A quantitative study of Ag-positive nucleolar segments revealed by silver staining in the interphase nuclei of trophoblast cells from the rat placental connective zone

A cytomorphological study was made of silver stained nucleoli in interphasic nuclei of trophoblast cells from the rat placenta connective zone, in addition to calculation of Ag-positive spherules in the nucleoli. The prevalent number of Ag-positive nucleolar spherules in the nuclei was 6, corresponding to the number of nucleolar organizers (NOR's) in the diploid chromosome complement of the rat. The mean number of Ag-positive spherules in the nucleoli progressively increase in the course of polyploidization from 2c to 32c; variability of the spherule number also increasing. The mean area of nucleoli is found to increase in proportion to the ploidy degree. A high correlation is found between the number of Ag-positive spherules and the area of nucleoli in the nucleus (r = 0.78). This appropriateness is exhibited at all the ploidy levels. The number of Ag-spherules and the area of nucleoli are found to depend slightly on the number of nucleoli. The possibility to use the number of Ag-positive spherules as a criterion of the activity of the NOR in interphasic nuclei is discussed.     

人卵母细胞前期染色体和核仁、微核仁的观察

多年来,许多生物学家采用压片法(Ohno等,1961,1962;Baker 1963),空气干燥法(Luciani等,1974;Vebele-Kaelhardt,1978;Speed,1985))连续切片的电镜观察(Baker和Franchi,1967)等相继对人卵母细胞染色体进行过研究。Hart-ung等(1978)曾经以~3H-尿嘧啶核苷标记人卵母细胞前期核,观察了RNA的进行性变化。近年来,银染法的广泛运用,不仅揭示了动物,而且还揭示了人的卵母细胞前期核     

Structure, development, and cytochemical properties of the nucleolus-associated "round body" in rat spermatocytes and early spermatids

The "round body," a spherical structure typically associated with a nucleolus in male germ cells of the rat, has been examined in the electron microscope using routine and cytochemical methods to determine its structure, composition, and mode of development. Cytochemical analysis indicates that the round body includes neither nucleic acid nor lipid, but is composed of nonhistone protein which appears in the form of 1.6-nm-wide fibrils. Development begins in late leptotene, when a single round body appears in each spermatocyte as an irregular spheroid located along the inner surface of the nuclear envelope. During subsequent stages of the meiotic prophase, the round body leaves the nuclear envelope, becomes a regular sphere, and gradually enlarges from a diameter of 0.4 micron in leptotene to 1.6 micron in diplotene. Concurrently, lacunae appear within its substance and enlarge. At each maturation division, the amount of round-body material is decreased by about half, presumably because the constituent proteins are dissociated at metaphase, distributed between the two daughter cells at telophase, and reconstituted into half-sized round bodies. As spermiogenesis proceeds, the round body shrinks gradually and disappears at step 8. Soon after its appearance at leptotene, the round body becomes associated with and is surrounded by the pars granulosa of one of the nucleoli. Moreover, 3H-uridine incorporation into nucleolar RNA is high as long as the size of the round body increases, but is low or absent when it decreases. It is possible, therefore, that the round body exerts some control on nucleolar activity in meiotic cells.     

Synaptonemal complexes with modified lateral elements in Sordaria humana: development of and relationship to the “recombination nodules”

Meiotic prophase in Sordaria humana has been analyzed by three-dimensional reconstructions of 3 leptotene, 2 zygotene, 10 pachytene and 3 diplotene nuclei. Several notable features emerged. The lateral components of the synaptonemal complexes (SC) are hollow tubes which show dilations of variable sizes from late leptotene to early diplotene. These bulges occur before pairing. Their number decreases as soon as the SC are completely formed, but their mean size increases. Bulges can be present in all parts of the lateral components including telomeres and nucleolar organizer region, but their distribution along bivalents is not random. The remarkably uniform width of the SC central region, normally observed in other species is not observed in S. humana. Although as a general rule the bulges rarely affect the homologous components at identical sites, they often either fill or partially cover the central region. The recombination nodules are not clearly connected with the bulges. This work provides also additional insight into the development of both SC and the nodules. At late leptotene, the homologues are aligned before SC formation. One case of interlocking has been observed at early pachytene. Nodules are present from zygotene to diplotene. They are not evenly distributed along the bivalents during pachytene. The mean number of nodules, constant from late pachytene to diplotene, is equal to the mean number of chiasmata.     

Nucleolar changes in maturing avian erythroblasts and erythrocytes

Maturing erythroblasts and erythrocytes were studied in chickens and adult hens to provide more information on the presence and frequency of various nucleolar types in these cells. Nucleoli were present at all stages of erythroblastic and erythrocytic development except in the case of a few reticulocytes and the mature erythrocytes. The number of nucleoli per cell (expressed as the nucleolar coefficient) reached a maximum at the stage of the polychromatic erythroblast. Early erythroblasts were characterized by the presence of compact nucleoli or nucleoli with nucleolonemata. Rings shaped nucleoli and micronucleoli increased in number with further maturation. Cells of the final erythroblast stage (orthochromatic erythroblasts) contained mostly micronucleoli, and micronucleoli alone were present in reticulocytes and mature erythrocytes.     

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.     

Development of the synaptonemal complex and the “recombination nodules” during meiotic prophase in the seven bivalents of the fungus Sordaria macrospora Auersw

Complete reconstruction of seven leptotene, six zygotene, three pachytene and three diplotene nuclei has permitted to follow the pairing process in the Ascomycete Sordaria macrospora. The seven bivalents in Sordaria can be identified by their length. The lateral components of the synaptonemal complexes (SC) are formed just after karyogamy but are discontinuous at early leptotene. Their ends are evenly distributed on the nuclear envelope. The homologous chromosomes alignment occurs at late leptotene before SC formation. The precise pairing starts when a distance of 200–300 nm is reached. Each bivalent has several independent central component initiation sites with preferentially pairing starting near the nuclear envelope. These sites are located in a constant position along the different bivalents in the 6 observed nuclei. The seven bivalents are not synchronous either in the process of alignment or in SC formation: the small chromosomes are paired first. At pachytene the SC is completed in each of the 7 bivalents. Six bivalents have one fixed and one randomly attached telomeres. The fixed end of the nucleolar organizer is the nucleolus anchored end. At diffuse stage and diplotene, only small stretches of the SC are preserved. The lateral components increase in length is approximately 34% between leptotene and pachytene. Their lengths remain constant during pachytene. From zygotene to diplotene the central components contain local thickenings (nodules). At late zygotene and pachytene each bivalent has 1 to 4 nodules and the location of at least one is constant. The total number of nodules remains constant from pachytene to diplotene and is equal to the mean total number of chiasmata. The observations provide additional insight into meiotic processes such as chromosome movements, initiation and development of the pairing sites during zygotene, the existence of fixed telomeres, the variations in SC length. The correspondence between nodules and chiasmata are discussed.     

Characteristics of karyosphere formation in the lake frog. Light microscopy data

Morphological peculiarities of the oocyte nuclear organization were examined in R. ridibunda during winter and spring (February-March). Numerous nucleoli were seen to be assembled around regressive lampbrush chromosomes in the centre of the nucleus, and a central body was formed to which the chromosomes were attached. As result, a structural complex is constituted that involves a karyosphere and a capsule. Nucleoli are characterized by segregation and intensive fragmentation of their material. In result, a considerable part of nucleolar DNA is eliminated in the form of ring and polymorphous structures (micronucleoli). Besides the membranous component of nucleoli (nucleolar threads or tails) is lost. Towards the end of this period, nucleoli with complicated morphology become spherical again. The formation of the central body is started from the appearance of some small optically-light protein structures 5-20 nm in diameter (central body precursors-CBP). CBP are closely surrounded with ring micronucleoli to make intimate contact with the chromosomes and nucleolar threads. CBP commonly lie in one region of the nucleus not far from each other. The formation of a definitive central body obviously occurs due to a fusion of some small CBP. A conclusion is made of the nucleolar origin of the ring and polymorphous structures and of their essential role in the central body formation. The participation of chromosomal and eliminated nucleolar DNA in this process is discussed.     

Gene amplification in oocytes with 8 germinal vesicles from the tailed frog Ascaphus truei Stejneger

In the last 3 oogonial mitoses in Ascaphus truei all daughter nuclei remain in the same cell. The oocyte is 8-nucleate at the start of meiotic prophase and remains so until late in oogenesis when 7 of the nuclei disappear. All 8 nuclei in a single oocyte resemble one another with respect to size and chromatin distribution at all stages of meiotic prophase. Much of the Feulgen-positive material in pachytene nuclei is concentrated into one region of the nucleus. — All of the 8 germinal vesicles of yolky oocytes have a full set of lampbrush diplotene bivalents. Germinal vesicles from oocytes of up to 0.8 mm diameter have less than 100 nucleoli, some of which are multiple nucleoli in the sense that they have more than one core region. Each of the 8 nuclei in oocytes from one animal had about the same volume of nucleolar material. — Two values have been obtained for the amount of DNA in a diploid nucleus from Ascaphus. A biochemical estimate utilizing erythrocyte nuclei and the diphenylamine reaction yielded a value of 7.1 pg per nucleus. Microphotometry of erythrocyte nuclei stained with Feulgen's reagent gave a value of 8.2 pg per nucleus. — Microphotometric measurements of Feulgen-stained nuclei at various stages of meiotic prophase up to diplotene indicate that each nucleus synthesizes up to 5 pg of extrachromosomal DNA during and immediately after pachytene. This DNA is considered to be nucleolar. Autoradiography of nuclei from oocytes which had been incubated for 6h in ³H thymidine showed silver grains over pachytene and early diplotene nuclei only. In pachytene nuclei the silver grains overlaid that part of the nucleus where Feulgen-positive material was most concentrated. Most of the chromosomal material was unlabelled. — The significance of the 8-nucleate condition in Ascaphus oocytes is discussed, and the amount of nucleolar DNA synthesized at pachytene and of nucleolar material present in germinal vesicles is compared with corresponding situations in other amphibians.     

Nucleolar structures in chromosome and SC preparations from human oocytes at first meiotic prophase

Summary We describe a comparative study of the behavior of nucleolar structures and their relationship with nucleolar chromosomes and synaptonemal complexes at first meiotic prophase of human oocytes in an attempt to elucidate the nature of this cellular organization and to learn more about maternal nondisjunction. The number of main nucleoli varies along the different stages of prophase I and is usually low. It shows an increase from leptotene to pachytene and a decrease from pachytene to diplotene related to a decrease and an increase of main nucleoli volume, respectively. The methodology employed has enabled us to analyze in detail dark bodies, round bodies, dense bodies, and main nucleoli in chromosome or synaptonemal complex spreads. The relationship between nucleolar chromosomes or synaptonemal complexes and the nucleoli implies the existence, in a very reduced space, of chromosomal regions that contain homologous sequences and that are often unpaired. This situation may facilitate the production of heterologous pairing and chromosomal exchanges between nonhomologous chromosomes and finally result in aneuploidy. THus, the situation explained above together with the differences between the oocyte and spermatocyte NOR cycles could be one of the reasons for the higher incidence of aneuploidies of maternal origin at meiosis I.     

Number and Nuclear Localisation of Nucleoli in Mammalian Spermatocytes

In seven mammalian species, including man, the position and number of nucleoli in pachytene spermatocyte nuclei were studied from electron microscope (EM) nuclear sections or bivalent microspreads. The number and position of the nucleolar organiser regions (NORs) in mitotic and meiotic chromosomes were also analysed, using silver staining techniques and in situ hybridisation protocols. The general organisation of pachytene spermatocyte nucleoli was almost the same, with only minor morphological differences between species. The terminal NORs of Thylamys elegans (Didelphoidea, Marsupialia), Dromiciops gliroides (Microbiotheridae, Marsupialia), Phyllotys osgoodi (Rodentia, Muridae) and man, always gave rise to peripheral nucleoli in the spermatocyte nucleus. In turn, the intercalated NORs from Octodon degus, Ctenomys opimus (Rodentia, Octodontidae) and Chinchilla lanigera (Rodentia, Cavidae), gave rise to central nucleoli. In species with a single nucleolar bivalent, just one nucleolus is formed, while in those with multiple nucleolar bivalents a variable number of nucleoli are formed by association of different nucleolar bivalents or NORs that occupy the same nuclear peripheral space (Phyllotis and man). It can be concluded that the position of each nucleolus within the spermatocyte nucleus is mainly dependent upon: (1) the position of the NOR in the nucleolar bivalent synaptonemal complex (SC), (2) the nuclear pathway of the nucleolar bivalent SC, being both telomeric ends attached to the nuclear envelope, and (3) the association between nucleolar bivalents by means of their NOR-nucleolar domains that occupy the same nuclear space. Thus, the distribution of nucleoli within the nuclear space of spermatocytes is non-random and it is consistent with the existence of a species-specific meiotic nuclear architecture.     

Studies on nucleoli in rosetting T and B lymphocytes of the human peripheral blood

The incidence of various nucleolar types was studied in human rosetting lymphocytes to provide an information on nucleolar types present in T and B lymphocytes of the peripheral blood. The results clearly demonstrate that both T and B lymphocytes of the peripheral blood mostly contain ring shaped nucleoli ("resting nucleoli") and less frequently other nucleolar types such as nucleoli with nucleolonemata or compact nucleoli ("active nucleoli") and micronucleoli ("inactive nucleoli"). Since all known nucleolar types and particularly micronucleoli may be observed in both T and B lymphocytes, nucleoli in these cells cannot indicate the type or origin of these cells but simply the state of the nucleolar RNA synthesis.     

Constitutive heterochromatin and micronucleoli in the human oocyte at the diplotene stage.

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.     

Meiotic chromosomes and nucleolar behavior in testicular cells of the grassland spittlebugs Deois flavopicta, Mahanarva fimbriolata and Notozulia entreriana (Hemiptera, Auchenorrhyncha)

Spittlebugs annually infest pastures and cause severe damage, representing a serious problem for the tropical American beef cattle industry. Spittlebugs are an important biotic constraint to forage production and there is a lack of cytogenetic data for this group of insects. For these reasons, we conducted this work, in which the spermatogenesis and nucleolar behavior of Deois flavopicta, Mahanarva fimbriolata and Notozulia entreriana were studied. The males possessed testes in the shape of a "bunch of grapes"; a variable number of testicular lobes per individual and polyploid nuclei composed of several heteropycnotic bodies. A heteropycnotic area was located in the periphery of the nucleus (prophase I); the chiasmata were terminal or interstitial; metaphases I were circular or linear and anaphase showed late migration of the sex chromosome. The chromosome complement had 2n = 19, except for N. entreriana (2n = 15); the spermatids were round with heteropycnotic material in the center and elongated with conspicuos chromatin. The analysis of testes after silver nitrate staining showed polyploid nuclei with three large and three smaller nucleolar bodies. Early prophase cells had an intensely stained nucleolar body located close to the chromatin and another less evident body located away from the chromatin. The nucleolar bodies disintegrated during diplotene. Silver staining occurred in two autosomes, in terminal and subterminal locations, the latter probably corresponding to the nucleolus organizer regions (NORs). The spermatids were round with a round nucleolar body and silver staining was observed in the medial and posterior region of the elongated part of the spermatid head.     

The nature of the Ag-staining of nucleolus organizer regions. Electron- and light-microscopic studies on human cells in interphase, mitosis, and meiosis.

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.     

Nuclear architecture of human pachytene spermatocytes: quantitative analysis of associations between nucleolar and XY bivalents

Summary Nucleolar association and heterochromatin coalescence have both been invoked as mechanisms involved in the origin of chromosomal associations between nucleolar bivalents themselves, as well as between these bivalents and the XY pair, during meiotic prophase in human spermatocytes. However, these mechanisms do not satisfactorily explain how associating bivalents meet each other within the nuclear space. To elucidate this problem, we have characterized different types of nucleolar-nucleolar and nucleolar-XY bivalent associations, and their frequencies, in light and electron microscope serial sections of spermatocyte nuclei. In the pachytene nucleus, nucleolar bivalent associations were found to involve only one nucleolar sphere of RNP granules connected through a fibrillar center to a chromatin mass composed of two, or more, nucleolar-bivalent short arms. Structural relationships between these elements were examined using 3D computer models of various nucleolar associations. XY and nucleolar bivalents were usually located towards the nuclear periphery associated with the inner face of the nuclear envelope. Some nucleolar bivalents, whether single or associated appeared beside or over XY chromatin. When nucleolar-bivalent short arms (BK) were found over nucleolar or over XY chromatin, their telomeres were unattached to the nuclear envelope and the corresponding synaptonemal complexes were not observed. Ninety nucleoli were found in sixty pachytene nuclei. Thirty six percent of these nucleoli were bound to associated BKs and the remaining 64% to single BKs. Over 40% of individual spermatocytes showed at least one cluster of associated BKs and about 20% presented single or multiple BKs associated with the XY pair. The frequencies of random BK associations, over the total or restricted areas of the nuclear envelope, were calculated according to a probabilistic nuclear model. A correspondence was found in comparing the observed frequencies of associated BKs with those calculated on the basis of bouquet formation. Such an analysis strongly suggests that the occurrence of associations between nucleolar bivalents may arise at random within the bouquet. Thus, the architecture of the meiocyte nucleus, particularly the organization of the bouquet, may be the primary mechanism by which nucleolar bivalents meet each other and, consequently, become associated either through common nucleolus formation or by heterochromatin coalescence.     

Nucleolar organization in oocytes of gryllid crickets: subfamilies Gryllinae and Nemobiinae

A cytological and cytochemical survey was made of nucleolar changes during oocyte development in several different species of crickets (Gryllidae) representing the subfamilies Gryllinae and Nemobiinae. A large mass of extrachromosomal DNA is characteristic of the pachytene stage nuclei of all species examined. Nucleolar material accumulates at the periphery of the DNA body as the cells proceed into the diplotene stage of development. As the oocytes proceed through diplotene, the nucleoli reorganize into many small masses which eventually disperse in the nucleoplasm. These changes reflect both an increase in number and in size of the nucleolar material during the diplotene stage and the mode by which dispersal of nucleolar material is accomplished. These differences probably reflect differences in the organization of extrachromosomal nucleolar DNA.