No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Localization and structure of nuclear organizers in the oocyte during meiotic prophase I.
Abstract
The localization, structure and activity of nuclear organizers were studied in quail and mouse oocytes during prophase I of meiosis using electron microscopy and in situ hybridization with tritium labelled 18S and 28S RNA. The newly synthesized nucleolus consists of a fibrillar centre surrounded by a layer of electron-opaque fibrils. DNP fibres with a diameter of about 50 Å extend from the nucleolar organizer to the fibrillar centre and penetrate into the latter. At the onset of nucleolar synthesis, the position of the fibrillar centre coincides with that of the ribosomal cistrons revealed by in situ hydridization. Following brief incorporation of tritiated uridine, labelling grains are localized over the electron-opaque fibrillar layer adjacent to the fibrillar centre. These observations indicate that rDNA is present in both the fibrillar centre and the dense fibrillar layer which are always in association, and that transcription of the ribosomal cistrons essentially occurs in the dense fibrillar layer.
... The main category of such sequences is rDNA and centromeres (Table 1). However, rDNA is expelled from the NPB mass upon termination of transcription by RNA polymerase I [11,24] and should therefore suffer only very limited damage in the transcriptionally silent oocytes. When analysed, metaphase II oocytes that had been previously enucleated show morphologically intact and lesion-free chromosomes with well-defined pericentric chromatin, centromeres and telomeres, suggesting that chromosomes remain intact after enucleolation [23]. ...
In mammals, mature oocytes and early preimplantation embryos contain transcriptionally inactive structures termed nucleolus precursor bodies instead of the typical fibrillo-granular nucleoli. These nuclear organelles are essential and strictly of maternal origin - if removed from oocytes, the resulting embryos are unable to replace them and consequently fail to develop. Historically, nucleolus precursor bodies have been perceived as a passive repository site of nucleolar proteins that are required for embryos to form fully functional nucleoli. Recent results, however, contradict this long standing dogma and show that these organelles are dispensable for nucleologenesis and ribosome biogenesis. In this article we discuss the possible roles of nucleolus precursor bodies and propose how they might be involved in embryogenesis. Furthermore, we argue that these organelles are essential only shortly after fertilization and suggest that they might actively participate in centromeric chromatin establishment.
... C'est en fin de pachytène que sa réorganisation et sa croissance reprennent. L'organisation du nucléole et l'étude des synthèses d'ARN et d'ADN ont fait l'objet de nombreuses descriptions ultrastructurales (rat : Schuchner, 1975 ;chat : Morato, 1965 ;champignons : Stockert et al., 1970 ;plantes : La Cour, 1975 ;souris et cailie : Mirre et Stahl, 1976homme : Très, 1975 ;Stahl et al., 1978Stahl et al., , 1980Hartung et al., 1979 ;Mirre et al., 1980). Cette évolution des composés nucléolaires est en relation avec les synthèses nucléaires. ...
... L'amplification des gènes ribosomiques multiplie par 1 000 la capacité de l'ovocyte àOakberg, 1967 ; Moore et al., 1974)Howell et al., 1975 ; Howell, 1977 ; Schwarzacher et al., 1977 Schwarzacher et al., , 1978).Ford,1966 ; Dev et al.,1971 ; Eicher, 1972). La présence de rDNA dans la constriction secondaire a été vérifiée par hybridation in situ dans les cellules somatiques (Elsevier et Ruddle, 1975 ; Henderson et al., 1976) et dans les cellules germinales (Stahl et al., 1977Stahl et al., , 1978) Nous avons constaté que la transcription du rDNA est très active dans l'ovogonie, active également au stade leptotène. Elle décroît ensuite au zygotène pour devenir nulle au pachytène. ...
... Certains auteurs relataient son absence en fin de leptotène et sa présence au diplotène (porc : Black et Erickson, 1968 ;hamster : Challoner, 1974). En étude ultrastructurale, le nucléole est cependant décrit tout au long de la méiose (Franchi et Mandl, 1962 ;Schuchner, 1975 ;Stahl et al., 1978). L'évolution des composés nucléolaires est en relation avec les synthèses nucléaires. ...
The structure of small ovarian follicle granulosa cells (450 μm mean diameter) of three duck categories was studied during two periods of the year: the month of January (weak ovarian activity) and the month of May (strong ovarian activity). Between the two activity phases a very distinct difference is observed in most cytoplasmic organelles but little variation in nuclear constituents. Indeed the types of nucleoli encountered do not vary from one season to another and the nucleolar activity is probably not very different in January and May. On the contrary, the number and size of all the cytoplasmic organelles vary considerably. They are scarce and little developed in resting phase whereas they are abundant and developed during the activity phase; during this phase, the granulosa cells are probably oriented towards protein synthesis.
This chapter summarizes current data concerning the ultrastructural localization of transcription sites and the subsequent distribution of newly synthesized ribonucleic acid (RNA) within the cell nucleus. It discusses the roles of ribonucleoprotein (RNP)-containing nuclear structures with respect to the synthesis and processing of nucleolar and extranucleolar RNA. RNA transcription in the nucleolar, as well as extranucleolar, areas takes place predominantly within the regions situated on the border of condensed chromatin. Perichromatin fibrils, considered as the first in situ detected structures containing newly transcribed extranucleolar RNA, originates in these perichromatin regions and the nucleolar fibrillar components are in intimate contact with intranucleolar chromatin areas. DNA replication sites also appear predominantly within this nuclear region. This particular nuclear area consequently plays a key role in the expression of chromatin functions and these findings lead to the conclusion that the layer of chromatin decondensing on the periphery of dense chromatin areas represents the most active chromatin fraction in the nucleus.
Methylation of nucleolar RNA was studied by autoradiography in HeLa cells using L-[methyl-3H]methionine and S-adenosyl-L-[methyl-3H]methionine as radioactive precursors. Pulse-labeling experiments show that nucleolar RNA methylation occurs on the newly synthesized RNA at the nucleolar fibrillar RNP component and mostly on the fibrillar ring of fibrillar centers, where pre-rRNA is being synthesized. Pulse-chase experiments show a shift of silver grains from the nucleolar fibrillar RNP component to the nucleolar granular component first and then to the cytoplasm. Labeling of nucleolar RNA via specific methylation permits the study of intranucleolar processing of pre-rRNA and confirms the sequence of labeling of the two nucleolar RNP components observed with radioactive uridine.
Mouse spermatocytes at pachytene stage have been examined by whole-mount electron microscope techniques complemented with autoradiography as an approach for visualizing their transcriptive activity. Structural elements of meiotic bivalents, such as synaptonemal complexes and chromatin fibers, have been satisfactorily displayed in the total set of autosomal and sexual bivalents in single spermatocytes. Adequate preservation of the entire set of bivalents has provided a basis for recognition of sites where presumptive preribosomal RNA and heterogeneous nuclear RNA species are being transcribed at different segments of autosomal bivalents. Nucleoli attached to the basal knob region where nucleolar organizer cistrons are assumed to be located and ribonucleoprotein fibrils associated with distinct chromatin loops have been recognized. These structural findings have been correlated with display of [(3)H]uridine incorporation sites in thin-section and whole-mount electron microscopy autoradiographic preparations. A low transcriptive activity of the sexual bivalent contrasted with extensive gene expression in autosomal bivalents. Each sex chromosome shows a double axial core. A short region of pairing with a synaptonemal complex joins the two chromosomes at one end. We conclude that variations in the rate of RNA synthesis throughout meiotic prophase stages in the mouse are expressed as fluctuations in the amount and distribution of distinct RNA species at specific segments of the bivalents.
Transmission electron microscopy is a powerful technique for studying the three-dimensional (3D) structure of a wide range of biological specimens. Knowledge of this structure is crucial for fully understanding complex relationships among macromolecular complexes and organelles in living cells. In this paper, we present the principles and main application domains of 3D transmission electron microscopy in structural biology. Moreover, we survey current developments needed in this field, and discuss the close relationship of 3D transmission electron microscopy with other experimental techniques aimed at obtaining structural and dynamical information from the scale of whole living cells to atomic structure of macromolecular complexes.
Monkey kidney cell cultures are labeled in pulses with tritiated uridine for 5, 10 and 30 min. At the end of these pulses the cells are fixed and embedded according to the technics of ultrastructural cytochemistry. These technics give a good differentiation of the associated and intranucleolar chromatin. Enzymic digestions (Pepsin or RNAase) are carried out on some specimens before the layering of the emulsion (NUC 307 Gevaert) for the electron autoradiography. The association of ultrastructural cytochemistry and electron autoradiography on the same cellular material demonstrates the synthesis of nucleolar RNA on the DNA linked to the nucleolus. This newly synthesized RNA is already present within the fibrillar zones of the nucleolus 5 min after the label is given. It begins to appear within the granular zones after 10 min and has spread over the nucleolus after 30 min. In the nucleus the regions of dispersed chromatin are the active sites of synthesis of the rapidly labeled RNA. The labeled RNA completely disappears in ultrathin sections treated with RNAase.The RNA present in the fibrillar zones of the nucleolus can represent the obligatory precursor of the ribosomal RNA. The RNA synthesized on the dispersed zones of chromatin in the nucleus could correspond to the messenger RNA.
Cytoplasmic polyribosomes from baby hamster kidney cells were isolated in high yield by homogenization in a high salt-nonionic detergent mixture. The polyribosomes were protected from degradation after deoxycholate solubilization of membranes by using dextran sulfate. As much as 80% of the total cytoplasmic ribosomes existed as polyribosomes, with more than 90% of the nascent polypeptides associated with polysomes sedimenting heavier than monomers.Cell-free incorporation of isotopic amino acids into proteins using purified baby hamster kidney polyribosomes ranged from 200 to 1200 pmoles/mg ribosomes per h depending upon the source of 100 000 × g supernatant enzyme.Additions of poly U or foot-and-mouth disease virus RNA to polyribosomes inhibited amino acid incorporation into protein. Stimulation of amino acid incorporation by poly U or foot-and-mouth disease virus RNA was observed only after preincubation or salt shocking the polyribosomes. Such dissociated polyribosomes appeared more responsive than intact polyribosomes to stimulation of amino acid incorporation by exogenous RNA.
The present study has been mainly focused on the nucleolar cycle in the slime mould Physarum polycephalum. The ultrastructural characteristics of the interphase nucleolus, in this species, are quite similar to those of nucleoli in other organisms: it is essentially constituted of large particulate zones surrounding denser regions which are predominantly fibrillar in texture. The latter nucleolar zones, following fixation with osmium tetroxide, are characterized by the presence of opaque granules approximately 25 nm in diameter. Contrary to the situation which generally prevails in other eukaryotes, the late prophase nucleolus fragments into numerous globular bodies which are recognizable by the presence of opaque particles. These fibrillogranular nucleolar fragments persist during mitosis and are observed to become incorporated in the newly formed nucleolus. High-resolution radioautographic observations reveal that these nucleolar remnants contain DNA. The present observations together with recent biochemical data from other authors on the characteristics and mode of duplication of nucleolar DNA in P. polycephalum have led us to the hypothesis that the nucleolus, in this organism, contains several distinct globular subunits each containing ribosomal DNA as a key component. The existence of such morphological subunits appears to account for the unusual behaviour of the nucleolus during the cell cycle.
The nucleolar organizers have been localized in the Japanese quail oocyte at pachytene and diplotene stages using hybridization in situ with rRNA. Autoradiography reveals that 8 microchromosomes, forming 4 bivalents at pachytene, contain ribosomal cistrons. The silver grains are located on the microchromosomes euchrpmatic segment, near the centromeric heterochromatin inserted in a chromocenter. This localization coincides with that of the nucleolar fibrillar center as revealed by electron microscopy. At advanced diplotene the silver grains, whose number is increased, are located over the central area of the nucleolus. This situation corresponds to that of the fibrillar centers which multiply at advanced diplotene and move to the central part of the nucleolus. Comparison of pachytene and diplotene grain counts suggests that moderate amplification of the ribosomal cistrons might take place during oogenesis in the quail.
Small, nucleolus-like structures were demostrated in the nuclei of human diplotene oocytes. At least some of these bodies were shown to be true micronucleoli by virtue of their ability to bind rRNA during RNA-DNA hybridization in situ.
Incorporation of 3H-uridine was studied during pachytene and diplotene stages of quail oocytes. No labelling could be detected during early pachytene. During advanced and late pachytene, labelling simultaneously appeared on the macrochromosomes and on certain michromosomes in the zone where they emerge from the chromocentric surface periphery. The latter localization corresponds to the region of ribosomal RNA synthesis. At diplotene the same localizations were labelled with a considerably increased intensity.
A new nucleolar fraction (P0) that is observed following drug treatment (actinomycin D, proflavine, camptothecin, and adenosine) of tissue culture cells has been identified and its properties are described. In addition, other previously described structural entities that appear as a result of drug treatment have further been analyzed and discussed.
All particulate fractions contain fibrils that on the basis of their digestion behavior and structural relationships are suspected to be chromatin in nature. They appear to form a part of a chromatin system that seems to include also the fibrils of fibrillar centers and the greater part of the condensed intra- and perinucleolar chromatin. The function of this chromatin system remains unknown. It is speculated that it might be linked to the processing of nucleolar granules.
Evidence which suggests that microspherules represent a condensed form of chromatin has been presented. The appearance of microspherules usually coincides with the inhibition of rRNA synthesis; therefore the question of whether they might contain inactivated ribosomal cistrons has been raised.
Stages of meiosis from the bluebell Endymion non-scriptus (L.) were studied by electron microscopy. The nucleolus went through the process of segregation at the beginning of meiosis with the movement to its surface of a pale-staining region. This region was shown to be the same as that called the 'L zone' or lacunae of nucleoli. Its chromosomal nature was strongly suggested by the presence of the synaptonemal complex within it. This demonstrated that the pale-staining region of nucleoli is the nucleolus organizer and almost certainly the chromosome region containing the ribosomal cistrons, and justifies the use of these terms to describe the structure when seen inside the nucleolus. The relationship between this zone and the heterochromatic knob called the nucleolar organizing body in maize by other workers is discussed.
The localization of nucleolar organizers in the quail oocyte was studied during meiotic prophase I.
During midpachytene, the oocyte nucleus contains chromocenters formed by association of the heterochromatic centromeric region of the microchromosomes. During late pachytene, the nucleolus always develops in strict relation to a chromosomal structure. The nucleolus may either be directly applied against the surface of a chromocenter or in contact with a specific region of a microchromosome emerging from a chromocenter. This microchromosome presents a juxtanucleolar knob of heterochromatic appearance. Two chromocenters, with their corresponding microchromosomes, may participate in nucleolar genesis, in which case the nucleolar constituents are arranged in a symmetrical manner with respect to the chromosomal structures.
Penetration of DNP fibers of nucleolar organizer always takes place in a pale fibrillar zone (fibrillar center) contained within the nucleolus and surrounded by electron dense fibrillar strands.
Ultrastructural data suggest that the nucleolar organizer may be situated in the pale fibrillar zone of the nucleolus. Nevertheless, no decisive argument exists excluding either the juxtanucleolar knob, which appears to be formed of facultative heterochromatin, or the DNP fibers of the nucleolonema.
The study of chromosomes in oocytes of the quail shows, at the pachytene stage, that microchromosomes are made of a euchromatic segment and a heterochromatic juxtacentromeric region. The heterochromatic regions of the microchromosomes amalgamate between themselves so as to constitute bulky chromocentres from which radiate the euchromatic segments which remain free. At late pachytene, nucleoli appear at the contact of these chromocentres. When the oocytes reach the diplotene stage, the nucleoli become quite large. They are stuck against chromocentres and establish a very close relationship with the euchromatic segments of the microchromosomes which surround or penetrate them. These observations lead one to think that the euchromatic segments of microchromosomes could be bearing nucleolar organizers. The close relations that the nucleolar organizers develop with the bulk of the nucleolus could explain its Feulgen-positive character in the quail.
In the diplotene stage of the human oocyte, the processes of elaboration of the nucleolar material are amplified. The principal nucleoli are more voluminous but their relations with the secondary constrictions and the satellites of the D and G chromosomes are not modified. Numerous micronucleoli, frequently to the number of 15-20 this stage. The most remarkable point is their association to various segments of constitutive heterochromatin: centromeric regions, secondary constrictions of the C9 and probably of the A1 and E16. These observations reveal that the human oocyte at the diplotene stage shows an amplification of the ribosomal cistrons. This phenomenon is homologous, to a more reduced scale, of this described from the inferior vetebrates. Besides, the role of heterochromatin in the synthesis of nucleolar material without the intervention of the classic nucleolar organizers is suggested.
Cytological detection of cistrons coding for 18S and 28S ribosomal RNA (rRNA) within the genome of Mus musculus inbred strain SEC/1ReJ was accomplished using the technique of in situ hybridization. Metaphase chromosome spreads prepared from cultured fetal mouse cells were stained with quinacrine-HCl and photographed. After destaining, they were hybridized to Xenopus laevis tritiated 18S and 28S rRNA, specific activity 7.5 X 10(6) dpm/mug. Silver grains clustered over specific chromosomes were readily apparent after 4 months of autoradiographic exposure. The identity of the labelled chromosomes was established by comparing the autoradiographs to quinacrine photographs showing characteristic fluorescent banding of the chromosomes in each metaphase spread. The 18S and 28S rRNA was found to hybridize to chromosomes 12, 18, and 16. Statistical analysis of the grain distribution over 26 spreads revealed that the three chromosomes were significantly labelled. Grains over these chromosomes were concentrated in an area immediately distal to the centromere, a region which in chromosomes 12 and 18 in this particular strain is the site of a secondary constriction. The relative size of the secondary constrictions, long and thus prominent on chromosome 12, obvious but shorter on 18, and indistinguishable on chromosome 16, correlated with the average number of grains observed over the centromeric region of these chromosomes, 2.5, 1.0, and 0.78, respectively.
Ehrlich tumour cell nucleoli contain fibrillar and granular components and low electron density areas called “fibrillar centres”. Analysis by high-resolution autoradiography using [3H]actinomycin D or [3H]TdR reveals that a small amount of DNA is present inside the fibrillar centres. Newly synthesized RNA is also present within the fibrillar centres or at their periphery, already 3 min after the precursor is given. According to these results, RNA is synthesized on DNA in the fibrillar centres. It is possible that the latter contain dispersed genetically active chromatin. These observations add further support to the hypothesis that fibrillar centres have a chromosomal origin and are related to the nucleolar organizers.
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) all 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 thearchitectural 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.
Excerpt
Recent studies on meiosis in male germinal cells have been able to provide data on all meiotic stages since it is now easier to obtain preparations of good quality, notably by the air-drying method introduced by Evans, Breckon & Ford (1964). Squash procedures applied to mammalian testicular material, though successful in demonstrating most stages of meiosis, do not always yield enough well-spread figures for detailed examination (for critical review, see Luciani, Capodano-Vagner & Devictor-Vuillet, 1972).
Only conventional squash methods have been used for the study of meiosis in fetal ovaries (Ohno, Klinger & Atkin, 1962; Baker, 1963; Kindred, 1963; Manotaya & Potter, 1963).
This paper presents a simple quick method for successful demonstration of all the stages of first meiotic prophase in a number of female fetal or neonatal mammals, including human, rabbit and cat.
One ovary is removed and placed in isotonic salt solution, such as Hanks or TC
The interphase nucleolus in Allium porrum meristematic cells is characterized by the presence of 1-4 dense fibrillar zones of rather complex organization. Each such zone appears to consist essentially of a convoluted, evacuolated, filamentous structure approximately 1.5 µm in diameter. At the ultrastructural level, these structures exhibit an intricate array of lacunar spaces each of which is surrounded by a dense coating. These lacunae are filled with a loose fibrillar material and the largest ones sometimes also show a dense central core. In appropriate preparations, certain of the peripherally located lacunae are found to be continuous with segments of chromosomes. High-resolution radioautography reveals, moreover, that DNA is present within both the dense and lighter portions of the nucleolar loops. These observations add further support to the hypothesis that the convoluted filamentous structures in question correspond to loops of chromosomal origin and are thus related to the nucleolar organizer.
A technique is described for forming molecular hybrids between RNA in solution and the DNA of intact cytological preparations. Cells in a conventional tissue squash are immobilized under a thin layer of agar. Next they are treated with alkali to denature the DNA and then incubated with tritium-labeled RNA. The hybrids are detected by autoradiography. The technique is illustrated by the hybridization of ribosomal RNA to the amplified ribosomal genes in oocytes of the toad Xenopus. A low level of gene amplification was also detected in premeiotic nuclei (oogonia).
The fine structure and macromolecular composition of a nucleolar constituent has been studied. For convenience of communication the term “fibrillar center” has been used to designate this constituent. Fibrillar centers are rounded structures that are associated with the fibrillar nucleolonema. They are composed primarily of pepsin digestible proteins and contain fine fibrils of 50 Å that resist pepsin and ribonuclease digestion. Individual fibrils of the fibrillar centers and the fibrillar nucleolonema under certain circumstances stain intensely with lead citrate. The substance reacting with lead has not been identified. It is likely hat the lead-positive material of the centers differs from that of the associated fibrillar nucleolonema because of slight differences in resistance of the material of the two nucleolar zones to pepsin and ribonuclease digestion.
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.
Primordial oocytes (oocytes in primordial follicles) from human ovaries aged 51/2 months post conception to 11 3/4 years post partum were examined in: (a) squash preparations of fresh and fixed tissue; (b) histological preparations; and (c) thin sections by electron microscopy, in order to study the structure of the chromosomes. — The light microscope shows that the chromosome consists of a thread bearing numerous fine lateral appendages. Cytochemical tests indicate that the thread contains DNA, and is surrounded by material containing RNA and protein. — The electron microscope shows that there are three main structural components in the chromosome: (i) an axis or “core” containing at least two longitudinal strands about 200 Å thick; (ii) a surrounding sheath composed of coiled fibrils which form symmetrically arranged columns and loops, and (iii) clusters of large granules which are associated with the outer parts of the sheath. Small nucleoli and other granular bodies are also present. — These observations indicate the presence of lampbrush chromosomes in the human oocyte. The significance of this type of chromosome in mammals is discussed in relation to the differential radiosensitivity of the oocytes, and to the form of chromosomes at the dictyate stage in rodents.
The structure, localization and activity of ribosomal genes were studied in quail, mouse and human oocytes. Meiotic prophase I is a favourable stage for these studies since the connections between the nucleoli and the chromosomes are analyzable by light and electron microscopy. In the newly-formed nucleolus at pachytene in the quail and mouse, the ribosomal genes were first localized in the fibrillar center, which was the nucleolar organizer at that stage. These genes were transcribed in the peripheral part of the fibrillar center. Superposition of the rDNA fibrils and the newly-synthesized rRNA led to the formation of a layer of electron-dense fibrils surrounding the fibrillar center. The development of the nucleolus in the mouse oocyte at diplotene constitutes an excellent model for understanding the nucleolar organization. We compared the results of autoradiography, after H3-uridine incorporation, to those obtained by three-dimensional reconstruction of the nucleolus after serial sections, and found that the rDNA, initially compacted in the fibrillar center, had at least partially unravelled to become situated in the dense fibrillar strands of the nucleolonema. These strands, like the dense fibrillar layer surrounding the fibrillar center, were zones of rDNA transcription. Small fibrillar centers could be seen scattered throughout the nucleolonema; the number of these centers no longer corresponded to that of the nucleolar organizers. They were thus considered as areas of localized rDNA packing with no active transcription in their inner part. The activity of the ribosomal genes in the human oocyte varied during the stages of meiotic prophase. rRNA synthesis, which was active in the oogonium, decreased considerably at zygotene and totally stopped at mid-pachytene; it resumed at the end of pachytene was accompanied by segregation of the nucleolar components. The ribosomal genes gathered in the fibrillar center, which was isolated from the other nucleolar components throughout pachytene. One outstanding feature of the human oocyte is that ribosomal genes originating from many acrocentric chromosomes were seen in juxtaposition in the same fibrillar center where they were wrapped in a silver-stained protein. The hypothesis is proposed that a proteolytic enzyme deficiency, perhaps related to advancing maternal age, would lead to non-disjunction of these genes. This mechanism could account for certain cases of trisomy, especially trisomy 21. At diplotene, the resumption of rRNA synthesis, enhanced by amplification of the rDNA, led to the presence of numerous micronucleoli.
Hybridization of 125-I-ribosomal RNA to mouse chromosomes in situ produced significant differences in grain count at known rDNA sites, depending on the strains from which they were derived. This is interpreted to mean that the number of rRNA genes in a given nucleolar chromosome, and in the entire genome, is polymorphic among strains and among outbred individuals.
Localization, structure and activity of ribosomal cistrons in the mouse oocyte during meiotic prophase I. Chromosomes to-day
- Stahl A
- C Mirre
- M Hartung
- B Knibiehler
- Navarro A
STAHL A., MIRRE C., HARTUNG M., KNIBIEHLER B., NAVARRO A., 1977. Localization, structure
and activity of ribosomal cistrons in the mouse oocyte during meiotic prophase I. Chromosomes
to-day, vol. 6, 255-264. Elsevier-North Holland Amsterdam.
Etude ultrastructurale des processus d'61abora-tion nucl6olaire dans l'ovocyte foetal humain en prophase I de m6iose
- J M Luciani
- M Guichaoua
- Stahl A
LUCIANI J. M., GUICHAOUA M., STAHL A., 1977. Etude ultrastructurale des processus d'61abora-tion nucl6olaire dans l'ovocyte foetal humain en prophase I de m6iose. Biol. cellul., 29, 36 0.
Mise en evidence en microscopie diectronique, dans les noyaux interphasiques des cellules L299, de filaments de DNA associés aux zones fibrillaires RNA des nucl6oles
- M Pouchelet
- Gansmuller A
- Anteunis A
- Robineaux R
POUCHELET M., GANSMULLER A., ANTEUNIS A., ROBINEAUX R., 1975. Mise en evidence en
microscopie diectronique, dans les noyaux interphasiques des cellules L299, de filaments
de DNA associés aux zones fibrillaires RNA des nucl6oles. C. R. Acad. Sci. Paris, s6r. D, 280,
2461-2463.
Mise en evidence ultrastructurale d'616ments sensibles d I'action de la désoxyribonucléase dans les zones fibrillaires des nucléoles des noyaux interphasiques des cellules L929
- Anteunis A
- M Pouchelet
- Gansmuller A
- Robineaux R
ANTEUNIS A., POUCHELET M., GANSMULLER A., ROBINEAUX R., 1975. Mise en evidence ultrastructurale d'616ments sensibles d I'action de la désoxyribonucléase dans les zones fibrillaires
des nucléoles des noyaux interphasiques des cellules L929. C. R. Acad. Sci. Paris, s6r. D, 281,
901-903.
Cytochemical and autoradiographic studies of the quail oocyte nucleolus and nucleolar organizers during meiotic prophase I
- C Mirre
- Stahl A
MIRRE C., STAHL A., 1978. Cytochemical and autoradiographic studies of the quail oocyte nucleolus
and nucleolar organizers during meiotic prophase I. J. Ultrastruct. Res. (submitted for publication).
Etude autoradiographique au microscope electronique des nucl6o[es de cellules tumorales d'Ehrlich. Mise en evidence d'ADN au niveau des centres fibrillaires
- Goessens G
GOESSENS G., 1974. Etude autoradiographique au microscope electronique des nucl6o[es de cellules tumorales d'Ehrlich. Mise en evidence d'ADN au niveau des centres fibrillaires. C. R.
Acad. Sci. Paris, s€r. D, 279, 991-993.
Transcriptional sites in spread meiotic prophase chromosomes in the mouse testis
- L Kierszenbaum A
- L Tres L
KIERSZENBAUM A. L., TRES L. L., 1974. Transcriptional sites in spread meiotic prophase chromosomes in the mouse testis. J. Cell Biol., 63, 923-935.
Localization of nucleolar organizers in the interphase nucleus of human fibroblasts
- M Vagner-Capodano A
- H Pinna-Delgrossi M
- Stahl A
VAGNER-CAPODANO A. M., PINNA-DELGROSSI M. H., STAHL A., 1977. Localization of nucleolar organizers in the interphase nucleus of human fibroblasts. Biol. cell. 29, 209-210.