The karyosphere during late oogenesis in Rana ridibunda.

The organization of the nucleus in the oocytes of Rana ridibunda was examined during late diplotene at the light and electron microscopic level. At this stage the chromosomes are relatively condensed and assembled in the centre of the nucleus, constituting a karyosphere. The chromosomes here are associated with the central "protein sphere" (15--20 microns in diameter), obviously at their telomeres. Numerous nucleoli are accumulated around the chromosomes, forming a karyosphere capsule and contain segregated fibrillar and granular components; structures resembling perinucleolar chromatin and fibrillar bodies (spherules) are associated with the nucleoli. Granules 30 to 40 nm in diameter are seen to surround the fibrillar spherules. "Nucleolus-like bodies" consisting of granules 10 to 15 nm in diameter which are embedded in finely fibrillar material are often associated in contact with the chromosomes. The central sphere is an accumulation of annular structures similar to those of the pore complexes of the nuclear envelope. These structures are bound to the chromosome material, the "nucleolus-like bodies" and the fibrillar bodies. A participation of "nucleolus-like bodies" in the formation of the central sphere is suggested. A possible role of the nuclear protein matrix in the construction of the karyosphere elements is discussed.     

Karyosphere of the oocytes of the common frog, Rana temporaria]

Karyospheres of ca. 200 mcm in diameter were isolated from the common frog oocytes of definitive size. An electron microscope study has revealed in the karyosphere fibrillar nucleoli and micronucleoli, modified synaptinemal complexes sometimes connected with chromatin and fibrillar material containing a great number of, mostly atypical, pore complexes resembling those of nuclear membrane and forming "pseudomembranes". An electrophoresis of the isolated karyosphere has revealed 12 distinct protein bands, of 3 which correspond to the protein triplet characteristic of the nuclear matrix and the rest 9 represent high molecular weight components with the molecular weight from 130 to 200,000 D.     

Karyosphere in oogenesis]

The purpose of this review is to draw attention to the peculiar phenomenon during gametogenesis: the formation of the karyosphere. This phenomenon is characterized by concentration of all chromosomes in the limited area of the nucleus and may be considered as alternative of the genome in the state of lumpbrush chromosomes. The formation of the karyosphere is a widely spread phenomenon during oogenesis of different animal classes. The karyosphere can be developed during different stages of oogenesis in different organisms; but as a rule the chromosomes of diploten stage of meiosis take part in its formation. As to functional identity of the karyosphere in different species, special investigations are to be done, but contemporary knowledge of the karyosphere formation reveals some common feature:1) in the karyosphere the chromosomes are in a relatively spiral state as demonstrated by the positive Feulgen reaction; 2) there is a low level of RNA synthesis or the absence of it in the karyosphere; 3) during the karyosphere formation the nucleus is enriched by the acid proteins and a lot of protein granules and structures appearing in a close contact with the karysphere. The more typical examples of the karyosphere formation can be observed in the insect oocytes belonging to the nutrimentary type of oogenesis. In the oocytes of some animals the peculiar protein substances are formed around the chromosome knot and appear as a fibrillar zone. Such karyosphere appears to be a kind of capsule inside the nucleus. The capsules are developed as a result of complex interaction between the main nuclear structures; chromosomes, nucleoli, and nuclear membrane as it is manifested by the analysis of some recent ultrastructural date obtained in some insect and amphibian oocytes. The function of the karyosphere capsule and the role of the nuclear structure (sinaptonemal complex, extrachromosomal DNA, and nuclear membrane) in formation of the capsule, are discussed as well as the ultrastructural and cytochemical similarity between the karyosphere capsule of oocytes and nuclear bodies of somatic cells.     

Nuclear structures in the telotrophic ovarioles of nocturnal ground beetles (Tenebrionidae, Polyphaga). III. The oocyte nucleus. Electron microscopic data]

The fine structural organization of nuclei was studied in the growing oocytes of Blaps lethifera, B. mortisaga and Gnaptor spinimanus. In the beginning of diplotene the nuclei contain primary fibrillar nucleoli and numerous electron dense globules dispersed all over the nucleus; the loose chromosome material (lampbrush chromosomes) is distributed all over the nucleus. With the oocyte growth the chromosomes are spiralized and join into the karyosphere. A capsule of fibrous material forms around the karyosphere. The karyosphere nucleoli appear on the chromosomes and, then, move to the capsule region and outside its limits, to the nuclear envelope. They are fibrillar and non-active with respect to RNA synthesis. The fibrous material of the capsule is represented by strands which consist of bundles of cross-striated filaments. These latter contact directly with the chromosomes in the karyosphere and with the surface of the karyosphere nucleoli. The fibrillar-granular bodies are distributed along the strands in the capsule; they contain both RNA and DNA. The nature of extrachromosomal DNA in the karyosphere capsule and its participation in the formation of the capsule material are discussed. A suggestion is put forward on the similarity of the capsule strands with the modified central elements of synaptinemal complex.     

The karyosphere capsule in oocytes of hibernating frogs <Emphasis Type="Italic">Rana temporaria</Emphasis> contains actin,lamins, and SnRNP

The nuclei of late vitellogenic oocytes of hibernating frogs Rana temporaria were studied. During this period of oogenesis, chromosomes are inactivated and surrounded by a fibrillar karyosphere capsule. Formation of the karyosphere capsule in grass frog oocytes has been investigated in detail at the light and electron microscopic levels, but the molecular composition of the capsule remains uncertain. Immunofluorescent staining of whole-mount preparations of oocyte nuclei revealed that the karyosphere capsule contained actin, lamins A, C, and B and snRNPs proteins. A putative role of these proteins in formation of the karyosphere capsule is discussed.     

A comparative study of male meiosis in Drosophila melanogaster and D. virilis

Male meiosis in D. melanogaster cytologically follows the usual pattern, whereas in D. melanogaster and in D. virilis oocytes the chromosomes clump into a karyosphere at early meiotic prophase and remain so up to metaphase I.Male meiosis in D. virilis spermatocytes has an intermediate character: a part of the chromatin clumps together in a karyosphere at early prophase, whereas the other part of the chromatin remains diffuse all through prophase. At the end of prophase, the diffuse chromatin becomes integrated into the karyosphere before metaphase I. During the meiotic divisions the chromosomes have the same clumped aspect as those in Drosophila oocytes and thus differ strikingly from the dividing chromosomes in D. melanogaster spermatocytes.In D. virilis spermatocytes the nucleolus exhibits changes during the meiotic prophase that may be related to synthetical activities. The DNA specific staining with the fluorochrome DAPI reveals the existence of extrachromosomal DNA in the later prophase. Other striking differences in meiotic events between the two Drosophila species concern the centrioles and spermiogenesis.     

Nuclear structures in the late oogenesis of Rana temporaria. III. Electron microscopic studies]

The ultrastructural organization of the vitellogenic oocyte nucleus (stage VI, according to Duryee, 1950) was studied in normal and in vitro hormone-stimulated maturing oocytes of Rana temporaria. At this stage, numerous nucleoli are gathered around the knot of highly contracted chromosomes (the karyosphere) thus making the karyosphere capsule. Light microscope observations reveal three zones in the capsule: a central fibrous zone separating the chromosomes from the nucleoli, a middle zone, consisting of numerous nucleoli and a distinct fibrous componen; in addition a fibrous zone on the capsule periphery is seen. The nucleoli are fibrillar, bear no proribosomal granules and do not synthesize RNA. This period is characterized by an intensive fragmentation and segregation of the nucleolar material. Numerous micronucleoli and nuclear bodies occur in the nucleus. The nucleoli are normally compound and irregular in shape to become spherical in hormone-stimulated maturing oocytes. In the central fibrous zone of the capsule, separating the chromosomes from the nucleoli, some peculiar abundant accumulations of annuli were detected lacking the membranes component. Annuli are linked with the fibrous material and are regularily packed making peculiar pseudomembranes (PMM). The chromatin is connected with PMM directly. In the middle zone of the capsule, accumulations of PMM are also seen, though less abundant and less regularly packed; along with annuli, membranous areas of various size and form are met in PMM. PMM are connected with the micronucleoli with filaments 20 nm thick. In the peripheral zone of the capsule, a variety of membranous structures is detected: intranuclear annuli lamellae, component of the capsule consists of different membranous and pseudomembranous (with annuli) structures. A question of the contribution of the chromatin material in the formation of the fibrous capsular component (PMM and membranous structures) is discussed.     

An immunoelectron study of karyosphere and nuclear bodies in oocytes of mealworm beetle, Tenebrio molitor (Coleoptera: Polyphaga)

The karyosphere and nuclear bodies (NBs) were studied in Tenebrio molitor oocytes using immunoelectron cytochemistry. During early diplotene (previtellogenic stage), oocyte chromosomes begin to unite in a small nuclear volume forming the karyosphere. In vitellogenic oocyte nuclei, the chromatin undergoes condensation, and the karyosphere acquires a ring-shaped structure. The karyosphere is the only structure containing DNA in the oocyte nucleus. Pre-mRNA splicing factors [small nuclear ribonucleoproteins (snRNPs) and SC35] are not found in the karyosphere itself. In previtellogenic oocyte nuclei, these factors are present in NBs and in a fibrogranular substance surrounding the chromosomes in the early stages of karyosphere formation. At this stage, larger fibrillar NBs contain the non-snRNP splicing factor SC35. Smaller roundish NBs were shown to contain snRNPs. Some NBs with the same morphology contain neither snRNPs nor SC35. In the vitellogenic oocyte, there are fibrogranular NBs containing both snRNPs and SC35 splicing factors, fibrillar NBs containing snRNPs only, and complex NBs containing both. Complex NBs are often connected with the ring-shaped karyosphere. Based on the obtained immunoelectron data, we suggest that T. molitor oocyte NBs containing both snRNPs and the non-snRNP splicing factor SC35 are homologs of the well-characterized B-snurposomes in amphibian germinal vesicles and clusters of interchromatin granules in mammalian oocyte nuclei. Other NBs containing only snRNPs are suggested to represent a special class of insect oocyte snurposomes. The nuclear organelles mentioned seem to play a role as storage domains for pre-mRNA splicing factors during T. molitor oogenesis.     

Ultrastructural Changes of the Nucleolus During Cell Cycle in Triticum aestivum

The ultrastructural changes of the nticleolus during cell cycle in common wheat (Triticum aestivum L. ) were studied by an "en bloc" silver-staining method. It was observed that in interphase, the nucleolus was heavily stained, within which fibrillar centres, dense fibrillar component, granular component and nucleolar vacuoles could be identified. A large quantity of argentine fine granules were distributed in the condensed chromatin. Dur-ing prophase, along with the disintegration of the nucleolus and condensation of the chromatin, the larger heavily-stained granules gradually appeared at the periphery of the chromatin. At late prophase, the materials derived from the nucleolus were spread and deposited on the surface of the chromosomes. The silver-stained, larger granules, deriving from the disintegrated nucleolus, accumulated at the periphery of the metaphase chromosomes and formed an uneven and discontinuous "sheath"-like structure. This "sheath"-like structure was also observed at anaphase. In telophase, the silver-stained nucleolar materials were progressively separated from the "sheath' and fused with each other to form prenucleolar bodies, and at last, participating in the formation of new nucleoli. The results showed that the nucleolar materials were transferred directly to the surface of the chromosomes and formed a discontinuous coat, but not incorporated into the interior of the chromosomes. The silverstained granules inside the chromosomes were neither related to the nucleolus nor to the materials from the disintegrated nucleolus.     

Cytochemical distinction of various nucleolar components in insect cells.

The fine structure of the insect Sf9 cell nucleolus has been investigated by means of different cytochemical and immunocytochemical techniques at the electron microscope level. Apart from a few perinucleolar condensed chromatin clumps, the insect cell nucleolus comprises two compartments. The first of these consists of a roundish compact zone formed of fibrillar material. The other is composed of fibrillar and granular structures organized into a network separated by interstitial spaces. But, unlike mammalian cell nucleoli, any fibrillar center has been observed in the Sf9 cell nucleolus, even after actinomycin D treatment. We also show that the compact fibrillar zone of Sf9 cell nucleoli contains silver-stainable material and DNA. In actinomycin D-treated cells, a preferential contact of this compact fibrillar zone with condensed chromatin has been visualized. Finally, silver-stainable material has been found to persist throughout the whole mitosis. These results suggest that the compact fibrillar zone at the insect Sf9 cell nucleolus should, at least partly, correspond to the fibrillar center of mammalian cell nucleoli.     

Intranuclear lipids in the late oocytes of the common frog

The complex of chromosomes and nucleoli, constituting the karyosphere with a capsule, was removed micro-surgically from the late oocyte nuclei of Rana temporaria. Lipids of nuclei and of karyosphere were investigated using biochemical and autoradiographical methods in hormone-stimulated maturing oocytes in vitro. Neutral lipids (triglyceride, diglyceride, cholesterol ester) were found in the karyosphere substance by thin-layer chromatography. During oocyte maturation the incorporation of a precursor (3H-glycerol) into triglyceride was seen to increase much more than into lecithin. The autoradiography on the sectioned oocytes showed that the intranuclear level of 3H-glycerol was more densely distributed in the nucleolar zone over the material of a fibrous component of the karyosphere capsule. The level was also detected over the central part of the karyosphere in close proximity to the chromosomes. The involvement of lipids in organization of the complicated intranuclear complex of the karyosphere with a capsule is discussed. It is suggested that lipid accumulation in the area of the karyosphere fibrous component may reflect their functional relation with the oocyte nuclear matrix.     

Dynamics of the growth and maturation of oocytes and follicles in the ovaries of the cow

Chromosome despiralization and nucleolus vacuolization have been studied during the oocyte intensive growth. Oocyte and nucleolus growth has been found to stop at the secondary antral follicles with the diameter more than 1000 mkm. Chromosomal and nucleolar activity decreases at this stage. Chromosomes condense and concentrate around the nucleolus and chromatine mass (karyosphere) forms.     

Electron Microscopic Studies on the Silver-stained Nucleolar Cycle of Physarum polycephalum

The dynamic changes of nucleolar ultrastructure in the cell cycle of Physarum polycephalum Schw. were studied by an en bloc silver-staining method. The results showed that the nucleolus was large in size and situated in the center of the nucleus in late G2-phase, and the fibrillar centers, dense fibrillar components and granular components could be observed in the nucleolus. During prophase, the nucleolus moved towards the periphery of the nucleus and in late prophase disintegrated near the nuclear envelope. In metaphase, the disintegrated nucleolar components were dispersed in masses and located at the periphery of the chromosomal region of the nucleus. No specifically silver-stained area and argentophilic protein sheath were observed on the chromosomes, but there were some big dispersed silver particles within the chromosomes. During telophase the nucleolar components moved towards the two poles along with the chromosomes and co-existed with the decondensing chromatin in daughter nuclei. The nucleolar components then gradually converged with one another and separated from the chromatin. A big nucleolus was formed in the nucleus about 120 min after the completion of mitosis.     

多头绒泡菌银染核仁周期的电镜研究

以同步化培养的多头绒泡菌（Physarum poldycephalum Schw.)原生质团为材料,应用整体银染技术,电镜下研究了核仁在细胞周期中的超微结构变化。结果变化：核仁成熟时比较大,位于细胞核中央,核仁内可区分出纤维中心、密集纤维成分和颗粒成分等。前期时,核仁向边缘移动,前期末在近核膜处解体,解体的核仁物质主要呈团块状散开。中期时,解体的核仁物质位于细胞核中央染色体区域的周围,染色体上没有特异的银染区域,染色体周边也看不到银染的“鞘”状结构,但在染色体中可见一些散在的银染大颗粒。末期时,核仁物质与染色体一起到达两极,在子细胞核中与正在解集缩的染色质共存一起,以后核仁物质逐渐汇合并与染色质分开。大约在有丝分裂结束120min后,在细胞核中形成一候 中央位置的大核仁,结果提示,低等真核生物的核仁结构和周期变化与高等真核生物的不完全相同。     

Nuclear structures in the ovarioles of the butterfly Laspeyresia pomonella. Sex chromatin in trophocytes

The nuclear structures in the ovarioles have been studied in Laspeyresia pomonella by means of light and electron microscopy, autoradiography (RNA and DNA synthesis) and molecular hybridization in situ. The karyosphere was shown to form in oocyte nuclei at the beginning of oocyte growth. Numerous protein granules appeared in close contact with the karyosphere chromosomes; the true nucleolus was absent and the whole nucleus was inactive in RNA synthesis. A special attention was paid to studying nuclear structures in trophocytes. Numerous complex nucleoli actively synthesizing RNA formed in highly endopolyploid nuclei of trophocytes. Besides, each trophocyte had a spheroid vacuolized body of DNA which developed from one of meiotic bivalents soon after trophocyte differentiation and increased in diameter up to 10-15 mu. The DNA body in trophocytes and follicle cells was in close contact with the nucleolar material. Ribosomal DNA was present in these bodies as was shown by molecular hybridization in situ. A suggestion is put forward to the effect that the DNA bodies take part in the formation of complex nucleolar apparatus of trophocytes. On the basis of both the author's and literary data, a conclusion is drawn that DNA spheres in trophocytes and follicle cells are sex chromatin bodies formed, however, by both the X- and Y-chromosomes, rather than by one Y-chromosome.     

Origin of nucleolus-like bodies found in the nucleoplasm and cytoplasm of Vicia faba meristematic cells

Cytohistochemical staining and RNase-gold labelling have been applied to root-tip meristematic cells of Vicia faba to study the origin and biological significance of 2 types of inclusions: one seen in the nucleoplasm and the other in the cytoplasm of early telophase cells. They have been termed "dense bodies" and "cytoplasmic nucleolus-like bodies" (NLB), respectively. Both types of inclusions respond positively to silver staining and ribonucleoprotein (RNP) staining in a similar fashion to nucleolus. Interestingly, the dense bodies label heavily with the RNase-gold complex, as does the nucleolus, while the cytoplasmic NLB have no affinity with the label. In most cases, the dense bodies label more heavily than the nucleolus. Light microscope surveys reveal that the dense bodies sometimes appear to be released from the surface of the nucleolus. On the other hand, prenucleolar material showing the same silver staining and RNP preferential staining characteristics as the dense bodies begin to accumulate on the surface of chromosomes in mid-anaphase. This material does not label with RNase-gold. These data are discussed in terms of the hypothesis that the dense bodies are derived from the nucleolus by direct budding or fragmentation, and the cytoplasmic NLB are composed of prenucleolar material that failed to attach to chromosomes.