusion, rRNA synthesis by nucleoli is low in young spermatocytes. During pachytene, while nucleoli enlarge and form a lacy nucleolonema, rRNA synthesis increases gradually to a high level by the end of mid pachytene. However, during the condensation and segregation of nucleolar components occurring from late pachytene onward, the synthesis gradually decreases and disappears. The small, compact spermatids arising from the second maturation division do not synthesize rRNA.     

Nucleolus-like morphology produced during the in vitro reassociation of nucleolar components

Nucleoli, the sites of rRNA synthesis, rRNA processing, and the assembly of ribosomes, are dynamic organelles that, in most cells, disperse and reform during mitosis. The mechanisms that regulate nucleolar formation are unknown as is the relationship between nucleolar morphology and the pathway of ribosome biogenesis. In this report we describe the in vitro formation of nucleolus-like particles (NLPs) from soluble extracts of nucleoli. NLPs, which reached sizes comparable to nucleoli (1-3 microns), were found to contain 40% of the nucleolar DNA, RNA, and protein. The ultrastructure of NLPs resembled that of a number of in vivo structures including compact nucleoli, prenucleolar bodies, and pseudonucleoli. The particles were composed of two morphologically distinct regions. The core resembled the dense fibrillar component (DFC) of nucleoli while the cortex resembled the granular component (GC) of nucleoli. The cortex of NLPs contained numerous 15-20 nm osmophilic granules that resembled the preribosomes found in the GC of nucleoli. The distribution of nucleolar proteins in NLPs also resembled that in nucleoli. BN46/51, a component of the GC of nucleoli, was restricted to the GC-like cortex of NLPs. A mAb that bound to the DFC of nucleoli, bound only to the DFC-like core of NLPs while a second mAb that bound to both the DFC and GC of nucleoli, bound to both the core and cortex of NLPs. Thus solubilized components of nucleoli can reassociate in vitro to produce particles that resemble nucleoli in their size, ultrastructure, and protein distribution.     

REPOPULATION OF POSTMITOTIC NUCLEOLI BY PREFORMED RNA : II. Ultrastructure

The reconstruction of the nucleolus after mitosis was analyzed by electron microscopy in cultured mammalian (L929) cells in which nucleolar RNA synthesis was inhibited for a 3 h period either after or before mitosis. When synchronized mitotic cells were plated into a concentration of actinomycin D sufficient to block nucleolar RNA synthesis preferentially, nucleoli were formed at telophase as usual. 3 h after mitosis, these nucleoli had fibrillar and particulate components and possessed the segregated appearance characteristic of nucleoli of actinomycin D-treated cells. Cells in which actinomycin D was present for the last 3 h preceding mitosis did not form nucleoli by 3 h after mitosis though small fibrillar prenucleolar bodies were detectable at this time. These bodies subsequently grew in size and eventually acquired a particulate component. It took about a full cell cycle before nucleoli of these cells were completely normal in appearance. Thus, nucleolar RNA synthesis after mitosis is not necessary for organization of nucleoli after mitosis. However, inhibition of nucleolar RNA synthesis before mitosis renders the cell incapable of forming nucleoli immediately after mitosis. If cells are permitted to resume RNA synthesis after mitosis, they eventually regain nucleoli of normal morphology.     

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.     

THE FINE STRUCTURE OF THE NUCLEOLUS IN MITOTIC DIVISIONS OF CHINESE HAMSTER CELLS IN VITRO

The nucleolus of Chinese hamster tissue culture cells (strain Dede) was studied in each stage of mitosis with the electron microscope. Mitotic cells were selectively removed from the cultures with 0.2 per cent trypsin and fixed in either osmium tetroxide or glutaraldehyde followed by osmium tetroxide. The cells were embedded in both prepolymerized methacrylate and Epon 812. Thin sections of interphase nucleoli revealed two consistent components; dense 150-A granules and fine fibrils which measured 50 A or less in diameter. During prophase, distinct zones which were observed in some interphase nucleoli (i.e. nucleolonema and pars amorpha) were lost and the nucleoli were observed to disperse into smaller masses. By late prophase or prometaphase, the nucleoli appeared as loosely wound, predominantly fibrous structures with widely dispersed granules. Such structures persisted throughout mitosis either free in the cytoplasm or associated with the chromosomes. At telophase, those nucleolar bodies associated with the chromosomes became included in the daughter nuclei, resumed their compact granular appearance, and reorganized into an interphase-type structure.     

Architectural reorganization of the nuclei upon transfer into oocytes accompanies genome reprogramming

The ability of cloned embryos to sustain full-term development depends on the ability of the recipient ooplasm to reprogram the donor cell genome. As the nuclear architecture has recently emerged as a key-factor in the regulation of gene expression, we questioned whether early embryos obtained from transfer of ES metaphasic chromosomes into mouse ooplasm would adopt the somatic or embryonic type of nuclear organization. We have particularly focused on the arrangement of chromosomal territories with respect to the nucleolar compartment, and the pericentric heterochromatin domains called chromocenters. We found that nuclear transfer triggers profound chromatin rearrangements including the dispersion of the donor cell chromocenters components. These rearrangements lead to a typical 1-cell pronuclear organization, namely a radial arrangement of the chromosome territories with centromeres attached to the nucleoli, which adopt the compact fibrillar structure of nucleolar precursor bodies (NPBs). Subsequently, during the second cycle, the cloned embryos undergo further reorganization with the establishment of new chromocenters, clustered in one part of the nucleus, as during normal embryogenesis. We could also establish that the adequate distribution of chromosomal territories at the pronuclear stage seems important for the development until blastocyst.     

NUCLEOLAR AND BIOCHEMICAL CHANGES DURING UNBALANCED GROWTH OF TETRAHYMENA PYRIFORMIS

Numerous nucleoli can be observed in the macronucleus of the logarithmically growing ciliated protozoan Tetrahymena pyriformis; at late log phase the nucleoli aggregate and fuse. In stationary phase this fusion process continues, leaving a very few large vacuolated nuclear fusion bodies in the nucleus. When these stationary phase cells are placed into fresh enriched proteose peptone medium, the large fusion bodies begin to disaggregate during the 2.5-hour lag phase before cell division is initiated. By 3 to 6 hours after inoculation the appearance of the nucleoli in many cells returns to what it was in logarithmic cells. In view of the possible role of nucleoli in ribosome synthesis, attempts were made to correlate the morphological changes to changes in RNA and protein metabolism. The beginning of an increased RNA synthesis was concomitant with the beginning of disaggregation of the large fusion bodies into nucleoli, which was noticed in some cells by 1 hour after the return to fresh enriched proteose peptone medium. Increased protein synthesis then followed the increased RNA synthesis by 1 hour. The supply of RNA precursors (essential pyrimidines) were removed from cultures which were grown on a chemically defined synthetic medium, in order to study the relation between nucleolar fusion and synthesis of RNA and protein. Pyrimidine deprivation drastically curtailed RNA and protein synthesis, but did not cause fusion of nucleoli. When pyrimidines were added back to this culture medium, RNA synthesis was immediately stimulated and again preceded an increased protein synthesis by 1 hour. These studies suggest the involvement of unfused nucleoli in RNA and protein synthesis and demonstrate the extreme plasticity of nucleoli with respect to changes in their environment.     

Nucleolar ultrastructure in bovine nuclear transfer embryos

Nuclear transfer experiments in mammals have attempted to reprogram a donor nucleus to a state equivalent to the zygotic one. Reprogramming of the donor nucleus is, among other features, indicated by a synthesis of ribosomal RNA (rRNA). The initiation of rRNA synthesis is simultaneously reflected in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout all three cell cycles. In the eight-cell stage embryo, a primary vacuole appeared as an electron lucid area originating in the centre of the nucleolar precursor body. In nuclear transfer embryos reconstructed from nonactivated cytoplasts, the nuclear envelope was fragmented or completely broken down at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary vacuoles. A nucleolar precursor body typical for the two-cell stage control embryos was never observed. None of the reconstructed embryos of this group reached the eight-cell stage. Nuclear transfer embryos reconstructed from activated cytoplasts, in contrast, exhibited a complete nuclear envelope at all time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear transfer embryos, which was one cell cycle earlier than in control embryos. Only nuclear transfer embryos reconstructed from activated cytoplasts underwent complete remodelling of the nucleolus. The reorganisation of the donor nucleolar architecture into a functionally active nucleolus was observed as early as in the four-cell stage nuclear transfer embryo. These ultrastructural observations were correlated with our autoradiographic data on the initiation of RNA synthesis in nuclear transfer embryos.