Formation Of Nucleoli Research Articles

The effect of actinomycin D on nucleolar formationin early Xenopus laevis gastrulae.

AbstractElectron microscopic examinations revealed drastic effects of actinomycin D on nucleolar formation in isolated cells of Xenopus laevis embryos. The nucleoli in newly isolated cells of early gastrulae were composed solely of a fibrous component. After culture for two to five hours in complete Stearns' solution without the antibiotic, a granular component appeared among the fibrous components in most of the nucleoli, which showed more elaborated structures. On the other hand, after being cultured for two hours in the presence of actinomycin D (l r/ml), by which inhibition of DNA synthesis was effected, all the nucleoli examined remained as spherical masses composed of fibrous component alone, lacking granular component. After five hours' treatment, inhibition of the synthesis of protein and DNA became biochemically detectable, and ultrastructural changes were observed in both the nucleoplasm and the cytoplasm: that is, fragmentation of nucleoli, condensation of chromatins and deformation of mitochondria. Relationships between the formation of nucleolar components and the RNA synthesis in amphibian embryogenesis are discussed.

Effects of benzamide on embryonic development of the housefly.

This paper deals with the effects of benzamide, a chromosomal RNA inhibitor, on embryonic development of the housefly Musca domestica nebulo. Eggs exposed to benzamide immediately after oviposition continued to develop until the blastema stage, but further development was arrested. Formation of cell boundaries and nucleoli failed to occur and the nuclei at the posterior pole did not differentiate into pole cells. This suggests that synthesis of new RNA is needed for development beyond the blastema stage. Treatment of eggs at different stages of development showed that as development progressed embryos became less sensitive to the drug. Introduction of benzamide into eggs during the pre-blastema period caused irreversible arrest of development, whereas, treatment during post-blastema stages resulted in reversible inhibition of development. The cortical cytoplasm thus appears to induce a significant change in the cleavage nuclei, which alters their sensitivity to the drug.

The idiogram of the sheep with particular reference to secondary constrictions.

Current improved techniques for making human and other mammalian mitotic chromosornc preparations have been used to show that the diploid chromosome number of the domestic sheep is 54 (Gimenez-Martin and LopezSaez, 1962; Borland, 1964; McFee, Banner and Murphree, 1965). This confirms the finding of a number of earlier worlters who used embryo and testicular material for thcir studies (Shivngo, 1930; Berry, 1938, 1941; Ahmed, 1940; Maltino, 1943; Melsnder, 1959). Latterly the X Chromosome has been identified as the largcst of the ncrocentric group of cl~romosomes by ~McFee et al. (1965), who also confirmed the Y to be the smallest chromosome and suggested that it was probably submetacentric. None of these workers has suggested, however, any means of identifying individual chromosomes of either the 3 pairs of large metacentric chromosomes or the 23 pairs of smaller acrocentric chromosomes. Nor have they described the modal chromosome number of counted metaphases from a number of narmal sheep of a similar age group. In man the presence of secondary constrictions on certain chromosomes of the human karyotype is now an accepted aid to identification The London conference on the normal Human Karyotype, 1963). There are, however, varying reports on the incidence of these and their value in cl~romosome recognition. The most constant constrictions in the human karyotype are the terminal satellites which occur on the short arms of all acrocentic chromosomes (Ferguson-Smith and Handmalter, 1961, 1963). These latter chromosomes are now known to be associated with nucleolus formation (FergusonSmith, 1964). Secondary constrictions have been described, however, on a number of the other chromoson~es at various positions on either the short or long arms (Muldal and Ockey, 1961; De la Chapelle, 1961; Ferguson-Smith, Ellis and Dicltson, 1962). The incidence of these secondary constrictions does show, however, considerable variations between different workers (Palmer and Funderburlt. 1965). This has been shown to be associated with various fdctors, including concentration of colchicine (Palmer and Funderburk, 1965), the use of calcium free medium (Sasalti and Maltino, 1963) or B.U.d.R. (5 Bromo deoxyuridine) (Kabaclt, Saksela and Mellman, 1964). None of these workers has discussed the effect of type of hypotonic solution used during Chromosome ~ r e ~ a r a t i o n . 1 1 Interest in disease of domestic animals associated with chromosomal abnormality has already been summarised by Pakes and Griesemer (1965) and Hare, Weber, McFeely and Tsu-Ju Yang (1966). Among the various reports there is only passing reference to secondary constrictions on the chromosomes of the domestic animals except in the cat in which one pair of the small group E chromosomes is satellited (Chu, Thuline and Norby, 1961; Jones 1965).

Membrane formation and membrane contacts during the development of the sea urchin embryo

Sea urchin embryos, 8-cell stage to pluteus stage, fixed in osmium tetroxide and embedded in Epon 812 were observed by electron microscopy. At no point in the development were syncytial junctions between the embryonic cells found. During the cleavage stages the membrane contact was closer than in later stages. In early blastula stages intercellular clefts appeared which in the gastrula stage demarcate every cell. At the same time a ringshaped desmosome structure develops at the outer cell surface. In the pluteus stage a closer cell contact is re-established. With proceeding embryogenesis endoplasmic membranes will attach to the cell membrane. These membrane structures may even be of nuclear origin. Gradually, long protrusions, vesicles and lamellae begin to be formed from the nuclear membrane. The commencement of this nuclear activity coincides in time with the formation of nucleoli. At cell division the new cell membrane seemed to arise partly independently of the cleavage furrow from a system of cytoplasmic vesicles.

The role of Lampbrush Chromosomes in the formation of Nucleoli in Amphibian Oocytes

Theories concerning the mode of origin of peripheral nucleoli in amphibian oocytes have been examined and tested. In Triturus cristatus the giant fusing loops of the 3 shortest lampbrush bivalents resemble nucleoli when viewed in phase contrast and may be considered as possible sites of production of nucleoli. Giant fusing loops, however, differ from peripheral nucleoli in certain important respects, and animals lacking giant fusing loops on their lampbrush chromosomes nevertheless have normal peripheral nucleoli. Therefore, similarity in appearance between objects attached to lampbrush chromosomes and free peripheral nucleoli may not be significant. In oocytes of T. c. carnifex, T. c. karelinii, and T. c. danubialis, peripheral nucleoli do not increase in number during the lampbrush phase of oogenesis, except by division of pre-existing nucleoli towards the end of oogenesis. There are about 1,000 nucleoli per oocyte nucleus in each of these sub-species. In T. c. cristatus there are more nucleoli in large oocytes than in small ones, and it seems likely that in this sub-species the giant fusing loops add to the existing population of nucleoli in an oocyte by successively growing and shedding new nucleoli. A similar situation probably holds in Plethodon cinereus. Hexaploid oocytes from triploid females of Ambystoma jeffersonianum have 3 times as many nucleoli as diploid oocytes from diploid females of the same species. The number of nucleoli in an amphibian oocyte nucleus is therefore related to the number of sets of chromosomes in the cell. In yolky oocytes from hypophysectomized newts most peripheral nucleoli are firmly attached to the inner surface of the nuclear membrane; whereas in similar oocytes from unoperated or gonadotrophin-treated animals none of the nucleoli is so attached. On the basis of these observations 2 mechanisms are suggested for the formation of amphibian oocyte nucleoli. The first of these mechanisms probably operates in T. c. carnifex, where all peripheral nucleoli are formed before or soon after the chromosomes assume the lampbrush form, and no part of a lampbrush chromosome is involved in a process which adds to the existing population of nucleoli. The second mechanism probably operates in T. c. cristatus, where most of the peripheral nucleoli are formed before the lampbrush phase of oogenesis but a nucleolar organizer on the lampbrush chromosomes continues to grow and detach nucleoli throughout oogenesis. Both these mechanisms are discussed in terms of what is known of the chemical composition and function of peripheral nucleoli.

Cytoplasmic regulation of RNA synthesis and nucleolus formation in developing embryos of Xenopus laevis

Pronounced cytological changes, as well as changes in the kinds of RNA synthesized, follow the transplantation of nuclei from embryonic or larval cells of Xenopus laevis to enucleated unfertilized eggs. Nuclei were transplanted into enucleated eggs which contained [ 32 P]phosphate, and the resulting nuclear transplant-embryos showed a pattern of RNA synthesis which was indistinguishable from that of normal embryos developing from fertilized eggs. Although ribo-somal RNA is actively synthesized by neurula endoderm cells and by swimming tadpole gut cells, no synthesis of ribosomal RNA was detected during the cleavage promoted by nuclei transplanted from these kinds of cells. However, synthesis of ribosomal RNA recommences during gastrulation in nuclear-transplant embryos in the same way as in embryos from fertilized eggs. DNA-like RNA and soluble RNA are synthesized during late cleavage by nuclear-transplant embryos as well as by embryos derived from fertilized eggs. Forty minutes after transplantation, nuclei have undergone a 30-fold increase in volume, reaching the same size as zygote nuclei during this period, and their definitive nucleoli disappear. At gastrulation, definitive nucleoli reappear in the nuclear-transplant embryos. These results show that the synthesis of RNA is regulated by the type of cytoplasm in which a nucleus lies.

Nucleoli and cytoplasmic male sterility

The paper describes the relation of cytoplasmic male sterility and the differentiation of nucleoli. Defects in the formation of nucleoli of pollen grains of maize with cytoplasmic male sterility were observed whereas the nuclei of control, i.e. fertile maize contained always only a single nucleole. In the nuclei of sterile pollen grains nucleoli of various sizes and numbers are formed.

THE SITES OF NUCLEOLUS FORMATION IN HUMAN PACHYTENE CHROMOSOMES.

Studies of the pachytene stage of human meiosis in young adult males show that the principal nucleoli are associated with the terminal chromomeres of certain bivalents. These findings are reconcilable with earlier observations on the principal nucleolar chromosome on the basis of the association of bivalents by their nucleolus-forming regions. The pattern of multiple associations suggests that three long and two short bivalents are capable of forming terminal nucleoli. The evidence suggests that these five bivalents are the five pairs of satellited chromosomes in the human mitotic idiogram.

SITES OF FORMATION OF THE EXTRA NUCLEOLI DURING EARLY OOCYTE GROWTH IN THE FRESHWATER TELEOST SALVELINUS FONTINALIS MITCHILL

The behavior of nuclear structures during early oocyte growth in Salvelinus fontinalis is described with special attention focussed on the sites at which the extra nucleoli first appear. In the species investigated, the process of extra nucleoli formation begins at about the mid-pachytene stage of meiotic prophase and proceeds in an uninterrupted fashion until the late strepsitene stage is reached; during that period of time, close to a 150 extra nucleoli form within the nuclear cavity. From the mid-pachytene up to about the mid-strepsitene stage, our observations suggest that, while some of the extra nucleoli arise by successive growth and detachment from a single large heterochromatic body adjacent to the oocyte's original nucleolus, the others develop in contact with minute chromo-centers located in the peripheral portion of the nuclear cavity. The relevant observational evidence would seem to favor the view that the heterochromatic body adjacent to the oocyte's original nucleolus contains one of the four alleged pairs of nucleolar organizing regions of the usual nucleolar chromosomes. During the mid-strepsitene stage, on the other hand, our observations reveal that, while the heterochromatic body and the chromocenters disappear from view, additional extra nucleoli develop at various points along the length of a number of chromosomes. These observations are taken to indicate that non-heterochromatic chromosomal regions distinct from the nucleolar organizing regions of the usual nucleolar chromosomes are, during that period of meiotic prophase, instrumental in the formation of extra nucleoli. The significance of the above findings is discussed in relation to the observations of other workers on the sites of formation of nucleoli in general.

Demonstration of a non-RNA nucleolar fraction by silver staining

Modifications are described of a technique for the silver staining of the nucleoli of plant and animal cells. The technique makes it possible to detect the sequences is the formation of nucleoli in cells. Autoradiographic studies of the same cells show that tritiated thymidine incorporation into micronuclei is correlated with the presence of silver stainable nuclear material. Applications of the technique for the study of nuclear formation and function in a variety of plant and animal cells are described. A non-RNA nuclear fraction was demonstrated by this technique.

Cytogenetic studies of South American diploid Solanums, section tuberarium

Meiosis is regular in a majority of the microsporocytes in a number of clones of six South American diploid species and their hybrids. Meiotic abnormalities such as the presence of univalents, quadrivalents, chromatin bridges and fragments, laggards, formation of restitution nuclei, micronuclei and other irregularities are extant in a small percentage of microsporocytes. The presence of occasional quadrivalents in certain clones of some species and hybrids suggests the existence of some structural differences between certain chromosomes. This probability is further supported by the occurrence of a bridge and fragment resulting from inversion heterozygosity and delayed separation of certain bivalents. The presence of univalents may be caused by segmental or genetic differences between homologous chromosomes as well as to other factors and cannot be entirely explained as being due to precocious separation of rod bivalents. However, the fact that the maximum possible number of bivalents are realized in a large number of the pollen mother cells suggests that such structural differences cannot be very extensive. An unusual type of nucleolus formation occurs. The frequency of various types of secondary groupings is variable.

Heterochromatic regions and nucleolus organizers in chromosomes of the mouse, Mus musculus

The heterochromatic regions of mouse spermatogonial chromosomes have been described. The 38 autosomes have heterochromatic regions adjacent to the kinetochore loci. The X and the Y chromosomes are entirely heterochromatic and are concerned with nucleolus formation.

On the Innervation of Glandulae Sublingualis et Submandibularis and their Surroundings in the Earlier Stage of Human Embryo

The gl. sublingualis and the gl. submandibularis in a human embryo of 3 or 4 months are found as simple branched duct systems sporadically arranged in the loose connective tissue, and their alveoli are formed by undifferentiated gland cells arranged in one row. In the gl. sublingualis, however, some mucosal cells are already formed. The large ducts of these glands are lined by a double-rowd cubic or cylindrical epithelium, but the intercalated and the secretory ducts of the submandibular gland are not yet differentiated and are lined by a single-rowed cubic epithelium. These glands are abundantly provided with blood vessels and nerves.The ganglion submandibulare is extremely well developed and stretching toward the sublingual gland, forms the so-called ganglion sublinguale. Small ganglia are also found migrated into many places in the n. lingualis. More or less thick nerve bundles are found running out of these ganglia into the submandibular and the sublingual glands, and small ganglia are frequently found in these bundles too.The nerve cells in the ganglia are already in a considerably high development. They are in the form of multipolar cells with 3-5 nerve processes and contain each one or sometimes two large nuclei, which are usually eccentric or marginal-standing and show as yet no formation of nucleoli within them. Fine nerve fibrillar nets are contained in the cell bodies.The short processes from the nerve cells are not yet in the stage of ramification, but running only a short course, and in sharp points. One or sometimes two long nerve processes are sent out per cell. The accessory cell plasmodium (STOHR) is as yet very poorly developed, being provided with one or two cell nuclei around a nerve cell. These nuclei are presumed to increse in number with the differentiation of the short processes. The connective tissue capsule covering such a plasmodium is also still very poorly developed.The nerve bundles running in the ganglia submandibulare and sublinguale are already very conspicuously formed. These always consist of fine vegetative fibres, which show mutual nervous anastomosis here and there. The bundles contain chromatin-poor club-shaped SCHWANN's nuclei rather frequently.The external vegetative fibres coming into the ganglia also undergo frequent anastomosis and gradually losing in size, develop finally into the pericellular terminal reticulum common to a number of nerve cells. This reticulum is formed of fine nerve fibrils showing frequent change in size and forming an irregular mesh-work. No direct connection seems to exist between the reticulum and the nerve cells.The fibres in the numerous bundles running into the two glands comprise both sympathetic and parasympathetic fibres, but it is impossible to distinguish between the two kinds. The development of the vegetative periarterial plexus formed around the arteries is already much advanced. This plexus here and there anastomosis with the vegetative nerve bundles mentioned above.The termination of the vegetative fibres distributed in the salivary glands is also represented by the STOHR's terminal reticulum formed of finest fibrills and extending cordwise. The mesh-work of the reticulum in the glands is finer and more regular than that of the pericellular terminal reticulum above. SCHWANN's nuclei are frequently found in this reticulum too. The terminal reticulum is in particularly good formation around the alveoli and the ducts of the glands and diffuses closely in contact of the epithelial cells, but apparently never send out side branches into their cell bodies. The terminal reticulum stands in tactile control over the smooth muscle fibres in the arterial walls, and is frequently found in the mucous membrane of the bottom of the oral cavity too.The sensory fibres originating in the n. lingualis, though as yet unprovided with myelin sheaths, are much thicker than the vegetative fibres.

Chromosomes and nucleoli in Pisum sativum.

SINCE the beginning of the century, reports have been published in which an ideogram has been given, or at least the morphology of the chromosomes has been described, of Pisum sativum. With improved techniques and with a better understanding of chromosome structure, it is reasonable to suppose that recent workers would be in agreement. In the latest observations1, all authors agree that there are fourteen chromosomes in a normal diploid cell; but here agreement ends. There is disagreement over the positions of the centromeres, the number of chromosomes with satellites, and the position of these satellites and their connexion with the nucleoli. In addition, a number of secondary constrictions (non-nucleolar) have appeared from time to time in nearly all the chromosomes. The majority of these constrictions can, we believe, be traced to the action of the drugs used on the cells prior to fixation. The doubts over the relationship of satellites and nucleoli have been prevalent since 1942, when Hakansson and Levan2 reported that nucleoli are formed at the centromeres of all the chromosomes and that the visible constrictions in the chromosomes are not associated with nucleolus formation.

Chromosomal control of nuclear composition in maize.

1. The composition of microsporocyte nucleoli of maize with different chromosomal constitutions was studied by the analysis of absorption spectra obtained from an ultraviolet microspectrophotometer. 2. The presence of ribonucleic acids (RNA) and proteins in the nucleolus was confirmed by the presence of two broad and overlapping absorption peaks around 2637 A and 2800 A. The former peak was removable by treatment of the tissue sections with cold perchloric acid. 3. The amount of RNA per nucleolus was determined by measuring the absorbance and the diameter and thickness of nucleolus sections at 2637 A. Non-specific light loss and absorption due to proteins were corrected for by the use of blank slides which were subjected to cold perchloric acid extraction. 4. By means of the above technique, the amount of RNA per nucleolus was found to be significantly different in the various strains of maize studied. 5. The size of and amount of RNA in the nucleolus were found to increase until mid-pachynema and then diminish and finally disappear at late diakinesis. The increase in volume was found to lag behind that in the RNA content. This observation, together with the observation that the RNA/protein ratio went down during the same period, was interpreted as indicating that during the growth of the nucleolus the RNA content increases faster than does the protein content, and that the synthesis or incorporation of proteins into the nucleolus is dependent upon RNA. The RNA content of the nucleolus was found to have doubled at some time between mid-leptonema and zygonema, an increase which was thought to be the result of the reduplication of the nucleolar, organizer during leptonema. 6. A linear relation was established between the RNA content of the nucleolus and the number of extra nucleolar organizers present on supernumerary B6 chromosomes. The extra organizers did not change the RNA/protein ratio of the nucleolus. Extra heterochromatin in the supernumerary B chromosomes was found to increase the RNA content of the nucleolus only very slightly. 7. Extra euchromatin was believed to have no appreciable effect on nucleolar composition since the RNA content of nucleoli from triploid plants, like that of nucleoli from plants trisomic for the nucleolar chromosome, was only three-halves as much as in their respective normal diploid siblings. 8. The entire nucleolar chromosome is probably involved in nucleolus formation since changes in the RNA content, though not in the RNA/protein ratio, were found in plants carrying a translocation involving the nucleolar chromosome.