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Abstract
Use of the silver-NOR method to study the nucleolar organizers in human oocytes demonstrates that topographic and quantitative variations occur during meiotic prophase. In the oogonia nucleolus the nucleolar organizers are dispersed, whereas beginning at leptotene and throughout the remaining stages of meiotic prophase they occupy a marginal position in the nucleolus. At leptotene, a modal number of seven nucleolar organizers can be observed, whereas this number falls to 2.5 at pachytene and rises to ten at diplotene, thus showing that there is intense rRNA synthesis during the latter stage of meiosis. During pachytene, one end of the bivalents containing the ribosomal cistrons is always associated with the Ag-positive zone of the nucleolus. Observation of pachytene in the electron microscope shows that the secondary constriction region of D and G bivalents is constantly associated with the fibrillar center of the nucleolus. Comparison of these two methods of investigation reveals that the silver-stained regions of the nucleolus correspond to the fibrillar centers. The latter are surrounded by a layer of electron-dense fibrils corresponding to the zone of rDNA transcription. This electron-dense layer is absent during pachytene when the nucleolus displays spontaneous segregation of its components; this absence is related to temporary arrest of rDNA transcription. The affinity of the fibrillar centers for silver-NOR staining confirms that these structures contain ribosomal cistrons. During the diplotene stage, numerous micronucleoli are formed outside the nucleolar organizers of D and G chromosomes. Most of these micronucleoli present an Ag-positive granule on one of their margins, thus indicating that they contain an actively transcribed sequence of rDNA. This observation confirms the existence of amplification of ribosomal genes in the human oocyte.
... The stages of prophase I were defined by the appearance of axial elements as previously described (Fig. 1B) (Beaumont and Mandl, 1962;Peters et al., 1997;Prieto et al., 2004). The dictyate stage was identified by the presence of two to four clearly visible nucleoli and chromosomes that were decondensed and diffuse (Bakken and McClanahan, 1978;Hartung et al., 1979). At 15.5 dpc, the majority of oocytes were in the zygotene stage of meiosis (Fig. 1C). ...
... Combined staining for SYCP1 and SYCP3 was used to determine whether SYCP1 disassembly occurred. The stages of meiotic prophase I were evaluated based on the appearance of axial elements according to previous studies (Hartung et al., 1979;Prieto et al., 2004). In total, 300 oocytes from two ovaries were counted on each slide, and repeated for three animals. ...
In mammalian ovaries, a fixed population of primordial follicles forms during the perinatal stage and the oocytes contained within are arrested at the dictyate stage of meiotic prophase I. In the current study, we provide evidence that the level of cyclic AMP (cAMP) in oocytes regulates oocyte meiotic prophase I and primordial folliculogenesis in the perinatal mouse ovary. Our results show that the early meiotic development of oocytes is closely correlated with increased levels of intra-oocyte cAMP. Inhibiting cAMP synthesis in fetal ovaries delayed oocyte meiotic progression and inhibited the disassembly and degradation of synaptonemal complex protein 1. In addition, inhibiting cAMP synthesis in in vitro cultured fetal ovaries prevented primordial follicle formation. Finally, using an in situ oocyte chromosome analysis approach, we found that the dictyate arrest of oocytes is essential for primordial follicle formation under physiological conditions. Taken together, these results suggest a role for cAMP in early meiotic development and primordial follicle formation in the mouse ovary.
© 2015. Published by The Company of Biologists Ltd.
... L'ovogonie présente plusieurs nucléoles de type réticulé, pourvus de nombreux centres fibrillaires. La technique à l'argent colore des structures arrondies dont la taille et la distribution correspondent à celles des centres fibrillaires (Hartung, Mirre et Stahl, 1979). Au stade leptotène, les centres fibrillaires du nucléole occupent une situation marginale. ...
... En effet, certains micronucléoles montrent sur l'un de leurs bords, une zone fortement colorée par l'argent qui correspond à une séquence de rDNA activement transcrite ( fig. 7) (Hartung, Mirre et Stahl, 1979). La signification biologique de cette amplification est obscure. ...
... One of these is round or oval in shape, and being connected with the secondary constrictions of the bivalents bearing the NORs, it is strongly argentophilic. The size of the argentophilic part of the nucleolus depends upon the number of adjacent chromosomes (Hartung et al., 1979;Mirre er al., 1980;Stahl, 1982). As far as the oocytes metaphases I and I1 are concerned, they have no Ag NORs (Mirre et al., 1980). ...
AgNOR data review concerning human chromosomes and interphase nuclei of bone marrow cells from healthy beings, leukemia and tumour patients including short-term cultured leukemic and tumor cell lines.
... The ovary sections were then incubated overnight with anti-SYCP3 antibody (dilution, 1:100) at 37 °C, followed by incubation with Alexa Fluor 488-conjugated secondary antibodies for 1 h at 37 °C and 5 μ g/mL Hoechst 33342 for 5 min. SYCP3 staining was performed to identify chromosomal axial elements during meiotic prophase I. Zygotene, pachytene, diplotene, and dictyate stages of meiotic prophase I were distinguished based on the appearance of axial elements 48,49 . In all, 300 oocytes from 2-3 ovaries were imaged for each slide and were counted using TCS SP8 STED downright microscope (Leica). ...
Well-timed progression of primordial folliculogenesis is essential for mammalian female fertility. Progesterone (P4) inhibits primordial follicle formation under physiological conditions; however, P4 receptor that mediates this effect and its underlying mechanisms are unclear. In this study, we used an in vitro organ culture system to show that progesterone receptor membrane component 1 (PGRMC1) mediated P4-induced inhibition of oocyte meiotic prophase I and primordial follicle formation. We found that membrane-impermeable BSA-conjugated P4 inhibited primordial follicle formation similar to that by P4. Interestingly, PGRMC1 and its partner serpine1 mRNA-binding protein 1 were highly expressed in oocytes in perinatal ovaries. Inhibition or RNA interference of PGRMC1 abolished the suppressive effect of P4 on follicle formation. Furthermore, P4-PGRMC1 interaction blocked oocyte meiotic progression and decreased intra-oocyte cyclic AMP (cAMP) levels in perinatal ovaries. cAMP analog dibutyryl cAMP reversed P4–PGRMC1 interaction-induced inhibition of meiotic progression and follicle formation. Thus, our results indicated that PGRMC1 mediated P4-induced suppression of oocyte meiotic progression and primordial folliculogenesis by decreasing intra-oocyte cAMP levels.
... 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. ...
... Other genomic regions also exhibit specific spatial organization in the nucleus that is related to biological function. For example, the ribosomal DNA is localized to the nucleolus (Hartung et al., 1979; Dujon, 1998; Kalmarova et al., 2007), whereas during interphase yeast centromeres cluster near 2012 Roberts et al., 2003) identified several of the same ETC loci, as well as other sites that recruit partial Pol III complexes. Recently ETC loci were shown to be able to function as chromatin insulators, blocking gene activation if artificially inserted between an upstream activation sequence (UAS) and its transcriptional start site (Simms et al., 2008; Valenzuela et al., 2009). ...
Chromatin function requires specific three-dimensional architectures of chromosomes. We investigated whether Saccharomyces cerevisiae extra TFIIIC (ETC) sites, which bind the TFIIIC transcription factor but do not recruit RNA polymerase III, show specific intranuclear positioning. We show that six of the eight known S. cerevisiae ETC sites localize predominantly at the nuclear periphery, and that ETC sites retain their tethering function when moved to a new chromosomal location. Several lines of evidence indicate that TFIIIC is central to the ETC peripheral localization mechanism. Mutating or deleting the TFIIIC-binding consensus ablated ETC -site peripheral positioning, and inducing degradation of the TFIIIC subunit Tfc3 led to rapid release of an ETC site from the nuclear periphery. We find, moreover, that anchoring one TFIIIC subunit at an ectopic chromosomal site causes recruitment of others and drives peripheral tethering. Localization of ETC sites at the nuclear periphery also requires Mps3, a Sad1-UNC-84-domain protein that spans the inner nuclear membrane. Surprisingly, we find that the chromatin barrier and insulator functions of an ETC site do not depend on correct peripheral localization. In summary, TFIIIC and Mps3 together direct the intranuclear positioning of a new class of S. cerevisiae genomic loci positioned at the nuclear periphery.
... Above all, the number of fibrillar complexes in lymphocytes differs from the number of nucleolar organizers. This can be explained in the following manner: nucleolar organizers in the nucleoli of nonstimulated lymphocytes are in close contact and form a common fibrillar center, similar to the situation in human oocytes [13,19]. The fact that this also applies to lymphocytes is testified by the findings of mitotic satellite associations formed by the fibriilar centers of mitotic chromosomes [30]. ...
Ultrastructure and three-dimensional distribution of nucleolus-organizing regions have been studied on ultrathin serial sections of PHA-stimulated human lymphocytes. During the 48 hr of activation the size of fibrillar centers (FCs) decreased from 0.6-0.9 microns to 0.2-0.3 microns and the number of FCs increased rapidly from one to 75-107 per cell. The number of fibrillar complexes (i.e. associations of a different number of FCs connected by the dense fibrillar component) also increased but did not reach the maximum number of nucleolar organizers presented here. Three-dimensional computer reconstructions of fibrillar complexes showed that lymphocyte activation was accompanied by early (2-4 hr) changes in the shape of the primary fibrillar center. Invagination of the dense fibrillar component on its surface occurred and division into two or more smaller FCs followed. Gradually, the typical structure of the nucleolus with several fibrillar complexes and many FCs was formed. These results confirm the hypothesis of fibrillar complex-nucleolar organizer correlation published recently.
... In the benign categories, three cases had high counts (8.5-11.4) but low scores (6)(7)(8). On the other hand, four benign cases with low counts (5-6.4) looked malignant, with scores of 10-13. ...
Benign and malignant breast lesions in cytologic preparations can be difficult to distinguish. The authors applied the silver nucleolar organizer region (AgNOR) technique to cytologic preparations obtained from surgical specimens to evaluate its diagnostic usefulness in making this distinction. Sixty-two benign and 36 malignant lesions were examined. AgNORs were counted and evaluated using a subjective scoring technique to examine AgNOR size, shape, and clustering. The benign lesions had a mean count of 4.44 AgNORs/nucleus (95% CI, 2.4-6.5) and the malignant cases, 9.52 AgNORs/nucleus (95% confidence interval, 7.4-11.7; P < .0005). The median score for benign cases was 7 and for malignant cases, 13 (P < .0001). With a few exceptions, cases with high counts had high scores. The diagnostic accuracy of combined counting and pattern assessment was 90%. The likelihood ratio for correct diagnosis using AgNOR counting was 14:1 and for AgNOR scoring, 13:1.
... The repression of the ribosomal genes during mitosis (Hernandez-Verdun et al., 2002) and after AMD (actinomycin D) treatment (Schofer et al., 1996) affects the structure and the integrity of the nucleolus, which results in changes in the relative abundance of several nuclear proteins and segregation of the nucleolar components (Andersen et al., 2002(Andersen et al., , 2005. In human meiotic oocytes, where the expression of the ribosomal genes is transitionally inactivated, the nucleolus also exhibits segregation of the nucleolar components (Hartung et al., 1979). ...
Background information:
The eurythermal fish carp (Cyprinus carpio) adjusts to the seasonal changes in the temperature and photoperiod of its habitat through diverse cellular and molecular mechanisms. We have observed that ribosomal biogenesis is modulated during the acclimatization process and correlates with profound phenotypic changes, reflecting a seasonal-dependent ultrastructural appearance of the nucleolar components. Previous studies using classical techniques showed that in winter-adapted carp the nucleolus appears to be segregated. In the present work, we have reassessed the nucleolar ultrastructural organization of the carp in summer- and winter-adapted fish by using more specific cytochemical and immunocytological techniques.
Results:
The acetylation method provided evidence that the nucleolar organization is different between winter- and summer-adapted carp. In winter-adapted fish the fibrillar component appears as a unique mass surrounded by several granular caps, whereas in summer-adapted carp the fibrillar component forms few cordons surrounded by granular masses. The nucleolar structure and distribution of the condensed chromatin observed varies upon seasonal acclimatization. In winter the nucleolar chromatin is densely packed in masses that surround the nucleolus, whereas during summer it displays a rather looser organization formed by filaments that not only surround the nucleolus, but also go through the nucleolar body. Using the TdT (terminal deoxynucleotidyl transferase)-immunogold labelling technique, we detected condensed and decondensed nucleolar chromatin, and found some labelling of fibrillar components in both seasons. When liver tissue from summer-adapted carp was treated with AMD (actinomycin D), we observed that the rearrangement of the nucleolar components and condensed chromatin were similar to that found in winter-adapted fish, with differences in the distribution of the perinucleolar chromatin.
Conclusions:
The acetylation and TdT-immunogold labelling experiments indicated that the rearrangement of the nucleolar components of winter-adapted carp is very similar to the AMD-treated summer-adapted carp nucleolus, with the latter representing the repression of the ribosomal biogenesis that occurs during the cold season. Nevertheless, the distribution of the condensed perinucleolar chromatin in winter-adapted carp compared with AMD-treated cells suggests that the transcription of rRNA genes in winter-adapted fish is less strongly inhibited and does not lead to the classical segregation of the nucleolus of that described after AMD treatment. In addition, we have confirmed that carp hepatocyte nucleoli comprise only two main structural compartments: a fibrillar component and a granular component. Fibrillar centres were not observed.
Nucleolus Organizer Regions (NORs) are the specific chromosome sites where ribosomal genes are located and highly expressed. We have applied the AgNOR staining to identify the distribution of NORs in the chromosomes of Korean Cattle. We have also studied the NORs pattern on the cells originated from different breeds, tissues and sex. Peripheral blood from forty-four Korean Cattle and Holstein was cultured for chromosme preparation. The fibroblast culture from biopsied ear skins was also conducted for chromosome analysis. The distribution of NORs was analyzed by sequential Ag staining and G-banding on metaphases of the cells. In Korean Cattle, the NORs are localized on the telomeres of the five chromosome pairs number 2, 3, 4, 11 and 28. The number of NORs per metaphase ranged from 2 to 10 giving a mean value of 5.6. The number of NORs per cell varied among individuals and cells within same individual. The size of NORs also differed in NO-chromosomes. The number of NORs was significantly different between Korean Cattle and Holstein, fibroblasts and lymphocytes, and male and female. However, the distribution and frequency of NORs were similar among the cells regardless of breeds, tissues, and sex.
The human acrocentric chromosomes, which contain a nucleolus organizer region (NOR) in their short arm, are often involved in numerical and structural anomalies. Trisomies of acrocentric chromosomes represent almost 40 % of all human lethal trisomies (Boué et al., 1975). In Down syndrome, the extra chromosome 21 originates in most cases as a result of nondis junction during the first meiotic division of the oocyte (Mattei et al., 1979). The incidence of the Robertsonian translocations, which are the most frequent form of chromosome rearrangement in man, is about 10/00 in the general population. In 90 % of Robertsonian translocations, nonhomologous acrocentrics are involved, the most frequently observed being the t(13;14) and t(14;21). These facts have led Hartung et al. (1979), Mirre et al. (1980) and Stahl et al. (1983) to investigate whether a structural arrangement exists at prophase I of human meiosis that might favor the failure of disjunction of acrocentrics and the formation of Robertsonian translocations. Using NOR-silver techniques and electron microscopy they showed that NORs belonging to several chromosomes are associated at pachytene within the fibrillar centre of the nucleolus. Mirre et al. (1980) observed the penetration into the fibrillar centre of chromatin fibers emanating from 1 to 3 bivalents and postulated that the ribosomal genes from 4 to 12 chromatids were temporarily juxtaposed inside the same nucleolar component.
To a large extent, the morphological organization of nucleoli reflects the degree of ribosomal RNA (rRNA) synthesis taking place in cells. Oocytes are cells in which ribosome formation is extremely active, but only at specific stages of oogenesis. Nucleoli of developing oocytes offer a wealth of morphological information related to the physiological state of the cell. Correlations of nucleolar structure and function during oogenesis can be made on the basis of new formation, in large numbers, intense growth and numerous morphological transformations.
Structure and function of the mammalian oocyte nucleus (germinal vesicle, GV) are reviewed in the light of the concept of nuclear architecture and compartmentalization. Some peculiarities of oocyte nuclear architectonics in different mammalian species are compared. Structural changes of chromatin configuration during mammalian oogenesis are discussed with special emphasis on the karyosphere formation. Besides, special attention is paid to the structure, dynamics and molecular composition of the nucleolus, nucleolus-derived structures, and non-nucleolar extrachromosomal domains in the mammalian GV.
Using the G-, C-, and NOR-banding techniques, a karyotyping for Korean Native Pig was performed. Blood samples were collected from 50 male Korean Native Pigs that had been bred at the National Livestock Research Institute and then blood cells were prepared from in vitro cultures followed by karyotyping; G-, C-, and NOR-banding patterns of metaphase chromosomes were analyzed. The karyotype of Korean Native Pig is 38, XX or XY which consists of 5 pairs of submetacentric chromosomes(Group I), 2 pairs of acrocentric chromosomes with short p-arm(Group II), 5 pairs of medium metacentric chromosomes(Group III), 6 pairs of acrocentric chromosomes(Group IV) and metacentric X and Y sex chromosomes. On GTG-banding, the Korean Native Pig exhibited a typical and identical banding pattern in each homologous chromosomes. Overall chromosomal morphology and positions of typical landmarks of the Korean Native Pig were virtually identical to those of Committee for the Standardized Karyotype of the Domestic Pig(CSKDP). However, numbers of G-bands of the Korean Native Pig chromosomes were more than those of CSKDP. In chromosomes 1, 3, 5, 6, 7, 8, 13, 14, 15, 16, 17, 18 and X, the Korean Native Pig exhibited more separated bands as compared with CSKDP. In C-banding patterns, although the quantity of heterochromatin was variable in each chromosome, most of the Korean Native Pig chromosomes had heterochromatic C-bands on centromeres. However, the heterochromatic C-band was constantly observed on the whole Y chromosome. In AgNOR staining, the NORs were located at centromeres on the chromosomes 8 and 10. The number of NORs per metaphase ranged from 2 to 4 giving a mean value of 2.13. The number of NORs were distributed on all chromosome pair 10 but not on chromosome 8. The sizes of NORs were also differed between homologous chromosomes 8. Numbers of NORs of Korean Native Pig were significantly higher than those of Yorkshire. The pattern of pig NORs was polymorphic in breeds, individuals and cells, especially on chromosome 8.
The zona pellucida (ZP), the extracellular glycocalyx that surrounds the oocyte, is well known to mediate homologous gamete interaction. In a previous study from our laboratories, we reported the qualitative characterization of the rat ZP. The ZP in this species, like the mouse, hamster, and human, was found to contain three glycoproteins, namely rZP1, rZP2, and rZP3 (Araki et al. [1992] Biol Reprod 46:912–919). In this study, cDNAs encoding whole rat ZP major components have been isolated and characterized. A rat ovary cDNA library was screened with the mouse ZP3 and ZP2 cDNA probes, respectively. For rZP1 cDNA cloning, cDNAs generated using reverse transcriptase-polymerase chain reaction and rapid amplification of 5' and 3' cDNA ends, were isolated and sequenced. The rZP3 cDNA showed 1338 bp with a coding region containing 1272 bp, that translates into 424 amino acids. The total translation of rZP3 peptide has a molecular weight of 45,820, containing six potential N-glycosylation sites and 75 Ser/Thr residues, possible O-glycosylation sites. The amino acid sequence derived from the cDNA sequence shares high sequence homologies to mouse (90%), hamster (78%), and human (65%) ZP3 (ZPC) glycoproteins, indicating that the rat and mouse ZP3 have quite a conserved amino acid sequence, including the potential glycosylation sites. The total transcript of the rZP2 was 2154 nucleotides and the largest open reading frame was 695 amino acids. This would translate into a protein of 78.4 kDa. In the case of rZP1, the cDNA clone consisted of 1960 bp, and the coding region contained 1851 bp translating into 617 amino acids. Significant homologies were observed between rZP2 and ZPA family from various mammalian species. The rZP1 also showed a sequence homology to mouse ZP1, known as a mouse orthologue of ZPB family, suggesting that the rZP2 and rZP1 are members of ZPA and ZPB families, respectively. The message distributions for each zona components were limited within the ovary and the signal was detectable in the growing oocytes. The present results will further our understanding of the structure of rat zona components and lead to a better understanding of species-specificity observed during sperm-egg interaction. Mol. Reprod. Dev. 51:454–467, 1998. © 1998 Wiley-Liss, Inc.
To provide more information on the number of silver-stained granules (SSGs) of active mitotic nucleolus organizer regions
(NORs) as well as interphase nucleoli, rat Yoshida ascitic sarcoma and Zajdela ascitic hepatoma cells were studied by means
of standardized one-step and two-step silver-staining procedures. The number of SSGs of mitotic active NORs was relatively
constant and corresponded to the number of active NORs of animals bearing the tumors investigated. Some anaphases and telophases
were “asymmetric”, i.e. chromosomal figures (future nuclei) in one and the same cell contained different number of SSGs (active
NORs). The incidence of such asymetric anaphases and telophases was higher in aneuploid (hypoploid) Zajdela hepatoma than
in euploid (diploid) Yoshida sarcoma cells. In the interphase, the number of SSGs was low in small or large cells with distinct
chromocenters or chromosomes condensation presumably representing postmitotic and premitotic cells. In contrast, the highest
number of SSGs was noted in nucleoli of large cells which were usually characterized by a fine chromatin structure.
This review addresses the problem of understanding nucleolar morphology in terms of nucleolar function. Nucleolar morphology and function are outlined to serve as a basis for reviewing in situ-cytochemical results that have, so far, not led to a generally accepted view on the structure-function correlation for nucleoli. Nucleoli in meiotic cells are dealth with in detail, because they illustrate the relationship between chromosomes and nucleoli particularly well. Spontaneous and induced changes in nucleolar morphology are presented and an attempt is made to explain particular morphologies in terms of functional states. The use of nucleolar morphology as a tool in diagnostics is critically evaluated.
Differential frequencies of C-band heterochromatin are reported in the cell nuclei of adult and larval Mytilus edulis L. Male mussels showed, on average, one extra small block of C-band heterochromatin in their gill cell nuclei. Mussel larvae (24-h-old), in contrast, showed no evidence of this relationship. Flow cytometry revealed no heterogeneity in DNA content in mussel spermatozoa; this is in agreement with previous cytological findings. The results of this investigation do not support the existence of a chromosomal mechanism of sex determination in M. edulis. It is suggested that this sex dimorphism was a reflection of rate differences in some fundamental activity, possibly rRNA synthesis, which was under the influence of the reproductive cycle. A previous study has indicated that the corresponding heterochromatic regions in metaphase nuclei are the sites of the nucleolar organizers: the chromosome regions containing the specific gene sequences responsible for coding for rRNA.
The ultimate goal in the study of gene expression in oocytes is to understand the special characteristics of the genetic program that confer totipotency on the egg. Totipotency includes both the ability to generate differentiated cells of every description among its progeny and the ability to develop as an organized whole organism. What switches and circuits among genes and gene products maintain or renew such potential at each generation? What special cytoplasmic organization underlies the pattern appearing in the early embryo? Obviously, we are a long way from answering this kind of question.
The localization, structure and function of two types of nuclear bodies have been investigated by cytological and cytochemical electron microscopy methods in oocytes from the Hoplonemertean, Amphiporus lactifloreus. Type I nuclear bodies differentiate in contact with the nucleolus-DNA body complex, whereas type II nuclear bodies develop close to the diplotenic chromosomal axes. The structure of type I and type II spherical nuclear bodies, 4-5 micron in width, results from the association of a fibrillar reticulum with some dense included regions. The cytochemical findings following the use of osmium-ammine reaction for DNA and silver reaction for NOR proteins support the hypothesis that type II nuclear bodies, derived from the extranucleolar area, as well as type I nuclear bodies, derived from the nucleolar complex, may be involved in ribosomal biogenesis.
The shape, relationships, relative DNA content, and nucleolar activity of the short arm of acrocentric bivalents were studied in human oocytes from early diplotene to dictyotene. At the beginning of diplotene, the short arms of the previously paired chromosomes were again separated and displayed the same morphological features as in mitotic prophase chromosomes. They were connected only with the nucleolus. In situ hybridization and silver staining showed that the nucleolar organizer regions (NORs) were located in the peripheral region of the nucleolus. Tritiated-uridine incorporation was active. At birth, the relationships of the acrocentric short arms showed increasing complexity. The chromosomes ended in nucleolus-associated chromatin blocks of irregular shape, containing large quantities of DNA as demonstrated by intense binding of 3H-actinomycin D. The number of chromosomes converging on these chromatin blocks exceeded the number of acrocentrics, suggesting that heterochromatic regions of other chromosomes were associated with the short arm of acrocentrics. In the electron microscope, the NORs were represented by fibrillar centers located on the periphery of the nucleolus and consistently connected with the blocks of dense chromatin. These relationships remained unchanged in the primordial oocyte in the adult ovary. Persistence of 3H-uridine uptake showed that the oocyte was not at a "resting" stage. The possible cytogenetic consequences of these observations are discussed.
The relative distribution of NOR proteins and chromatin fibers in the nucleoli was visualized in human cell line. The chromatin was revealed by a Feulgen-like procedure using osmium-ammine as DNA tracer. This selective staining was combined with NOR-silver staining. We provide morphological evidence for constant overlapping of the silver deposit sites with dispersed intranucleolar chromatin fibers. Silver stained proteins were sometimes observed in contact with the chromatin fibers, suggesting that at least some of the Ag-NOR proteins might be closely connected with the dispersed nucleolar DNA.
Meiotic nuclei preparations obtained from human fetal ovaries were studied with the silver-NOR technique. At leptotene, the NOR's were located at the periphery of the nucleoli. The mean number of NOR's per nucleus was 6.4. Moreover, 2-12 micronucleoli, each containing an Ag-positive zone, were observed. At pachytene, each nucleolus was divided into two zones, one of which was Ag-positive. The mean number of Ag-positive zones was 2.1 per nucleus. The subterminal region of the short arm of nucleolar bivalents was in contact with the argyrophilic nucleolar zone. In most cells, micronucleoli were dispersed between the bivalents. A single rounded body which stained heavily with silver was consistently observed. At early diplotene, the NOR's separated, appearing again as distinct rounded Ag-positive structures embedded in the peripheral part of the nucleoli. The silver-stained round body was still visible. Eight to twelve micronucleoli were observed. At a more advanced stage of diplotene, the number of micronucleoli increased, varying from 20 to 50 per nucleus. These observations suggest that the micronucleoli, already visible at the leptotene stage, are the morphological expression of an early transcription of amplified rDNA. The biological significance of the Ag-positive round body remains obscure, but the consistency of its appearance suggests that it has a specific function somehow related to nucleolar proteins.
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.
The spatial relationships of acrocentric chromosomes were studied during prophase I of meiosis in human oocytes and spermatocytes by using cytogenetic techniques, electron microscopy, and in situ hybridization. Ultrastructural investigations revealed an ordered arrangement of nucleolar bivalents at the zygotene and pachytene stages. The end of the bivalent corresponding to the cytological satellite was consistently attached to the nuclear envelope. The fibrillar center of the nucleolus always contained rDNA chromatin fibers emanating from the secondary constriction region. Association of ribosomal genes from two bivalents in the same fibrillar center was frequently observed. Ultrastructural studies demonstrated the close proximity of chromatids in the short arm region of the involved nonhomologous acrocentrics. A breakage/reunion model based on our data can explain the formation of all observed types of Robertsonian translocations: monocentrics and dicentrics with or without rDNA.
Microsporogenesis of Allium flavum was investigated by light microscopy using a silver impregnation technique. Ag-positive structures were present at the nucleolus organizing regions (NORs) in all stages of the meiotic cycle. During prophase I the nucleoli were found to be composed of a strongly impregnated central and a weakly impregnated peripheral component, probably corresponding to the pars fibrosa and pars granulosa, respectively. A heteromorphism with regard to the presence of a NOR allowed the determination of the crossing-over frequency in the chromosome arm concerned.
Prophase I meiosis was studied in the human oocyte obtained from 16- to 24-week-old fetuses. Electron microscopy and silver stainihg showed that, at pachytene, the ribosomal genes belonging to several chromosomes are gathered in the same nucleolar fibrillar center, where they are embedded in an argyrophilic protein. The nucleolus showed spontaneous segregation of its components due to temporary inactivation of the ribosomal genes. The fibrillar center, separated from the other nucleolar components, was penetrated as midpachytene by chromatin fibers containing rDNA emanating from one to three nucleolar bivalents. Thus, the ribosomal genes from 4-12 chromatids are temporarily juxtaposed inside the same structure. Such a structural arrangement is completely different from that observed in the pachytene-stage mouse oocyte, where two independent and active nucleoli, each displaying its own fibrillar center, were formed on the bivalents containing paired ribosomal genes. These different structural patterns are correlated with the high frequency of nondisjunction in the human oocyte and the relative infrequency of such in the mouse oocyte. The pattern observed in the human oocyte may be a cause of translocations.
The emergence of newly formed nucleoli and their development have been studied in mouse oocytes from pachytene to diplotene stages. At mid-pachytene, the nucleolus first appears as a fibrillar centre surrounded by a layer of electron-dense fibrils and penetrated by chromatin fibres emanating from the secondary constriction region of the nucleolar bivalent. Since this bivalent contains 2 paired nucleolar organizers, 2 nucleoli are formed in a symmetrical fashion. At advanced pachytene, the nucleoli are extended by strands of fibrillar component which become fibrillogranular distally. The 2 nucleoli fuse together at late pachytene. At diplotene, the nucleolus becomes large and reticulated. The development of the nucleolonema coincides with the appearance of numerous secondary fibrillar centres. Three-dimensional reconstruction of the reticulated nucleolus shows that the number of fibrillar centres largely exceeds that of nucleolar organizers. Radioautography after [3H]uridine incorporation demonstrates that during the first step of nucleologenesis the labelling is limited to the layer of electron-dense fibrils surrounding the fibrillar centre. Study of the time course of tritiated uridine incorporation from pachytene to diplotene shows that the labelling extends with the extending strands of fibrillar component. In the fully developed nucleolus, all fibrillar strands are labelled and contain, therefore, actively transcribed rDNA. These observations suggest that the rDNA, which is initially compacted in the primary fibrillar centre at the onset of nucleogenesis, progressively unravels and becomes distributed throughout the fibrillar parts of the nucleolonema. The lack of labelling of the secondary fibrillar centres suggests that they are zones of inactivity of the ribosomal genes where the rDNA remains locally compacted. A model of the ultrastructural organization of the nucleolus is proposed based on our observations.
Activity of nucleolar organizer regions (NORs) was studied in cultured blood lymphocytes from 20 monozygotic (MZ) and 20 dizygotic (DZ) twin pairs. The number of Ag-stained NORs, the degree of staining, and the frequency of acrocentric associations were used as criteria of the NOR activity, the acrocentric chromosomes being identified by G-banding. Analysis of intrapair concordance as well as of intrapair variance showed the number of Ag + NORs and the size of Ag-deposits to be highly heritable traits. Intrapair differences in acorocentric association frequency were not significantly higher in DZ compared with MZ twins.
Polymorphisms of the NORs as tested by Ag-staining of metaphase G-banded chromosomes were investigated in cultured blood lymphocytes of karyotypically normal individuals from the Moscow population.
The study of cell-to-cell variability in the number of Ag-stained NORs carried out on 14 monozygotic twin pairs showed the phenomenon to have some features of real intercellular variation.
In 40 unrelated individuals the individual acrocentric chromosomes were compared by the number of Ag-stained NORs, their degree of staining, and their participation in acrocentric association. Chromosome 21 was found to be significantly more active than four others by all the criteria, and chromosome 15 was less active compared with the others by the size of the Ag deposits and the frequency of participation in NOR associations. The frequency distribution of homozygotes and heterozygotes for Ag-stained NORs in the same group of 40 individuals was in accordance with the Hardy-Weinberg law.
Nucleoli are the sites of biosynthesis of the ribosomal precursors. They contain may copies of the genes for the main rRNAs (18S- and 28 S-rRNA) in the form of tandemly arranged repeats at the chromosomal nucleolar organizer regions (NORs). They also contain the small rRNA (5S-rRNA) that is synthesized outside the nucleolus, specific nucleolar proteins, among them the factors and enzymes necessary for transcription and transcript processing, and the precursor units of the ribosomes. In man as in may vertebrate species, three main components of nucleoli, besides chromatin, can be detected: fibrillar centres (FC), dense fibrillar component (DCF), and granular component (GC). Within a nucleolus the FCs are in many cases situated in its central region. The DFc forms a network of strands surrounding the FCs, but may sometimes reach for out towards the periphery of the nucleolus. The GC is usually situated in the peripheral regions of the nucleolus. In cells with a low level of ribosomal biosynthesis the nucleoli are small, usually with a single FC and little surrounding DFC and GC ("ring-shaped nucleolus"). In active cells the DFC forms a large network enclosing several, sometimes up to hundreds of FCs, and the GC covers a large area in the periphery ("compact nucleoli"). In cells at the onset of a new stimulation, the DFC is very prominent whereas the FCs are few and small, and the GC is also not very extensive ("reticulate nucleoli"). In some special cell types that are very active other arrangements of the structural components are found. In Sertoli cells, for instance, only one nucleolus is found, or occasionally two, each with a single large FC and a distinct area of GC, both areas being engulfed by DFC intermingled with some peripheral GC. Immunocytological and in situ hybridization studies to localize the rRNA genes within the nucleolus have so far led to divergent results. Both fibrillar components, the FCs and the DFC, have been claimed as the most probable candidates. Transcription of rDNA and the subsequent early steps of ribosome biosynthesis are localized in the DFC, whereas later steps (mature rRNA, preribosomes) are localized in the GC. The FCs may also serve as sites for the preparation of the rDNA for transcription, and as a store for certain nucleolar proteins. During mitosis, parts of the nucleolar proteins remain at the NORs. A direct contact between the nucleolus and the nuclear envelope is frequently observed but is not dependent on nucleolar activity.(ABSTRACT TRUNCATED AT 400 WORDS)
In the present study we analyse the nature and the functional significance of the spherical and fibrillo-granular structures appearing in the oocyte nucleus of the lizard Podarcis sicula, following the disappearance of the typical nucleolus. By LM and TEM approaches, we demonstrate that the fibrillo-granuli, containing DNA, RNA and nucleolar proteins, are micronucleoli transcriptionally active and that their DNA is probably derived from nucleolar fragmentation. By contrast, we could not explain the origin and role of the so-called spherical bodies, appearing earlier in oocyte growth; these, in fact, do not contain nucleic acids or nucleolar proteins and do not incorporate uridine. Different possible explanations of their significance are discussed.
The locations of genes coding for 18S and 28S ribosomal RNA have been mapped on metaphase chromosomes of the Indian muntjac M. muntjak by in situ hybridization with (3H)rRNA from the toad X. laevis. The results show that, in the muntjac, rDNA clusters are associated with the prominent secondary constrictions on the X and the Y1 chromos. In addition a cluster of rDNA is found near the tip of one arm on the longest pair of autosomes. The autosomal cluster of rDNAs usually does not express as a secondary constriction at metaphase.
The transcriptional activity during meiotic prophase in the mouse testis is studied with light microscopy and high-resolution autoradiographic techniques using [(3)H]uridine as a labeled precursor. In the present study, two types of RNA synthesis are detected during meiotic prophase: an extranucleolar RNA synthesis of perichromosomal localization and a nucleolar RNA synthetic activity. In some of the autosomes and close to the basal knobs, the activity of the nucleolar organizers is evidenced by the incorporation of [(3)H]uridine into nucleolar masses from zygotene on and at earlier labeling times. The evolution of nucleoli and the formation of a nucleolus attached to the sex pair are described during the different meiotic stages. Perichromosomal labeling, from leptotene on, reaches a maximum during middle pachytene and falls progressively to a low level at longer incorporation times. Sertoli's cell, the most active RNA synthetic cell in the seminiferous epithelium, rises to a maximum of labeling and drops at earlier times compared with the meiotic prophase cells. The condensed sex chromosomes show some scattered silver grains especially at middle pachytene. The axial chromosome cores and synaptonemal complexes are devoid of silver grains during the meiotic prophase. The observations suggest that a control mechanism operates during meiotic prophase to regulate transcriptional activity in the sex chromosomes and to provide differential RNA synthesis in autosomal bivalents at various stages of prophase and within certain segments of the chromosomes.
Hybridization of (3)H-labeled ribosomal RNA to human chromosomes on slides resulted in specific labeling of the satellite regions of chromosomes 13, 14, 15, 21, and 22, with an over-all efficiency of about 5%. Differences between D and G chromosomes, and between associated and unassociated satellites, were not significant. Labeling of all other parts of the preparations was nonspecific, and increased in the order: extrachromosomal regions < chromosome arms < centric regions.
A large DNA-containing body is present in addition to the chromosomes in oocytes of the house cricket Acheta domesticus. Large masses of nucleolar material accumulate at the periphery of the DNA body during the diplotene stage of meiotic prophase I. RNA-DNA hybridization analysis demonstrates that the genes which code for 18S and 28S ribosomal RNA are amplified in the ovary. In situ hybridization indicates that the amplified genes are localized within the DNA body of early prophase cells. As the cells proceed through diplotene the DNA which hybridizes with ribosomal RNA is gradually incorporated into the developing nucleolar mass.
Ribosomal cistrons for 28S and 18S RNA have been located in the mitotic chromosomes of several species at the nucleolus organizer region, but when amplification occurs at meiosis the number of chromomeres per site and the number of sites involved in this process have not been determined. In Acheta domesticus where amplification occurs for these genes every chromosome of the complement has been identified at pachytene by its size and characteristic chromomere markers both in oocytes and spermatocytes. In oocytes there are 5 amplification sites. Of these, two are major chromomeres, or DNA bodies, which are localized in the autosomes Nos. 6 and 11. Since these major chromomeres are absent from the chromosomes of spermatocytes, a comparison of the chromosomes of the two tissues at pachytene makes it possible to determine the size of the amplicons (the regions of amplification) in this species. The amplicon of chromosome 6 consists of 2 chromomere pairs and that of chromosome 11 of one chromomere pair. The total chromomere number of the autosomes is 185.3 in the oocytes and 178.4 in the spermatocytes. The X chromosome is an exception since it has 41.7 chromomeres in the oocytes, whereas in the spermatocytes it has none. The significance of these data for the understanding of gene amplification is discussed.
The synaptonemal complexes of the 22 autosomal bivalents have been reconstructed in 22 human spermatocyte nuclei at pachytene.
The mean total length of the autosomal synaptonemal complexes has been measured and amounts to 231 μm (s.d.=16μm). On the
basis of absolute and relative lengths and the position of the centromeric heterochromatin it was possible to identify 14
of the 22 autosomal bivalents and to allocate each of the remaining 8 bivalents unequivocally to a major group. The relative
synaptonemal complex lengths and centromere indices of the autosomal bivalents exhibit a good correlation with light microscopical
data on relative lengths and centromere indices of bivalents at diakinesis and of somatic metaphase chromosomes. Significant
differences in total mean length were not found, neither among nuclei from the five individuals analyzed nor amng nuclei in
different substages of pachytene. The absolute lengths of the individual bivalents were however found to vary among different
nuclei, the maximum difference exceeding the estimated reconstruction and measuring error. It is furthermore shown that each
of the five acrocentric bivalents is capable of organizing a nucleolus but that in most cases less than five nucleoli are
present in each nucleus. A short piece of synaptonemal among nuclei in different substages of pachytene. The absolute lengths
of the individual bivalents were however attachment site of the telomeres on the nuclear envelope.
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.
Electron micrographs reveal that the Ag-stainable substance is located on the outside of NOR’s or around them but not in the chromosomes themselves. In association figures, the Ag-positive material lies between the acrocentric chromosomes. Light-microscopic studies show that the Ag stainability of the nucleolus in interphase is correlated with the function of the NOR, as seen from inactive and activated lymphocytes. Much more Ag-positive material is seen in prophase than in meta- and anaphase. It starts to increase again in late telophase. In male meiosis the NOR’s remain Ag-positive until pachytene. First and second metaphase figures are negative. Experiments using RNase, TCA, and trypsin indicate that the Ag-stainable substance is an acidic protein. The precipitation of Ag granules in interphase nuclei seen in the electron microscope is greatest over the fibrillar component of the nucleolus. The most likely interpretation is that the Ag-stainable material is a component of ribonucleic protein accumulating around active NOR’s. In mitosis some of this material remains at the NOR’s. In first meiosis it is completely removed before diakinesis.
1.
Die Schleifenkerne der Malpighischen Gefe von Bibio hortulanus enthalten stets fnf Kernschleifen. Diese Zahl entspricht der haploiden Chromosomenzahl. Jede Kernschleife besteht aus zwei lngsgepaarten Elementen. Die Lngen der Schleifen eines Kerns sind untereinander konstant verschieden in der gleichen Weise, in der sich auch die Lngen der prophasischen Chromosomenpaare untereinander unterscheiden.
2.
Drei der Schleifen sind auer durch bestimmte Lnge auch durch konstante Formbesonderheiten gekennzeichnet. Eine mittellange trgt nahe einem Ende einen Nukleolus, der in seinem Innern chromatische (Nuklealfrbung) Einschlsse enthlt. Eine andere, die lngste, besitzt eine scheibenartige Endverdickung. Eine weitere, die zweitkleinste Schleife, ist terminal zu einem Endbumchen aufgespalten.
3.
Aus der Gesamtheit der Tatsachen ergibt sich, da die Kernschleifen die stark vergrerten Chromosomenpaare darstellen.
4.
Die Kernschleifen besitzen chromomerenartige, qualitativ ungleiche Scheibenstrukturen, deren Auftreten an bestimmten Stellen der jeweiligen Schleifen konstant ist.
The behaviour of the NOR material in mitotic and meiotic cells of Acheta domesticus was studied by silver staining. — In mitotic chromosomes black silver staining is observed in the centromeric region of 2 pairs of acrocentric chromosomes. Additionally a polymorphic silver positive region is found at the telomere of a large submetacentric chromosome. — The Ag-pattern of the amplified rDNA material in various stages of oogenesis was followed. During pachytene the extra DNA body shows dark brownish staining and only a few black spots. One distinct black precipitate, however, is found in association with meiotic chromosomes. In early diplotene the central core of the extra DNA body is heavily stained with silver. The outer shell shows only brown staining. In the following stages of diplotene the compact structure of the outer shell is loosened and small brown extra nucleoli are found in the remaining nucleus. These nucleoli show black Ag-precipitates in their centres. During the desintegration of the extra DNA body the nucleus becomes filled with small extra nucleoli. The black stained central core is reduced in size and finally disappears.
3H-rRNA obtained from Xenopus laevis tissue cultured cells, or a 3H-cRNA made from Xenopus ribosomal DNA, was used for heterologous in situ hybridisation with human lymphocyte metaphase chromosomes. Prior to hybridisation, chromosome spreads were stained with Quinacrine and selected cells showing good Q-banding photographed; the same cells were then rephotographed after autoradiography and pairs of photographs for each cell were used to make dual karyotypes. The chromosomes within each karyotype were divided into equal sized segments (approx. 0.7 ), with a fixed number of segments for each chromosome type. The distribution of silver grains between segments showed that the 3H-RNAs hybridised specifically to the nucleolar organising regions of the D and G group chromosomes with no other sites of localised labelling in the complement. Control experiments showed no localisation, with insignificant labelling, when metaphase spreads were incubated in a mixture containing Xenopus
3H-rRNA and competing cold human (HeLa) rRNA. Filter hybridisation experiments on isolated human DNA showed that the Xenopus derived 3H-RNAs hybridised to a fraction of human DNA which was on the heavy side of the main DNA peak and that these RNAs were competed out in the presence of excess cold human rRNA, confirming the specificity of the heterologous hybridisation. In situ hybridisation experiments were also carried out on cells from individuals with one chromosome pair showing heteromorphism for either a very long stalk (nucleolar constriction) subtending a satellite, or a large satellite. It was shown that the chromosome with the large stalk hybridised four times as much 3H-rRNA as its homologue, whereas differences in the sizes of the subtended satellites did not materially affect hybridisation levels indicating that rDNA is located in the stalks and not the satellites. The amount of 3H-rRNA hybridised differs between chromosomes and individuals; these differences are heritable and rDNA can be detected by in situ hybridisation in all three chromosomes number 21 in cells from Down's patients and in translocated chromosomes conta.ining a nucleolar constriction. Different D and G group chromosomes which hybridised equal amounts of 3H-rRNA participated in rosette associations at metaphase in a random fashion in some individuals and in a non-random fashion in others. In all individuals studied chromosomes with large amounts of rDNA were not found to be preferentially involved in association. It was therefore concluded that the probability of a chromosome being involved in the formation of a common nucleolus is not a simple function of its rDNA content and other possible factors are considered.
Es wird eine neue Ammoniak-Silber-Methode für das Färben menschlicher Chromosomen in der Metaphase beschrieben, wodurch die Satelliten aller 10 akrozentrischen Chromosomen zu unterscheiden sind.
A karyotype based on banding pattern and chromosome length is presented for the white-handed gibbon, Hylobates lar. Little homology with the banding patterns of the chromosomes of the other Hominoidea can be seen, confirming the early evolutionary separation of Hylobatidae and the other apes. Hybridization in situ with ribosomal RNA shows that the secondary constriction of a submetacentric chromosome (15) is the only site of the nucleolar organizer, as in the Cercopithecoidea. The correlation of polymorphic variation in size of this secondary constriction with grain density suggests differences in the number of gene copies per chromosome.
A simple ammoniacal silver staining procedure, designated Ag-AS, differentially stains the chromosomal locations of ribosomal DNA in certain mammalian species. This was critically demonstrated by Ag-AS staining of the nucleolus organizer regions in karyotypes of the same species and cell lines used for locating the ribosomal cistrons by DNA/RNA in situ hybridization. With Ag-AS, silver stained NORs (Ag-NORs) are visualized as black spherical bodies on yellow-brown chromosome arms. Ag-NORs were visualized throughout mitosis at the secondary constrictions in the rat kangaroo, Seba's fruit bat, Indian muntjac, and Rhesus monkey. The Chinese hamster and cattle have telomeric Ag-NORs, the mouse subcentromeric Ag-NORs, and the field vole Ag-NORs as minute short arms or choromosomal satellites. Ag-NORs occur at both secondary constrictions and at telomeres in the cotton rat. Variability in Ag-NOR pattern included differences in the number of Ag-NORs per cell within a cell population, size of Ag-NORs among chromosomes of a complement, and presence of Ag-NOR on particular chromosomes in two cell lines of the Chinese hamster. The available cytochemical data suggest that the Ag-AS reaction stains chromosomal proteins at the NOR rather than the rDNA itself.
The D and G group chromosomes from cultured human lymphocytes exhibit single and multiple satellite associations when stained with silver. Unlike earlier methods this simple and highly repeatable procedure shows physical attachments between satellited regions of various acrocentric autosomes. After studying 1,000 satellite associations from 118 normal individuals, it was found that both single and multiple associations occur with frequencies that correlate with random expectancies.
Cells from four different mouse-human somatic cell hybrids were stained with quinacrine to identify each metaphase chromosome and with ammoniacal silver by the Ag-AS method to locate nucleolus organizer regions. Each of the hybrids contained human acrocentric chromosomes. None of these human acrocentric chromosomes was stained with silver in any hybrid cell. Diploid cells were available from the human parent of one of the hybrids. In these cells both copies of nos. 13 and 15 stained with silver; the same chromosomes in the hybrid cell were not stained. These results support earlier reports that the expression of human ribosomal RNA (rRNA) genes is suppressed in mouse-human hybrid cells. Further, they suggest that silver staining by the Ag-AS method reflects activity of rRNA genes rather than just the presence of these genes.
Cricket oocyte chromosomes were stained with silver at pachytene when certain chromosome regions are active in rDNA amplification and rRNA transcription. The silver preferentially stained the known locations of 18S + 28S ribosomal cistrons. Cytochemical tests revealed that the silver binds neither to the rDNA nor transcribed rRNA, but rather to proteins which rapidly associate with the freshly-transcribed rRNA. As rRNA transcription proceeds, the quantity of silver stainable proteins progressively increases. The silver procedure can be used to visualize gene activity at the rDNA sites with conventional light microscopy.
Electron micrographs reveal that the Ag-stainable substance is located on the outside of NOR's or around them but not in the chromosomes themselves. In association figures, the Ag-positive material lies between the acrocentric chromosomes. Light-microscopic studies show that the Ag stainability of the nucleolus in interphase is correlated with the function of the NOR, as seen from inactive and activated lymphocytes. Much more Ag-positive material is seen in prophase than in meta- and anaphase. It starts to increase again in late telophase. In male meiosis the NOR's remain Ag-positive until pachytene. First and second metaphase figures are negative. Experiments using RNase, TCA, and trypsin indicate that the Ag-stainable substance is an acidic protein. The precipitation of Ag granules in interphase nuclei seen in the electron microscope is greatest over the fibrillar component of the nucleolus. The most likely interpretation is that the Ag-stainable material is a component of ribonucleic protein accumulating around active NOR's. In mitosis some of this material remains at the NOR's. In first meiosis it is completely removed before diakinesis.
The mitotic chromosomes of six specimens from Triturus vulgaris meridionalis have been examined by both in situ hybridization with 3H 18S + 28S rRNA and AS-SAT staining method. The results of these two sets of experiments can be summarized as follows: 1) in each specimen the NORs and the additional ribosomal sites, which react positively to in situ hybridization with 3H 18S + 28S rRNA, are also stained by silver; 2) other chromosomal regions, which do not hybridize in situ with 3H 18S + 28S rRNA, are on the other hand stained by the AS-SAT method. These latter AG-positive sites show a species-specific pattern of chromosomal distribution.
Silver staining of mammalian spermatocytes revealed, in light microscopy, synaptonemal complex and structures within the sex vesicle. It is feasible to follow the chromosome pairing phenomenon from zygotene to pachytene by examining the behavior of synaptonemal complexes. Nucleolus organizer regions take heavy silver stain in pachytene but are no longer detectable in later stages of meiosis.
The nucleolus organizer regions can be selectively stained in metaphase chromosome preparations by the Goodpasture and Bloom's technique which was adapted to electron microscopy analysis of cells during interphase. Using this technique, a selective accumulation of silver grains was observed over nucleolus light areas. This selective accumulation allows the identification of the interphase fibrillar centers as the nucleolus organizer regions. Ultrastructural relationships between fibrillar centers and dense fibrillar component are discussed.
Of 61 families of children with trisomy 21, polymorphism of chromosome 21 elucidating the origin of the extra chromosome was found in 42. Nondisjunction was of paternal origin in 8 cases (19.04%) and the anomaly occurred with equal frequency during the first and second meiotic divisions. Maternal nondisjunction was demonstrated in 34 cases (80.95%), in which nondisjunction occurred by far the most often during the first meiotic division (29 cases).
These results are in agreement with data from the literature, and suggest the existence of at least two different causes for chromosomal nondisjunction, the first being the same in both sexes and occurring in both meiotic divisions and the second specifically limited to the first meiotic division in the mother.
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.
In the quail, the somatic ovarian cell and oocyte nucleoli are composed of a fibrillar center which is constantly surrounded by a layer of electron-opaque fibrils and a fibrillo-granular region. Enzymatic digestion using Pronase, RNase, and DNase demonstrated that the fibrillar center contains DNA and proteins. Incorporation of tritiated actinomycin D followed by autoradiography confirmed the presence of DNA in the fibrillar center but also demonstrated a smaller quantity of DNA in the layer of electron-opaque fibrils. Staining by the technique of Cogliati and Gautier revealed that the fibrillar center contains a network of DNA fibrils. Following incorporation of tritiated uridine, the labeling is localized over the layer of electron-opaque fibrils surrounding the fibrillar center. These results suggest that the fibrillar center contains the rDNA fibrils and that their transcription occurs in the peripheral electron-opaque layer. The morphological features of the latter result from superposition of rDNA fibrils and newly synthesized rRNA.
Several experimental observations provide evidence for the existence of an amplification-type phenomenon involving the genes for ribosomal RNA (rRNA) during meiotic prophase in the human oocyte. In previous investigations multiple micronucleoli, in addition to primary nucleoli, were shown to be present in late pachytene and in diplotene human oocyte nuclei. In the present study, detailed quantitative analysis of grain counts from more than 1 000 cells following hybridization in situ indicated the presence of greater than the expected 4C number of ribosomal genes in the oocyte nuclei, most notably in these same stages. The extent of this increase over the expected 4C amount of DNA complementary to rRNA was approx. 2-fold in oocytes early in meiotic prophase and rose to approx. 4-fold in late pachytene and early diplotene oocytes. These results constitute the first evidence for the presence of extra rDNA in mammalian oocyte nuclei, the occurrence of which is clearly consistent with earlier cytological and ultrastructural observations.
The incorporation of 3H-uridine in oogonia and oocytes during meiotic prophase I was studied in three human fetuses 13, 18, and 19 weeks old. Following a 40- or 60-min pulse, intense nuclear and nucleolar labeling was observed in oogonia. During the preleptotene chromosome condensation stage, the heteropycnotic masses were unlabeled, while numerous silver grains were seen on the filaments persisting around these masses. During leptotene, chromosomal and nucleolar RNA synthesis was significant, but less than that in the oogonia. The rate of incorporation declined rapidly during zygotene and fell to a very low level at early pachytene. Throughout pachytene no nucleolar RNA synthesis was observed. Chromosomal RNA synthesis progressively recovered during middle pachytene, was of moderate intensity at late pachytene, and increased again at early diplotene. Nucleolar RNA synthesis was very intense at early diplotene, at the same time as nucleolar size and basophilia increased.
The mouse oocyte is the site of nucleolar synthesis during pachytene. The chromosomes containing a nucleolar organizer are attached to the nuclear envelope by their paracentromeric heterochromatin, either alone or by taking part in the formation of a chromocentre. The nucleolus appears at the junction of the paracentromeric heterochromatin with the euchromatic portion of the bivalent. In this zone, 5·0-nm-diameter fibres, thinner than those of the rest of the chromosome (10·0 nm), extend from the lateral element of the synaptonemal complex up to the nucleolar fibrillar centre in which they penetrate. At the onset of its synthesis, the nucleolus only contains the fibrillar centre and an electron-dense fibrillar component in continuity with the latter. Growth of the nucleolus often takes place in the form of a strand whose proximal end, in contact with the fibrillar centre, is formed by preribosomal fibrils and whose distal end is at first fibrillo-granular then granular.
Following brief incorporation of tritiated uridine, nucleolar labelling is active in oogonia. No ribosomal RNA-synthetic activity is revealed during leptotene and zygotene. Incorporation resumes at mid-pachytene, with labelling located over the electron-dense fibrillar component adjacent to the fibrillar centre.
These observations suggest that the rDNA is located in both the fibrillar centre and its associated electron-dense fibrillar component and that the rDNA transcription occurs in the latter.
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.
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.
Use of specific stains permits analysis of the frequency of nucleolus-associated heterochromatin in chromosomes 1 and 9 from human fibroblasts. In 81% of interphase nuclei the heterochromatic segment of both No. 1 chromosomes is associated with the nucleolus, while in 19% only one heterochromatic segment shows such an association with the other occupying a random position in the nucleoplasm. The nucleolar association of chromosome 9 heterochromatin is less constant: in 42.3% of the nuclei both segments are associated with the nucleolus, in 39% of the nuclei only one heterochromatic segment presents such an association, and in 18.7% neither of the two heterochromatic segments is in nucleolar association. In 6% of the cells, one or two chromosome 9 heterochromatic segments are in contact with the nuclear membrane.
In situ hybridization using tritium-labeled 28S and 18S RNA shows that in the interphase nucleus the acrocentric short arms, carriers of ribosomal cistrons, are associated with the nucleolus.
These observations demonstrate the complexity of the nucleolus-associated chromatin which, in addition to segments of chromosomes 1, 9, 13, 14, 15, 21 and 22, may include the Y chromosome. They also confirm that the nucleolus constitutes one of the orientation points determining the relative localization of chromosomes in the interphase nucleus.
In situ hybridization with 3H 18S and 28S ribosomal RNA from Xenopus laevis has been used to study the distribution of DNA sequences coding for these RNAs (the nucleolus organizing regions) in the genomes of six mammals. Several patterns of distribution have been found: 1) A single major site (rat kangaroo, Seba's fruit bat), 2) Two major sites (Indian muntjac), 3) Multiple sites in centromeric heterochromatin (field vole), 4) Multiple sites in heterochromatic short arms (Peromyscus eremicus), 5) Multiple sites in telomeric regions (Chinese hamster). - The chromosomal sites which bind 3H 18S and 28S ribosomal RNA correspond closely to the sites of secondary constrictions where these are known. However, the correlation is not absolute. Some secondary constrictions do not appear to bind 3H ribosomal RNA. Some regions which bind ribosomal RNA do not appear as secondary constrictions in metaphase chromosomes. - Although the nucleolus organizing regions of most mammalian karyotypes are found on the autosomes, the X chromosomes in Carollia perspicillata and C. castanea carry large clusters of sequences complementary to ribosomal RNA. In situ hybridization shows that the Y chromosome in C. castanea also has a large nucleolus organizing region.
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.
Human meiotic prophase spermatocyte nuclei were studied by electron microscope autoradiography after a 3 hours 3H-uridine labeling pulse, followed by postincubation in non-radioactive medium. In autosomes, 3H-uridine nucleolar labeling reaches a peak during early-middle zygotene prior to the peak labeling of chromosmal RNA species at middle pachytene. Transcription activities of sex chromosomes are inconspicuous when compared with that of autosomes. An increasing condensation of nucleolar-associated chromatin in acrocentric bivalents contributes to the formation of basal knobs in human pachytene spermatocytes. Upon completion of knob formation, nucleolar components segregate and the uptake of 3H-uridine decreases. These findings suggest that the template capability of ribosomal DNA cistrons, located next to the basal knob region, is largely associated with a dispersed state of chromatin whereas increased chromatin condensation is correlated with a restriction of ribosomal RNA transcription.
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.
A modification of the silver impregnation method used by Fernandez Gomez, et al (1969) for light microscopy is described. It has proved highly effective for the purpose of discerning, at the level of electron microscopy, the argyrophilic part of the nucleolus observed under light microscopy. By means of this simple technique the fibrillar part of the nucleolus, can be detected, both in plant and animal cells (meristematic and rat liver cells respectively) in a specific manner, which appears in strong contrast with the other components of the cell. Enzymatic digestion and the extraction of the phosphate ions show that the argyrophilia is due to linkage between the silver and a protein molecule, and that the silver precipitate is a macromolecule insoluble acid which is strongly attached to the nucleolus, and not some diffusible substance.
Ultrastructural study of the nucleoli of Ehrlich's tumor cells shows areas of low electron density called 'fibrillary centers'. The authors studied the nucleolar constituents after successive treatment with pepsin and ribonuclease or desoxyribonuclease. Their observations suggest that these 'fibrillary centers' consist mainly of proteins but could equally contain a small quantity of DNA.
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The karyotypes of two species of toads, Xenopus laevis and Xenopus mulleri, are almost indistinguishable (Tymowska and Kobel, 1972) and matings between individuals of the two species produce fertile hybrid animals. Yet, in spite of this evidence that X. laevis and X. mulleri are closely related, certain groups of repeated DNA sequences show marked evolutionary divergence between the two species (Brown and Sugimoto, this volume). The spacer DNA interspersed between the repeated genes coding for 18 and 28 S ribosomal RNA and also the spacer DNA interspersed between the genes coding for 5 S RNA are almost entirely species-specific. X. mulleri has a family of repeated AT-rich DNA sequences which is not found in the genome of X. laevis (Stern, 1972). An understanding of the spatial relationships between members of a family of repeated sequences might help to explain the mechanism by which the sequences evolve together within a species.
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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
In the Feulgen stained nuclei of Acheta domesticus the amplification regions show up at early pachytene as 5 major chromomeres. In the light and electron microscopes, they are structurally alike, containing both a central core consisting mainly of DNA and an outer region rich in RNA. Of these five major chromomeres three release their DNA copies rapidly. The two remaining major chromomeres do not release their copies so rapidly and for this reason can be localized in the autosomes Nos. 6 and 11 respectively. The frequency and the diameter of these chromomeres was recorded. The amount of DNA released from the chromomeres has been measured directly on the Feulgen stained pachytene chromosomes. In the major chromomere of chromosome No. 6 the amount of DNA from early to late pachytene remains practically constant: the values are 2.00, 2.33, 2,16 and 2.20 picograms respectively. In the major chromomere of chromosome 11, which releases its copies during these stages, the values in picograms are: 1.48 (early), 0.27 (middle), 0.11 (late) and 0.05 (late 2 pachytene). The present results support the view that given chromomeres in Acheta are not only spiralized regions of the chromosome but are also regions containing amplified DNA copies. Moreover, these findings have three genetic implications: (1) chromomeres release DNA copies during different stages of meiosis, (2) they contain amplified genes and (3) are regions which show 'puff' formation essentially similar to that of the bands of salivary gland chromosomes.
Photometric measurements of DNA in Acheta domesticus were made on single nuclei and on each pachytene chromosome, chromomere by chromomere. The extinction values were processed by a computer. The background is negligible and there is good agreement between the chromomere pattern and the amount of DNA found.The 2C value of Acheta brain nuclei is 4.0 pg. The following DNA values were obtained for oocyte nuclei at early pachytene. (1) All DNA bodies or major chromomeres 4.18 pg. (2) The chromosomes only, 9.11 pg. (3) The amplified major chromomere of chromosome 6, 2.00 pg. (4) The major chromomere of chromosome 11, 1.48 pg.The 11 pachytene chromosomes of the oocytes contain 0.4, 0.3, 0.3, 0.2, 0.2, 0.2, 0.1, 0.1, 0.1, 0.1 and 0.1 X 109 nucleotide pairs respectively (haploid value). The amplicon (the site of amplification) of chromosome 6 contains 0.02 pg whereas that of chromosome 11 has 0.01 pg (haploid value); this corresponds to 0.02 X 108 and 0.01X 109 nucleotide pairs respectively.In the case of chromosome 6, the amplified major chromomere contains 1.9-2.5 X 109 nucleotide pairs, which is circa 100 times the DNA of its amplicon (haploid). An average pachytene chromomere (haploid) in Acheta has 0.01 X 109 nucleotide pairs.The location in specific chromomeres of well-defined genes such as the cistrons for 28S and 18S ribosomal RNA, coupled with knowledge of the number of nucleotide pairs in these chromomeres helps to elucidate the organization and function of the genetic units in eukaryotes.
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.
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.