Protein synthesis in the livers of aging mice studied by electron microscopic radioautography.

The application of 3H-leucine results in labeling of the liver cells of mice in which protein is synthesized at various ages of the animals. Quantitative changes of protein synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. The silver grains in the hepatocytes were mainly located over the rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, cytoplasmic matrix, and a few over the nuclei. The number of silver grains in the cytoplasm and nuclei of the hepatocytes gradually increased after birth, reached the maximum at 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the hepatocyte cytoplasm was more than that in nuclei at various ages. The number of silver grains in the rough surfaced endoplasmic reticulum and mitochondria gradually increased from embryo to 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the Golgi apparatus showed almost no change from fetal stage to 6 months after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the cytoplasmic matrix gradually increased from fetal stage to 2 months after birth, then decreased with aging until the 24th month. These changes reflect the quantity of protein synthesized in each cell organelle at various ages of animals.     

Macromolecular synthesis in the livers of aging mice as revealed by electron microscopic radioautography

For the purpose of studying the aging changes of macromolecular synthesis in animal cells, we studied many groups of aging mice during development and aging from fetal day 19 to postnatal newborn, juvenile, young adults, aged and senescent adults up to 12 and month 24 (2 years). They were injected with ³H-thymidine, ³H-uridine or ³H-leucine, precursors for DNA, RNA and proteins, as well as ³H-glucose, ³H-glucosamine, ³⁵S-sulfuric acid, or ³H-glycerol for glucide and lipid precursors, respectively, then sacrificed and the liver tissues were taken out, fixed and processed for light and electron microscopic radioautography. On many radioautograms the localization of silver grains demonstrating DNA, RNA and proteins in hepatocytes in respective aging groups were analyzed qualitatively. The number of silver grains and the number of cell organelles in each cell of each animal in respective aging groups were analyzed quantitatively in relation to the aging of individual animals. The results revealed that the localization of respective precursors as well as the number of silver grains in cell nuclei, cell organelles, changed with the aging of animals. The numbers of labeled nuclei and cell organelles, as well as the numbers of silver grains in nuclei and cell organelles changed due to aging of individual animals. The number of mitochondria, the number of labeled mitochondria and the mitochondrial labeling index labeled with silver grains were counted in each hepatocyte. It was demonstrated that the numbers of mitochondria, the numbers of labeled mitochondria and the labeling indices showing DNA, RNA and protein synthesis at various ages from embryonic day 19 to postnatal newborn day 1, 3, 9, 14, adult month 1, 2 and 6, reaching the maxima, then decreased to senile year 1 to 2, indicating the aging changes. The results indicated that mitochondria in hepatocytes synthesized nucleic acids and proteins independently from the nuclei, but their synthetic activities were affected from the aging of the individual animals.     

Electron-microscopic study of silver staining of nucleoli in growing oocytes of rat ovaries

The nucleoli of dictyate-stage growing oocytes in rat ovaries were examined both with routine electron microscopy and electron microscopy after silver nitrate and ammoniacal silver nitrate (Ag-AS) staining. The nucleoli of the unilaminar follicular oocytes consist of twisted strands of dense fibrillar components, aggregates of granular components, and small fibrillar centers. After Ag-AS staining, silver grains are numerous on the dense fibrillar strands, fewer on the fibrillar centers, and very sporadic on the granular aggregates. The same stainability of three nucleolar components with the Ag-AS method was also confirmed in the nucleoli segregated by actinomycin D. During the transition of growing oocytes from bilaminar to plurilaminar follicle stage, the nucleolar dense fibrillar strands gradually conglomerate and are transformed into large and compact spherules. The stainability of dense fibrillar components with the Ag-AS method was lost along with this nucleolar transformation. These results may provide some new clues on the functional significance of Ag-AS-positive proteins in the nucleoli.     

LIGHT AND ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF HEPATIC CELL NUCLEOLI IN MICE TREATED WITH ACTINOMYCIN D

Nucleolar partition induced by actinomycin D was used to demonstrate some aspects of nucleolar RNA synthesis and release in mouse hepatic cells, with light and electron microscopic radioautography. The effect of the drug on RNA synthesis and nucleolar morphology was studied when actinomycin D treatment preceded labeling with tritiated orotic acid. Nucleolar partition, consisting of a segegration into granular and fibrillar parts was visible if a dosage of 25 µg of actinomycin D was used, but nucleolar RNA was still synthesized. After a dosage of 400 µg of actinomycin D, nucleolar RNA synthesis was completely stopped If labeling with tritiated orotic acid preceded treatment with 400 µg of actinomycin D, labeled nucleolar RNA was present 15 min after actinomycin D treatment while high resolution radioautography showed an association of silver grains with the granular component. At 30 min after actinomicyn D treatment all labeling was lost. Since labeling was associated with the granular component the progressive loss of label as a result of actinomycin D treatment indicated a release of nucleolar granules. The correlation between this release and the loss of 28S RNA from actinomycin D treated nucleoli as described in the literature is discussed.     

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.     

FINE STRUCTURAL ALTERATIONS OF INTERPHASE NUCLEI OF LYMPHOCYTES STIMULATED TO GROWTH ACTIVITY IN VITRO

This report describes fine structural changes of interphase nuclei of human peripheral blood lymphocytes stimulated to growth by short-term culture with phytohemagglutinin. Chromatin is found highly labile, its changes accompanying the sequential increases of RNA and DNA synthesis which are known to occur in lymphocyte cultures. In "resting" lymphocytes, abundant condensed chromatin appears as a network of large and small aggregates. Early in the response to phytohemagglutinin, small aggregates disappear during increase of diffuse chromatin regions. Small aggregates soon reappear, probably resulting from disaggregation of large masses of condensed chromatin. Loosened and highly dispersed forms then appear prior to the formation of prophase chromosomes. The loosened state is found by radioautography to be most active in DNA synthesis. Small nucleoli of resting lymphocytes have concentric agranular, fibrillar, and granular zones with small amounts of intranucleolar chromatin. Enlarging interphase nucleoli change chiefly (1) by increase in amount of intranucleolar chromatin and alteration of its state of aggregation and (2) by increase in granular components in close association with fibrillar components.     

SITES OF NUCLEOLUS PRODUCTION IN CULTURED CHINESE HAMSTER CELLS

Chinese hamster cell strains in the early passages in culture display wide variation in number of nucleolus-like bodies per cell, though such strains are characteristically euploid. A variety of criteria indicate that the nucleolus-like bodies are true nucleoli. Their Azure B- and fast green-staining properties indicate the presence of RNA and protein; they have typical nucleolar fine structure, including both fibrous and granular components; radioautography reveals that their patterns of uptake of uridine-³H into RNA are similar to those reported for nucleoli of other cell types; actinomycin D, at a level which selectively inhibits ribosomal RNA synthesis, greatly reduces their RNA synthesis and also causes segregation of fibrous and granular nucleolar components. Colchicine was used to experimentally fragment the nuclei of these cells into a number of separate karyomeres, each presumably containing some, or only one, of the chromosomes of the complement. Almost all the karyomeres contain nucleolus-like bodies which, by the same criteria applied to the multiple nucleolus-like bodies of uninuclear cells, appear to be true nucleoli. The nucleoli of individual karyomeres of the same cell often differ from each other in fine structure while the multiple nucleoli of a uninuclear cell generally resemble each other. The evidence presented in this study indicates that Chinese hamster cells contain many nucleolus-producing sites scattered through the genome.     

A radioautographic study on RNA synthesis in aging mouse spleen after 3H-uridine labeling in vitro.

EM radioautographic study on RNA synthesis in aging mouse spleen was conducted after 3H-uridine labeling in vitro. The localization of radiolabelled precursor was used to determine the site of RNA synthesis. The site of the radiolabelled uridine uptake was localized in the haematopoietic cells, particularly in the lymphoblasts. In the labelled cells, most of the silver grains were localized in the nucleus, specifically in the euchromatin. Few cytoplasmic organelles such as the mitochondria and endoplasmic reticulum were labelled with 3H-uridine. Silver grains were also observed over the nucleoli. The labeling index was expressed as the percentage of labelled cells over the total number of cells counted. The labeling index increased from day one after birth and progressively until the 14th day. Thereafter, the labeling index decreased gradually until the 10th month. A significant difference of p less than 0.05 was noted. In all the EMRAG analyzed, it was observed that the number of silver grains per cell increased proportionally with the labeling index. The result of the quantitation of the changes in RNA synthesis correlated well with the maturational development/aging of the animal.     

Localization of ribosomal RNA genes by high resolution autoradiography

In the myxomycete Physarum polycephalum, the genes for ribosomal RNA replicate to a large extent in the G2 phase of the mitotic cycle. In mitotically synchronous plasmodia this fact allows the rRNA genes to be localized by electron microscopic autoradiography. After labelling with thymidine in the G2 phase, silver grains were concentrated over the fibrillar and not over the granular regions of the nucleoli. This shows the presence of DNA in these fibrillar regions or their immediate vicinity. Following labelling and chase during the G2 period, nucleoli are dispersed during mitosis and silver grains concentrate over some regions of condensed chromatin.     

Ultrastructure of trophocyte nuclei in polytrophic ovarioles of Chrysopa perla (Neuroptera)]

The light and electron microscope and autoradiographic studies (H3-uridin incorporation) were carried out on the trophocyte nuclei of imago polytrophic ovarioles of Chrysopa perla (Neuroptera), from the trophocyte differentiation up to their degeneration. Like the oocytes, one of the seven nurse cells o every ovariole chamber contains extrachromosomal DNA bodies. This nurse cell is formed during differential mitoses in the germarium as one of two prooocytes. In contrast to extrachromosomal DNA of oocytes the trophocyte DNA bodies are less active structures. Several (2--4) complex nucleoli develop in the trophocytes of Chrysopa in the early stages of oogenesis. They consist of three main components: the chromatin mass, fibrillar bodies and granular strands. Such nucleoli grow, through increasing in number of fibrillar bodies and granular strands. They are most developed by the start of the vitellogenesis. At the middle vitellogenesis the general nucleolar structure modify due to the beginning of trophocyte degeneration. The consecutive stages of nuclear degeneration are described. The trophocyte nucleoli synthesize RNA still in germarium. The most intensive RNA synthesis is observed at the beginning of the vitellogenesis to decrease by the beginning of trophocyte degeneration.     

Electron microscopic radioautographic studies on DNA synthesis in the hepatocytes of aging mice as observed by image analysis

Quantitative changes of DNA synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. Ultrastructural changes of labelled and unlabelled hepatocytes were estimated quantitatively by the image analysis. The results revealed that at various ages, the area of the cytoplasm and nuclei of labelled hepatocytes were more than those of the unlabelled cells. No significant changes were observed in the nucleoli. The area of the endoplasmic reticulum and mitochondria were less in the labelled hepatocytes than in the unlabelled hepatocytes. The number of the mitochondria was less in the labelled hepatocytes than in the unlabelled hepatocytes. The cell organelles of the hepatocytes that were synthesizing DNA were not well developed, as compared to those not synthesizing DNA during the postnatal development.     

Differential Uptake of Tritiated Thymidine into Hetero- and Euchromatin in Melanoplus and Secale

Grasshoppers of the species Melanoplus differentialis were injected with tritium-labelled thymidine. At intervals thereafter autoradiographic stripping film was applied over Feulgen squashes and sections. In this species during early prophase of meiosis the sex chromosome forms a heterochromatic block large enough to be resolved in tritium autoradiographs. A study of the squash preparations reveals that the sex chromosome is synthesizing DNA at a different period of time from the euchromatic autosomes. Since there is a developmental sequence of spermatocyte cysts along the testicular tubes it is possible from the sections to show that the heterochromatin synthesizes DNA later than does the euchromatin. To find out whether the results obtained in Melanoplus were characteristic of heterochromatin in general, young seedlings of rye were grown in a tritiated thymidine solution and Feulgen squashes were made as for Melanoplus. In rye leaf nuclei there is a large block of heterochromatin constituted by the proximal regions of the chromosomes and a euchromatic one formed by the median and distal regions of the same chromosomes. Here also the heterochromatin synthesizes DNA at a different period of time from the euchromatin. It is concluded that in rye the asynchrony of synthesis occurs within each chromosome. Counts of silver grains over the two types of chromatin in nuclei of Melanoplus and Secale disclosed that the number of grains per unit area was two to three times higher over the heterochromatin. To check the DNA content, Feulgen photometric measurements were made of Melanoplus nuclei at the same stage. The Feulgen and grain counts agree in showing that the heterochromatin contains two to three times more DNA per unit area than the euchromatin.     

NUCLEOLAR AGING IN TETRAHYMENA DURING THE CULTURAL GROWTH CYCLE

Nucelolar morphology was studied by electron microscopy in control and actinomycin D-treated populations of Tetrahymena pyriformis (W) during the cultural growth cycle. Nucleoli exhibit an "aging" cycle concomitant with the cultural growth cycle, but independent of the individual cell cycle. Four different stages in the course of this aging process have been defined. Stage 1 occurs upon inoculation (low number of cells per milliliter) and lasts through lag and accelerating growth phases. In this stage, many small nucleoli are found at the nuclear periphery. In stages 2 and 3, nucleolar fusion begins. Stage 2 dominates the first half of logarithmic growth, and stage 3 dominates the second half. In late decelerating growth phase, the nucleoli enter stage 4. In this stage, only a few large nucleoli are present and these are apparently inactive in ribosome production. In stationary phase, where total RNA remains constant, only stage 4 nucleoli are present. The relative preponderance of granular vs. fibrous components in the nucleoli changes during this cycle, the granular component dominating stage 1 nucleoli and the fibrillar, stage 4 nucleoli. There is a shortening of the intermediate nucleolar stages in the treated cultures; fusion occurs early and is now pronounced. Not enough ribosomes accumulate to carry the treated cultures through the number of generations equivalent to those of the control, which produces a premature stationary phase.     

A further contribution on nucleoli of human lymphocytes.

Cultured human lymphocytes were investigated by means of light as well as electron microscopic procedures to provide more information on the structural organization of their nucleoli. The transformation of ring shaped nucleoli to nucleoli with less or more distinct nucleolonemata in PHA stimulated cells was characterized by a marked increase of granular RNP components in number indicating the activation of their production. This phenomenon seems to be related not only to the activation of the ribosomal RNA synthesis but also to its processing. The appearance of fibrillar RNP components in the central area of the ring shaped nucleoli apparently represents the first sign of the nucleolar RNA synthesis in these cells. The proportion of fibrillar and granular nucleolar RNP comonents in PHA inresponsive lymphocytes was similar to that in lymphocytes from patients with the usual type of lymphocytic leukemia. The intranucleolar chromatin areas appeared to be larger in PHA stimulated lymphocytes but the proportion of these areas to the nucleolar body did not show substantial difference as compared to the resting cells.     

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.     

Silver staining in pinealocyte nuclei: Observations and quantitative analysis during the rat oestrous cycle

Summary Morphology, distribution and number of different argyrophilic aggregates appearing in pinealocyte nuclei have been studied during the rat oestrous cycle. Using the Ag-NOR reaction we have found two types of argyrophilic aggregates in pinealocyte nuclei. One of them, corresponding to the fibrillar centres and the surrounding fibrillar component, appears in the nucleoli. The silver grains are more loosely packed in the dense fibrillar component than in the fibrillar centres. A decrease in the number of these nucleolar argyrophilic aggregates was obtained at oestrous. A second type of silver grain aggregate was observed in the pinealocyte nucleoplasm. We call them Ag-granule clusters because they are similar to the interchromatin granule clusters and constitute the only silver deposit forming aggregates apart from the nucleolus. The number of Ag-granule clusters is significantly smaller at oestrous and meta-oestrous than at dioestrous and pro-oestrous.