Nucleoli Of Oocytes Research Articles

Uptake and cellular localization of 3H-1-methyladenine and 3H-adenine in the starfish gonad and oocytes

Incorporation and distribution in vitro of radioactive purines ( 3H-1-methyladenine and 3H-adenine) into the various components of the starfish ovary were studied in relation to spawning induced by the hormone 1-methyladenine. Uptake of radio-active hormone from the medium was proportional to its concentration in the medium and the quantity of ovarian tissue present in the incubation mixture, but was in great excess of that needed for physiological effects. More than 95% of the incorporated isotope was localized in the ovarian wall, with only a small proportion accumulating in the oocytes. Isolated oocytes incorporated 3H-1-methyladenine independent of ovarian tissue. Disruption of the cellular structure of the ovarian tissue resulted in rapid loss of the radioactivity retained in the ovary. Most of the incorporated radioactivity released from ovarian tissue was dialyzable and not precipitated by TCA. This suggests that 1-methyladenine remains as a small molecular weight compound during its incorporation into the ovary. The distribution of 3H-adenine and 3H-1-methyladenine in the gonad was compared autoradiographically. 3H-1-Methyladenine was primarily localized in the outer ovarian epithelium and in the nucleoli of a few immature oocytes in the peripheral region of the ovary. Follicle cells were unlabeled, as were the oocytes which had undergone germinal vesicle breakdown. 3H-Adenine in contrast was localized in the ovarian wall, follicle cells, and the nucleoli of all the oocytes following a 4-hr incubation. These concentrations of isotope were observed in the absence of oocyte maturation. Follicle cells and possibly the oocytes, therefore, appear to exhibit a high degree of selectivity with regard to the structure of the purine incorporated. Specificity of purine retention was not correlated with physiological activity.

Maturational cleavage of nucleolar ribosomal RNA precursor can be catalyzed by non-specific endonuclease

A cell-free system has been developed that allows incubation of prelabeled oocyte nucleoli, with no spontaneous processing of the 32-S rRNA precursor. It is shown that the addition of a nuclear lysate of embryos, of a stage that in vivo shows a fast rRNA maturation, causes the specific cleavage of the 32-S rRNA precursor into the 27-S and 21-S products. This specific cleavage can also be induced by the addition of a non-specific endonuclease such as the bovine pancreatic ribonuclease. Specific cleavage of the purified 32-S RNA with pancreatic RNAase is much more difficult to achieve. It is inferred that the nucleolar proteins protect the 32-S rRNA precursor from the endonuclease attack, leaving it much more exposed at the site where it has to be cut for the maturational processing.

Transcription of ribosomal RNA cistrons: Correlation of morphological and biochemical data

Electron microscopic spread preparations of oocyte nucleoli (lampbrush stage) of various amphibians are quantitatively evaluated and the length distributions of repeat-, matrix-, and spacer-units along the rRNA cistron containing axes are given. The correlation of the matrix unit data with the gel electrophoretic pattern of labelled nuclear RNA from the same oocytes is examined. The mean value of the matrix unit corresponds fairly well to a 2.6 million D peak of pre-rRNA but the distribution of both matrix units and labelled pre-rRNAs shows an asymmetrical heterogeneity indicating the existence of some larger primary transcription products of rDNA. Novel structural aspects are described in the spacer regions which suggest that transcription does also take place in DNP regions between the matrix units. A special ‘prelude piece’ coding for approx. 0.5 million D of RNA is frequently visualized in the spacer segments at the beginning of a matrix unit. Possible artifacts resulting from the preparation, the relative congruence between the data obtained using both methods, and the functional meaning of the findings are discussed against the background of current concepts of structural organization and transcription products of nucleolar DNA.

Proteins of the newt germinal vesicle nucleus.

THE major chemical constituent of the lampbrush chromosomes and nucleoli of newt oocytes is known to be protein1,2. We report here the semi-bulk fractionation of germinal vesicles, isolated manually from oocytes of Triturus cristatus carnifex, into nucleoplasm and a “chromatin” preparation consisting of lampbrush chromosomes, nucleoli, nucleoplasmic granules and nuclear membranes. Two properties of the proteins in the chromatin have emerged. First, protein–disulphide-bond crosslinks make a definite contribution to the morphological integrity of the isolated lampbrush chromosomes and nucleoli. Second, the non-histone proteins of oocyte chromatin consist predominantly of two major polypeptide species.

Nuclear pore flow rate of ribosomal RNA and chain growth rate of its precursor during oogenesis of Xenopus laevis

The number of ribosomal RNA molecules which are transferred through an average nuclear pore complex per minute into the cytoplasm (nuclear pore flow rate, NPFR) during oocyte growth of Xenopus laevis is estimated. The NPFR calculations are based on determinations of the increase of cytoplasmic rRNA content during defined time intervals and of the total number of pore complexes in the respective oogenesis stages. In the mid-lampbrush stage (500–700 μm oocyte diameter) the NPFR is maximal with 2.62 rRNA molecules/pore/minute. Then it decreases to zero at the end of oogenesis. The nucleocytoplasmic RNA flow rates determined are compared with corresponding values of other cell types. The molecular weight of the rRNA precursor transcribed in the extrachromosomal nucleoli of Xenopus lampbrush stage oocytes is determined by acrylamide gel electrophoresis to be 2.5 × 10 6 daltons. From the temporal increase of cytoplasmic rRNA (3.8 μg per oocyte in 38 days) and the known number of simultaneously growing precursor molecules in the nucleus the chain growth rate of the 40 S precursor RNA is estimated to be 34 nucleotides per second.

The nucleolar organizer of Plethodon cinereus cinereus (Green). I. Location of the nucleolar organizer by in situ nucleic acid hybridization.

The 7th longest lampbrush bivalent in oocytes of Plethodon cinereus cinereus has a region of attached oocyte nucleoli near to the centromere on the shorter arm of each half bivalent. When squash preparations of P. c. cinereus spermatocytes are treated with NaOH to denature chromosomal DNA, and subsequently incubated in a solution of (3H) ribosomal RNA from Xenopus cell cultures, the ribosomal RNA binds specifically to a region near the centromeres on the shorter arms of the 7th longest bivalent, and to a region near the end of the shorter arm of the 14th bivalent. The amount of ribosomal RNA bound to the 7th bivalent at diplotene and 1st meiotic metaphase is regularly different on the 2 halves of the bivalent. Each half of the 14th bivalent is usually labelled more heavily than the less heavily labelled half of the 7th bivalent. These observations are discussed in relation to the involvement of nucleolar organizers in gene amplification and rectification.

Chromosomal localization of repetitive DNA in the newt, Triturus.

The repetitive DNA sequences of the newt, Triturus viridescens, have been studied by nucleic acid hybridization procedures. Complementary RNA was synthesized enzymatically from unfractionated newt DNA. This RNA hybridized strongly to the centromeric regions of both somatic and lampbrush chromosomes It also bound to other loci scattered along the lengths of the chromosomes The amplified ribosomal DNA in the multiple oocyte nucleoli was demonstrated by in situ hybridization

The nucleolus and its genes in amphibian oogenesis.

The nucleolus and its genes in amphibian oogenesis.

The effect of actinomycin D in vivo upon peripheral nucleoli and other nuclear organelles in oocytes of Triturus cristatus.

ABSTRACT Exposure of the ovaries of Triturus cristatus to actinomycin D at a concentration of 100 /tg/ml causes characteristic changes in the peripheral nucleoli and other nuclear organelles in oocytes of o·6–1·1 mm diameter. Viewed with the light microscope untreated oocytes contain nucleoli that stain uniformly with a variety of dyes. They also appear homogeneous under phasecontrast optics. After 2 or 4 h of in vivo incubation with actinomycin D, oocyte sections stained with Haidenhain’s haematoxylin or viewed under phase-contrast optics show nucleoli composed of 2 regions. The more heavily stained or contrasted zone is crescent-shaped and directed away from the nuclear membrane. Neither sections stained with azure B bromide nor gallocyanin chrome alum show this feature. Ribonuclease digestion does not eliminate or alter it. Autoradiography with [3H]uridine indicates that all recently synthesized RNA is lost from the nucleolus during actinomycin D treatment. The zonation is not therefore a reflexion of RNA distribution. During recovery from actinomycin D poisoning there is a reduction in the degree of zonation shown by nucleoli which re-establish a normal appearance some 48 h after treatment. Electron microscopy of peripheral nucleoli in oocytes sampled during this treatment indicates that the zonation is not associated with reorganization of ultrastructural components. During incubation with actinomycin D the coarse granules (20 nm diameter) are completely lost from the nucleolus. There is associated shrinkage of the nucleolus which after treatment is found to consist entirely of fibrils (5 nm thick) and small granules. The reappearance of the coarse granules during recovery is completed in about 48 h. It is thought that the loss of the granular component during treatment represents the movement of the 30-s precursor and the 18-s ribosomal unit from the nucleolus. Some 20–30 μm inside the nucleus of untreated oocytes is a region containing many spheroidal bodies, less than 1·0 μm diameter. They have been termed micronucleoli and consist of granules 2·5–5 nm in diameter and fibrils of similar thickness. Actinomycin D treatment causes these components to segregate and eventually (within 24 h of treatment) the granular component is extruded. This component reappears during the second day after treatment. It is postulated that these micronucleoli represent the sites at which the 30-s ribosomal precursor undergoes its final maturation. The segregation of components induced by actinomycin D is probably the morphological manifestation of an abnormal metamorphosis of this precursor. Treatment with actinomycin D also induces the immediate formation within the nucleus of crystalline bodies composed of lamellae 16 nm wide, 4 nm thick and with a centre-to-centre spacing of 8–10 nm. They are not present 24 h after treatment. They are thought to represent a protein fraction normally associated with periods of intense RNA synthesis.

The formation of "accessory nuclei" and annulate lamellae in the oöcytes of the flour moth Anagasta kühniella.

Oocytes in various stages of maturation were studied with the electron microscope. Stacks of annulate lamellae were found attached to large ribosome studded vesicles. The outer nuclear membrane was found to form blebs and it is considered that these produce the large vesicles. It is suggested that mRNA enters the cytoplasm in membraneous vesicles which collapse, liberating the mRNA, and thus produce annulate lamellae which may later form E.R. Oocyte nuclei were examined in the E.M. as a potential source of accessory nuclei. Membrane bound organelles containing granules were found in the periphery of the nucleus. The granules were shown to be extruded from the oocyte nucleolus. The timing of the disappearance of these organelles from the nucleus, together with their structure, suggests that they are precursors of accessory nuclei.

Interlocking and knotting of ring nucleoli in amphibian oocytes.

Various configurations of interlocking and knotting of ring nucleoli from amphibian oocytes have been identified in material taken from 2 females of Eurycea bislineata and one female of Plethodon cinereus. The simplest configuration is a simple interlock between 2 rings of similar or different lengths. More complex interlocks have been seen, in which 3 rings are linked together in such a way that they cannot be extended into a chain. A third configuration involves complex knotting of single long rings. It is suggested that the interlocked configurations arise through the replication of rings of DNA that have low levels of supercoiling, and that the knotted rings arise by misjoining of the ends of linear molecules that have become wound around one another. Models are suggested in support of these proposals and the significance of interlocking of ring nucleoli in relation to the mechanism of gene amplification is discussed.

A light- and electron-microscope study of the nucleolus during growth of the oocyte in the prepubertal mouse.

ABSTRACT The ordered changes which occur in the structural organization of the nucleolus during growth of the mouse oocyte have been studied by both light and electron microscopy. All observations have been made on those oocytes whose growth is initiated on the day of birth and completed by postnatal day 14 in prepubertal animals of the ICR albino mouse strain. During that period the oocyte nucleolus undergoes an approximate 90-fold increase in volume. During the unilaminar follicle stage (from birth to postnatal day 4), the growing nucleolus exhibits an overall reticulated-type of structure consisting of: (1) a moderately electron-dense fibrillogranular component occupying most parts of the nucleolar framework; (2) an electron-transparent nucleoplasm-like component filling the numerous interstices of the nucleolar framework; (3) an electron-dense fibrillar component located in the peripheral portion of a number of small islands widely and uniformly scattered within the nucleolar framework, and (4) a slightly less-dense fibrillar component situated in the central portion of these same islands and referred to as fibrillar centres. Increase in nucleolar volume during that stage is brought about mainly through an increase in the overall dimensions of the fibrillogranular framework, accompanied by a parallel increase in the number and, to a certain extent, the size of its electron-transparent interstices. During the bilaminar follicle stage (postnatal day 5 through 8), the following structural and organizational changes take place more or less concomitantly within the still enlarging nucleolar mass: (1) the fibrillogranular framework becomes predominantly fibrillar in texture as a result of what appears to be an unravelling or unfolding of its constituent granules of ribosomal dimensions; (2) the nucleolar interstices decrease rapidly both in number and size because of the accumulation within their interior of a material the texture and density of which match that present in the nucleolar framework itself; and (3) a number of rounded electron-transparent spaces, the nucleolar vacuoles, make their appearance in the regions formerly occupied by some of the fibrillar islands and adjacent interstices. Increase in nucleolar volume during that stage is largely due to the appearance and subsequent enlargement of the nucleolar vacuoles in question. During the plurilaminar follicle stage (postnatal day 9 through 14), the following sequential events take place within the nucleolar mass: (1) a moderately electron-dense fibrillogranular material accumulates within the nucleolar vacuoles; (2) this fibrillogranular material, which eventually fills all vacuolar spaces, undergoes degranulation and a concomitant increase in density, eventually matching that of the rest of the nucleolar mass; (3) ail remnants of the lightly stained nucleolar interstices disappear from view; and (4) the fully grown rounded nucleolus finally appears as a dense, compact mass, exclusively fibrillar in texture, and exhibiting no internal structural organization. An attempt is made to interpret these changes in the light of current knowledge concerning the architectural and functional organization of the mammalian nucleolus in general. The observations are consistent with the view that the nucleolus, during growth of the primary oocyte, is the site of massive synthesis and storage of nucleolar material.

Some measurements on amphibian oocyte nucleoli.

The dry mass of nucleoli from yolky oocytes of T. cristatus, as determined by interference microscopy is about 150 μμg. Less than 10% of this is attributable to RNA that is removable by ribonuclease from formalin-fixed nucleoli. There is a linear relationship between nucleolar dry mass and size. There are basic proteins in oocyte nucleoli and these are bound to the nucleolar RNA. The concentrations of cytochemically detectable RNA and basic protein are higher in the nucleolar core than in the cortex. The ratio of RNA to basic protein is higher in the core than in the cortex, as determined by microdensitometry of nucleoli stained with gallocyanine and fast green. The significance of these observations in relation to the role of the nucleolus in ribosome biosynthesis is discussed.

Observations on the membranous components of amphibian oocyte nucleoli.

ABSTRACT Nucleoli in some large yolky oocytes of Plethodon cinerens and Ascaphus truei have attached to them a filament which may or may not have a beaded appearance. These filaments have been called ‘nucleolar tails’. One nucleus may contain several hundred of them. They may be up to 200 μm long and are usually about 1 μm wide. The beads which are sometimes attached to the filament are round, refractile and up to 3 μm in diameter. The tails are a feature of nuclei in which the nucleoli have left the nuclear envelope and migrated inward to cluster around the chromosomes in the centre of the nucleus. The tails project radially outwards from the central mass of nucleoli. Tails may be attached to round solid nucleoli, as is usually the case in A. truei, or they may be attached to ring nucleoli, as is often the case in P. cinereus, or they may lie free in the nuclear sap. Those nucleoli which remain attached to the nucleolar organizing site on the lampbrush chromosomes of P. cinereus never have tails. Nucleolar tails are Feulgen-negative and do not stain with gallocyanine when it is used under the proper conditions for staining nucleic acids. Tails are unaffected by deoxyribonuclease and ribonuclease but are destroyed by proteolytic enzymes. Thin sections of oocytes whose nuclei contained nucleolar tails showed chains of membranous vesicles lying near to many of the nucleoli. These vesicles appeared empty, some of them were confluent with their neighbours, all were bounded by a unit membrane and all were coated on their outer surfaces by a fine fibrous material. The chains of vesicles commonly ended near a depression on the surface of the core of a nucleolus. Chains of vesicles sometimes extended into a long membranous tube. We have interpreted these chains of vesicles and tubes as sections through nucleolar tails. It is suggested that nucleolar tails may be associated with the replication of nucleolar DNA, or with the division of nucleolar cores and nucleoli, or with the production of nuclear membrane at a time when the nucleus is enlarging rapidly. Each of these suggestions is discussed. A likeness between nucleolar tails and the mesosomes of bacteria is proposed and discussed.

Cytodifferentiation in the Rana pipiens oocyte : I. Association between mitochondria and nucleolus-like bodies in young oocytes

Small oocytes (approximately 125 μ in diameter) in adult Rana pipiens contain a number of cytoplasmic bodies which resemble nucleoli in size, shape, and staining reactions. These cytoplasmic nucleolus-like bodies are, however, more finely granular than nucleoli. The nucleolus-like bodies are surrounded by “satellite” bodies, “cytoplasmic cyclinders”, both “empty” and “yolk-containing” vesicles, and numerous mitochondria. Some of the mitochondria associated with the nucleolus-like bodies have small deposits of intracristal, crystalline yolk protein. The nucleolus-like bodies appear to contain thin filaments as well as small granules (40–80 A) as are also apparent in some of the associated mitochondria. Filaments are also present between the inner and outer mitochondrial membranes at a point where the mitochondria abut upon the nucleolus-like bodies; this condition may reflect an exchange mechanism. Structural variations in the nucleoli of young oocytes are also described. The possible significance of the observations in terms of informational exchange between the nucleus and cytoplasm and mitochondrial function in the frog oocyte is discussed.

Visualization of nucleolar genes.

The presence of extrachromosomal nucleoli in amphibian oocytes has permitted isolation and electron microscopic observation of the genes coding for ribosomal RNA precursor molecules. Visualization of these genes is possible because many precursor molecules are simultaneously synthesized on each gene. Individual genes are separated by stretches of DNA that apparently are not transcribed at the time of synthesis of precursor rRNA in the extrachromosomal nucleoli.

The origin of multiple oocyte nucleoli from accessory DNA bodies in Gryllus domesticus

The origin of multiple oocyte nucleoli from accessory DNA bodies in Gryllus domesticus

Gene amplification in the oocyte nucleus of mutant and wild-type Xenopus laevis.

DURING the development of the amphibian oocyte, the germinal vesicle becomes enlarged and can be seen to contain very many nucleoli1,2 instead of the four which would be expected from the tetraploidy of the egg nucleus. The RNA of these nucleoli is similar in base composition to ribosomal RNA3 and each nucleolus contains a small amount of DNA4–6. Amphibian oocyte nucleoli can be hypotonically disrupted to reveal DNP rings in the shape of beaded circles or necklaces2,6. There is compelling evidence in amphibia2,6,7 as well as in the insect Tipula8 that this DNA is derived from the chromosomal nucleolar organizer region which contains the ribosomal cistrons9,10. This conclusion is supported by the recent finding that in ovarian tissue of Xenopus there is a disproportionately greater synthesis of ribosomal cistrons11 which leads eventually to a higher proportion of them than that found in DNA of somatic tissues11,12. In Bufo, the nucleolar DNA migrates as granules from the chromosomal bouquet to the periphery of the nucleus where, at later stages, the granules give rise to nucleoli5. A similar process occurs in Xenopus where Feulgen-positive material appears in the oocyte nucleus as a “nuclear cap” at leptotene13. After zygotene this cap disperses and numerous nucleoli are formed which contain one or several DNA cores or rings14.

Spheroidal and ring nucleoli in amphibian oocytes. Patterns of uridine incorporation and fine structural features.

In maturing oocytes of the newt Triturus viridescens, the nucleoli undergo a series of morphological changes that are very similar to those described by Callan for the axolotl, Ambystoma mexicanum. The nucleoli first assume the form of spheroids which then become extended into ring or necklace shapes that are DNase-sensitive; in mature oocytes the nucleoli revert to a spheroidal form. Short term in vitro incorporation studies with uridine-(3)H on both species show that RNA synthesis occurs in a restricted, eccentric portion of the spheroidal nucleoli, thereby producing an asymmetrical pattern of labeling. In the ring forms, however, the localization of the radioactivity suggests that synthesis takes place symmetrically throughout their entire length. The changes in nucleolar morphology apparently reflect the fact that the component DNA has undergone a redistribution from a localized region in the spheroidal nucleoli to an extended circle in the rings; the patterns of uridine-(3)H incorporation, therefore, parallel the distribution of DNA in both the spheroidal and the ring nucleoli. Ultrastructurally, the nucleoli contain a fibrillar component that corresponds in position to that of the DNA. The typical spheroidal nucleolus consists of a fibrillar core situated eccentrically and surrounded by a hull of granular, ribonucleoprotein material. The ring nucleoli are composed of a central fibrous region that is ensheathed all around its circumference by a layer of similar granular material. This granular substance is thicker at intervals along the length of the rings, representing the "beads" of the necklaces.

The occurrence of ribosomal proteins in nucleoli of starfish oocytes

The occurrence of ribosomal proteins in nucleoli of starfish oocytes