Ultrastructural localization of silver-staining nuclear proteins at the onset of transcription in early bovine embryos.

Argyrophilic nuclear proteins, known to be functionally associated with ribosomal genes, were localized, in four-, eight-, and 16-cell bovine embryo blastomere nuclei using two different silver-staining procedures. Within the eight-cell cleavage stage by the process of embryonal nucleologenesis in the cow embryo the full-capacity ribosome-producing machinery is established. In the four-cell embryo, many patches and islands of argyrophilic (Ag+) material were detected in the nucleoplasm. The nucleolus-precursor bodies (NPBs), composed uniformly of a homogeneous compact mass, were completely devoid of any silver staining. On the other hand, clear-cut localization of argyrophilic proteins was detected during the eight-cell stage either inside the transforming NPBs or in the close vicinity, or in the already differentiated nucleolus. In compact, nonvacuolated NPB, an intensive Ag+ area was detected, in the form of a lenticle, at the periphery of the NPB. During and following vacuolation of the NPB, no Ag+ was detected inside these vacuoles. It was seen, however, in the dense fibrillar nucleolar component surrounding the smaller vacuoles formed at the time of the establishment of nucleolar structure. Ag+ areas were seen repeatedly in the vicinity of NPBs, probably a part of the nucleolus-associated chromatin or, alternatively, representing the extranucleolar bodies. In blastomere nuclei of 16-cell embryos, already possessing reticulated nucleoli known from intensively synthesizing somatic cells, the silver-staining pattern corresponded to the usual situation in differentiated cells: slight staining of fibrillar centers, heavy labelling in the dense fibrillar component, and absence of silver deposits in the granular component.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA synthesis in the ultrastructural and biochemical components of the nucleolus of Chinese hamster ovary cells

A correlated autoradiographic and biochemical study of RNA synthesis in the nucleoli of chinese hamster ovary cells has been made. Quantitative analysis of the labeling indicates that the fibrillar ribonucleoprotein (RNP) component is labeled faster than 80S RNP and 45S RNA molecules, but approaches simultaneously a steady-state 3H to 14C ratio or grains/mum2 after 30 min of [3H]uridine incorporation. On the other hand, the 55S RNP, the 36S + 32S RNA, and the granular RNP components have the same kinetic of labeling with [3H]uridine. These results suggest that the fibrillar and granular RNP components of the nucleolus are the ultrastructural substratum of, respectively, the 80S RNP (45S RNA) and 55S RNP (36S + 32S RNA). The possibility that precursors to 80S RNP exist also in the fibrillar region of the nucleolus is strongly suggested by the rapid labeling of the fibrils on the autoradiographs.     

Ultrastructural Studies of H-1 Parvovirus Replication VI. Simultaneous Autoradiographic and Immunochemical Intranuclear Localization of Viral DNA Synthesis and Protein Accumulation

The localization of H-1 viral replicative-form double-stranded DNA and progeny single-stranded DNA replication in parasynchronously infected, simian virus 40-transformed newborn human kidney cells was studied with high-resolution electron microscope autoradiography (80-nm silver grains). We analyzed wild-type H-1 and ts1 H-1 (a conditional mutant defective in progeny single-stranded DNA synthesis). The proportion of the total DNA synthesis that was viral was estimated to be >90% by comparing the amount of [(3)H]thymidine uptake in cultures infected with wild-type H-1 versus ts14 (an H-1 mutant defective in DNA replication). Simultaneous staining with cytochrome c-conjugated anti-H-1 immunoglobulin G was performed to ensure that cells incorporating [(3)H]thymidine (2- to 60-min pulses) were H-1 infected. The sites of H-1 replicative-form (in ts1-infected cells) and progeny (in wild-type-infected cells) DNA synthesis were identical. Immunospecifically labeled nuclei at the earliest stages of infection exhibited dense clusters of silver grains over material extruded from nucleolar fibrillar centers. These foci became larger with increasing cellular damage, forming a limited number of H-1 DNA synthetic centers in the euchromatin. Each island-like focus was surrounded by tufts of heterochromatin containing high concentrations of unassembled H-1 capsid proteins. In late phases of infection, the heterochromatin became completely marginated, and the nucleoplasm contained only euchromatin that exhibited randomly distributed sites of H-1 DNA replication. This indicates that H-1 DNA synthesis begins at localized euchromatic or nucleolar sites and then spreads outward. Immunostained heterochromatin and nucleolar chromatin never incorporated [(3)H]thymidine. Our results suggest that H-1 proteins and cellular cofactors associated with the fibrillar component of the nucleolus and the euchromatin may play a role in the regulation of H-1 DNA synthesis.     

Evolution of the rat oocyte nucleolus during follicular growth

The ultrastructural evolution of the nucleolus was followed during follicular growth by means of a silver staining procedure. The oocyte nucleolus in the primordial and primary follicles consists of strands of dense fibrillar silver-stained component and aggregates of granules which are devoid of silver grains. Small fibrillar centres are also recognized and appear to have less silver stainability. At the secondary follicle stage, a new nucleolar component appears in the reticulated oocyte nucleolus. This component is devoid of silver grains. During follicle growth, at the antral follicle stage, this new component seems to fuse and the nucleolus becomes constituted of a compact homogeneous mass which exhibits a vacuole at the end of the oocyte maturation. The results obtained suggest that this nucleolar mass is essentially made of proteins and particularly of acidic proteins.     

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.     

The nucleolus and its modifications during oogenesis of Torpedo marmorata

The structural organisation of the nucleolar apparatus during oogenesis of the spotted ray Torpedo marmorata was investigated. The observations showed that unlike other cartilaginous fishes, in T. marmorata the nucleolar apparatus was always represented by one or two conspicuous nucleoli, whose organization significantly changed during oocyte development. In the smallest follicles (follicles <300 μm in diameter) the nucleolus was made up of granular and fibrillar components, and actively incorporated ³H uridine; later it becomes more and more electron‐dense so in follicles of 400 μm in diameter its components and ³H uridine incorporation were no longer evident. These results indicate that in T. marmorata the nucleolar apparatus significantly changes and undergoes a possible impairment in rRNA synthesis. After nucleolus inactivation, the synthesis of rRNA may be substained by granulosa.     

Timing of nucleolar DNA replication in Amoeba proteus.

Light- and electron-microscope autoradiography have been used to follow the incorporation of [3H]thymidine at different stages during the interphase of synchronously growing populations of Amoeba proteus. Two main patterns were found for tritiated thymidine incorporation, i.e. DNA synthesis. The major incorporation was in the central region of the nucleus, but a lesser degree of incorporation occurred in the nucleolar region. The bulk of this nucleolar DNA was found to be late replicating, i.e. it replicated during the G2 phase.     

Correlation of nucleolar activity and nucleolar vacuolation in plant cells

In root meristematic cells nucleolar structure varies with the cell cycle. Apart from normal meristematic nucleoli one finds nucleoli with a big central vacuole surrounded by a loose cortex with individual fibrillar centres [22] clearly visible within it. There are also intermediate structures between both nucleolar types. In Pisum sativum nuclear tissue, the structure of the vacuolated nucleoli is similar and appears in periods of high metabolic activity during megasporogenesis. In both tissues, vacuolated nucleoli incorporate tritiated uridine more actively than 'normal' nucleoli. In this work the structure of spontaneous nucleolar vacuoles is compared with that induced by drugs such as cordycepin, and FUdR. The vacuolated nucleolus with its increased surface corresponds to a transient structure which not only shows higher metabolic activity but also supplies a storing and/or transporting mechanism for nucleolar products.     

Initiation of transcription and nucleologenesis in equine embryos

The time of activation of the embryonic genome (maternal-embryonic transition) in equine embryos was investigated by assessing incorporation of ³H-uridine and nucleolar development. In Experiment 1, embryos were recovered from the oviduct (n = 15) and the uterus (n = 3). Recovered embryos were assessed for morphologic development and quality score. Recovered embryos with less than 8 cells (two cells, n = 4; four cells, n = 5; five cells, n = 2) were incubated with ³H-uridine (560 μCi/ml) for 10 hr, while eight-cell embryos (n = 2), morulae (n = 2), and blastocysts (n = 3) were incubated with 280 μCi/ml for 0.5–1 hr. At the end of incubation, embryos were washed twice in PBS with 10% FBS and incubated for 30 min with 2.5 mg/ml of unlabelled uridine. Embryos were spread onto glass slides, dipped into emulsion, and exposed for 8 d, then developed and counter-stained with Giemsa and propidium iodide. Embryos at the blastocyst, morula, eight-cell, and five-cell stages incorporated ³H-uridine into their cell nuclei as detected by autoradiography. In a second experiment, nucleologenesis in equine embryos was examined by transmission electron microscopy. Nucleoli or nucleolar precursors were found in 12 of 23 embryos examined. Most embryos in the four- to six-cell stage (n = 7) had nucleolar precursor bodies (npb) consisting of homogeneous fibrillar structures. Two five- to six-cell embryos also possessed reticulated nucleoli with both fibrillar and granular components as did all eight-cell embryos (n = 3). Nucleoli in one morula and one blastocyst were reticulated with prominent granular components, fibrillar components, and apparent fibrillar centers. These results indicate that incorporation of ³H-uridine and the formation of functional nucleoli with typical fibrillar and granular components occurs between the four- to eight-cell stage in equine embryos. © 1995 wiley-Liss, Inc.     

Fate of the nucleolar vacuole during resumption of cell cycle in pea cotyledonary buds

Summary Meristematic cells of pea cotyledonary buds blocked in G_0–1 state contain a small nucleolus with a large central clear area surrounded by a fibrillar rim. The nucleolar structure varies according to the cell cycle from the G_0–1-blocked state until the first mitoses occurring between 24 and 27h after removal of the main stem. In order to better identify and understand the role of the central area in the nucleolar function, its content was investigated by cytochemical and terminal deoxynucleotidyl transferase-immunogold methods. The central area showed the characteristics of a vacuole commonly constituted of the condensed chromatin, ribonucleoprotein granules, and lack of argyrophilic proteins. 3 h after decapitation, a thickening of the fibrillar rim occurred, accompanied by an increase of granules in the vacuole. After 6h, the unique vacuole broke up into two to four small vacuoles in which the granules are more abundant. After 12 h the nucleolus acquired compact structure with few minute vacuoles dispersed over the fibrillar component. During the whole cell cycle, the condensed chromatin is always observed in the vacuole. Our findings suggest that the appearance of the vacuoles is subsequent to the output of preribosomes from nucleolus. These vacuoles might play a role in condensation and decondensation of the chromatin.     

Autoradiographic detection of the earliest stage of [3H]-uridine incorporation into the cow embryo

Cow embryos, obtained from superovulated heifers on days 3 and 4 after oestrus, were cultured for 20 min in Ménézo's complete culture medium (B2), enriched with 200 microCi/ml of 5-[3H]-uridine. Semi-thin Epon sections of this material were investigated by autoradiography for sites of RNA synthesis. It was found that 5-[3H]-uridine was incorporated into the nucleoplasm and nucleoli only at the end of the 8-cell stage. This suggested that synthesis of hnRNA and rRNA occurred from this stage onwards. Ultrastructural studies were performed on these embryos as well as on other non-incubated 4-cell embryos recovered on day 2. The transformation of dense fibrillar primary nucleoli into functional reticulated nucleoli appeared sooner in the development of cow embryos than in other mammalian species hitherto studied and took place generally during the 8-cell stage. An unusual step in this transformation was represented by the development of a single vacuole in nucleoli at the beginning of this stage (day 3 post-oestrus).     

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.