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.     

The nucleolar organizer of Plethodon cinereus cinereus (Green)

The lampbrush nucleolar organizers in P. c. cinereus are on the shorter arms of the 7th longest bivalent, near to the centromeres. The organizer consists of a non-chromomeric, Feulgen-negative section of the half bivalent axis inserted between typically chromomeric regions with normal lampbrush loops. The main axis of the organizer may appear double or single. The distributions and lengths of the double regions are variable. The axial strand of the organizer can be broken with DNase. Nucleolus-like objects are attached at irregular intervals along the organizer axis. The attached nucleoli closely resemble the free nucleoli in the same nucleus. Where free and attached nucleoli appear as beaded rings, the general characteristics and range of lengths of the attached rings are similar to those of the free rings. The attached rings occur singly, in pairs, or in clusters. The point of attachment of a ring is sometimes marked by a granule on the organizer axis. Pairs of attached nucleolar rings sometimes form double bridges in which the nucleoli extend linearly across a gap in the organizer axis. The length of the organizer varies from 20 μm to 300 μm, depending, at least in part, on the stage of oocyte development. The difference is a function of the length of the axial strand. Nucleolar organizers similar to that described for P. c. cinereus have been seen on the lampbrush bivalents of 7 other species of Plethodon and 2 species of Eurycea. In all of these, the 2 organizers of the nucleolus bivalents differ in length.     

Unusual prophase structures and multiple nucleoli in male meiosis of Drosophila species of the virilis group

With silver nitrate (Ag-NOR) staining, unusual fibrillar structures, apparently coupled to the nucleolus, were found is several species of the D. virilis group. In D. littoralis, beaded strings appear in connection with these structures, whereas the late prophase is characterized by the appearance of multiple nucleoli in the nucleoplasm. In D. virilis, the nucleus has a prominent pointed protrusion in the region of the nucleolus and often a fibril protrudes from this point. Small nucleoli are budding from the nucleolus during prophase. The multiple nucleoli at late prophase are smaller and fewer. A nucleolar body with black spots appears at prometaphase and persists through metaphase and anaphase. In D. lummei, the nucleolus becomes surrounded by fibrils, which are released into the nucleoplasm and on which multiple nucleoli are synthesized.These phenomena are similar to the events described in oocyte meiosis of many animals, where rDNA amplification, coupled to the synthesis of multiple nucleoli in late prophase, has been established.     

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.     

A chromosome rearrangement in Neurospora that produces segmental aneuploid progeny containing only part of the nucleolus organizer

In translocation T(ILVL)OY321 of Neurospora crassa a distal portion of the nucleolus organizer chromosome, including ribosomal DNA sequences and the nucleolus satellite, is interchanged with a long terminal segment of IL. When OY321 is crossed by Normal sequence, one-fourth of the meiotic products are segmental aneuploids that contain two copies of the long IL segment and that are deficient for the distal portion of the organizer. Each such product forms a nucleolus and is viable. The complementary aneuploid products are deficient for the IL segment and are therefore inviable. — In crosses of OY321xOY321, each product is capable of making two nucleoli; nucleoli formed by the separated nucleolus organizer parts usually fuse, but most 8-spored asci contain some nuclei in which two separate nucleoli can be seen. One nucleolus is then terminal on its chromosome while the second is interstitial and somewhat smaller. — In crosses of OY321 x Normal, half of the meiotic products are capable of making two nucleoli. However, only about 15% of 8-spored asci have one or more nuclei containing separate nucleoli. At pachytene and later in prophase I, the single fusion nucleolus is associated with three bivalent chromosome segments. Each nucleus of every ascus contains at least one nucleolus, even in asci where some nuclei display two nucleoli. — Crosses of Aneuploid x Normal are usually semibarren, producing a reduced number of ascospores, some of which are inviable. Some aneuploid cultures become fully fertile by reverting to a quasinormal sequence lacking a satellite. In some crosses of Aneuploid x Normal, individual asci may show at prophase I either complete loss, partial loss, or pycnosis of the translocated IL segment. This observation of pycnosis suggests chromosome inactivation. — Growth from aneuploid ascospores is initially slow, but can accelerate to the wild-type rate.     

Synaptonemal complexes of normal and mutant yeast chromosomes (Saccharomyces cerevisiae)

Synaptonemal complex (SC) analysis of six laboratory yeast strains showed the SC karyotypes to be repeatable within strains. Chromosomal differences were found between strains. In five of the strains, two SCs insert into the nucleolus. This represents a single bivalent with a nucleolar organizer in a medial position as is suggested by genetic data or two bivalents each with a terminal nucleolar organizer. In the first interpretation, n=16; in the second, n=17. Strain Tris has a single nucleolar SC and n=17. In strains DCx374, DCx416 and x 8366a the genetically determined rearrangements of linkage group III could not be identified. Presumably the short SC (0.33 m) associated with linkage group III cannot accommodate an inversion loop or a translocation configuration. The strains however were found to harbour a reciprocal translocation involving the nucleolar chromosome. Trisomy for one of the longer chromosomes was observed in Tris and spo10. It is concluded that rearrangements of the medium and long but not short yeast chromosomes can be detected cytologically. — Measurements of nuclear volumes show SC length to vary with artifactually induced swelling of the nucleus. Linear regression of SC length over nuclear radius indicates that actual SC length is only about one-half the observed length. As a result the DNA packing per SC unit length is higher then previously estimated.     

Studies on nucleoli of maturing frog erythroblasts

Summary Frog erythroblasts were studied in summer animals with a very active as well as reduced erythropoiesis due to experimental hibernation, the latter being administered in order to get more information on the frequency of various nucleolar types in maturing cells. The results suggest that nucleoli with nucleolonemata are a transitional nucleolar type between compact and ringshaped nucleoli. Since micronucleoli represent final nucleolar maturation changes and compact nucleoli are present in most immature cells, the sequence of nucleolar changes based on the frequency of investigated nucleolar types is as follows: compact nucleolinucleoli with nucleolonemataringshaped nucleolimicronucleoli. The experimental hibernation produces a shift of nucleoli to less active and maturer nucleolar types in all stages of the erythroblastic maturation. In addition, the experimental hibernation produces the formation of ringshaped nucleoli in the first stages of the erythroblastic maturation which in summer animals usually contain compact nucleoli and/or nucleoli with distinct nucleolonemata.     

Accessory nucleoli in microsporocytes of hybrid Phlox

Studies were conducted on the number of nucleoli present during diplotene and diakinesis in plants adjacent to, at the edge, and at the center of a zone of secondary intergradation between Phlox pilosa subsp. pilosa and P. pilosa subsp. fulgida. Assessory nucleoli were present in some cells of about 75% of the plants examined. Nucleolar numbers varied from 1 to 5. Where 2 or more nucleoli were present they were usually attached to different chromosomes. As the number of nucleoli increased, the volume of single nucleoli decreased so that volume of all nucleoli was roughly that of a normal single nucleolus. — Only 1% of the microsporocytes from populations adjacent to the zone had accessory nucleoli as compared to 4.6% of the cells in populations at the edge of the zone, and 7.7% in populations in the center. The correlation between population hybridity and incidence of accessory nucleoli is r = 0.80.     

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.     

Pachytene mapping in the female silkworm,Bombyx mori L. (Lepidoptera)

Pachytene preparations of the chromosome complement of female larvae of Bombyx mori were improved to give a distinct chromomere pattern of the bivalents suitable for chromosome mapping. Six of the 28 bivalents are described and can be identified regularly in the bivalent complements, among them the bivalent containing the nucleolus organizer. The relative lengths of these bivalents compared with one another change during development of pachytene. In contrast to other members of the Lepidoptera there is no conspicuous heterochromatic W-chromosome, which corresponds to the female-specific heterochromatin body present in the nuclei of somatic tissues. This tissue-specific heteropycnosis indicates a different functional state of the responsible chromosome or chromosomal segment in germ line and somatic cells.     

Non-relationship between nucleolus and sex chromosome system Xyp in Chelymorpha variabilis boheman (Coleoptera: Chrysomelidae)

Nucleolar-organizer region, nucleolus and mode of association of the sex bivalent were analyzed in spermatecytes of Chelymorpha variabilis Boheman. This species (2n=10II+Xy_p) shows the typical sex chromosome system of the group Polyphaga. The results of silver staining techniques showed the nucleolar organizer region localized in a subterminal position of an autosomal bivalent. During meiotic prophase the nucleolus was distinguished with the silver staining and acridine orange fluorescence technique up to diakinesis. The independence of nucleolus and sex bivalent Xy_p during meiosis is demonstrated. The positively silver staining but negatively orange-red material found within the parachute could be involved in the regular co-orientation of both sex chromosomes. After a longer hypotonic treatment, sex bivalents were observed elongated and paired only at one end during the pachytene stage. Along these sex chromosomes, C-bands showed positive blocks located in the pericentromeric and telomeric regions. Heterochromatic association of both sex chromosomes was suggested.     

The effect of colchicine on meiosis in Lilium speciosum cv. “Rosemede”

Chromosome pairing and chiasma frequency were studied in meiocytes at diakinesis of Lilium speciosum cv. Rosemede fixed up to 21 days after the start of either continuous or 3 day pulse colchicine treatment. The two treatments gave similar results. In pulse treated pollen mother cells (PMCs) the mean chiasma frequency per cell fell from 26.4 in controls to 8.5 after fourteen days while the mean number of univalents per cell increased from 0.05 to 17.58. There was a negative correlation between mean chiasma frequency per bivalent and per PMC in colchicine treated buds; univalents were preferentially induced in bivalents with one chiasma, and preferentially excluded in bivalents with 4 chiasmata. Some chiasmata were redistributed to surviving bivalents despite the concurrent reduction in chiasma frequency per meiocyte. — Colchicine sensitivity began in premeiotic interphase and extended to mid or late zygotene in PMCs; ongoing synapsis was unaffected. However, susceptibility to univalency was asynchronous between bivalents occurring at zygotene in short chromosomes but at late premeiotic interphase in the longest chromosomes. The number of chiasmata per bivalent could be altered by colchicine without inducing univalents, but the ultimate effect was to reduce the number of chiasmata per bivalent (or per chromosome arm) directly to zero. The major factors determining the order and extent of reduced pairing and chiasma number were total chromosome length and arm length. Pairing and chiasma formation in embryo sac mother cells were less sensitive to colchicine than in PMCs, but their behavior was otherwise similar.     

On the nucleoli of the dinoflagellate Prorocentrum

To date no nucleolus had been observed in Prorocentrum under the light microscope. The author failed to show the nucleoli of P. micans and P. cassubica with eosin in 70% alc or with methyl green-pyronin. But when these dinoflagellates were treated with an Ag-1 technique which had been improved for demonstrating NORs in unicellular organisms, nucleoli were stained dark brown or black, while all other parts showed no colour. When the materials were stained well, only the central part of the nucleolus was stained. Under the electron microscope, it was observed that all the silver grains were concentrated in the pars fibrosa of the nucleolus. P. cassubica had only one small oblate nucleolus attached to the nuclear envelope, with NOR usually in the shape of the letters O or C. P. micans had 1–7 nucleoli of various sizes and shapes with NORs in various complicated forms. The number of nucleoli bore a certain relationship to the living state of the dinoflagellate. One day after fresh medium was added, cells with 3 nucleoli were most common, and 28.5% of the individuals had 4–6 nucleoli. Cells having only one nucleolus accounted for 8.6%. 3 days after, cells with 2 nucleoli became dominant, and those with 4–6 decreased to 18.4%. After a month, cells with 1 nucleolus became most abundant, cells having 4 nucleoli decreased to 2.4%, and no cells had 5 or 6 nucleoli.     

Stereoscopic back-scattered electron imaging of silver-stained proteins in nucleoli

By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode and stereo-tilt in scanning electron microscopy (SEM), it is possible to observe the nucleus through the cell surface, the nucleolus, and the tri-dimensional distribution of the AgNOR-associated acidic proteins. In C3H10T1:2 cells and their 7-12-dimethylbenz--anthracene-treated transformants, the staining demonstrates several intranucleolar silver-staining granules (SSG), surrounded by a weakly staining region. The SSG may represent the fibrillar center (FC) and the weakly staining region, the fibrillar dense component (FD). This component can link several SSG together to form a rope-like structure. In cells with no visible nucleolus and inactive nucleolar organizer regions (NORs) the silver-staining granules are less numerous, close together and the presumed fibrillar dense components are not visible. The SSG are located more peripheraly, and the weakly staining region and the rope-like structure are less prominent in control cell nucleoli than in transformed cells with a comparatively high rate of RNA synthesis.     

Nucleolar transformation in plants grown on clinostats

Summary Cells of carrot calli (Daucus carota L.) grown on clinostats (simulated weightlessness) exhibit increases in nucleolar number and volume. In clinostat-grown whole barley plants (Hordeum vulgare L. cv. Steptoe), nucleoli in 70% of root meristem and root cortical cells in the 1 mm root apex exhibit multiple nodulations after one day of growth. The nucleolar nodules (1.1 m mean diameter) are densely and finely fibrous, distinctly different from the nucleolus in which the content is so compact that the granular component is masked. Control nucleoli (from vertically rotated and stationary seedlings) rarely exhibit nodule-like protrusions, are not compact, and contain a well defined granular component. Proteins that are heat soluble, characteristic of many stress responses, rapidly increase in barley grown on clinostats. Barley growth on clinostats is slowly and steadily inhibited. There is no difference between vertically rotated and stationary controls for any of the parameters measured, indicating that clinostat motion per se does not affect significantly barley development. The evidence taken together suggests that barley plants germinated and grown on clinostats are stressed, the effects of which are expressed sequentially by alteration of nucleolar morphology, increased production of heat-soluble proteins, and decreased plant growth. Similar stress-related changes may be expected to occur in plants subjected to weightlessness during space flight. It is therefore of interest that nucleoli in wheat roots (Triticum aestivum L. cv. Broom) obtained from the space flight IML-1 mission show irregularity that is not observed in any of the ground controls for the flight experiment.Abbreviations Act D actinomycin D - C clinostat rotation - EM electron microscopy - LM light microscopy - R vertical rotation - rDNA ribosomal DNA - S stationary     

Induced Bivalent Interlocking and the Course of Meiotic Chromosome Synapsis

WHEN chromosomes pair at meiosis the bivalents so formed do not normally interlock. Heat-treatments can, however, induce bivalent interlocking in the locust Locusta migratoria. Only the longest bivalents interlock and usually only two are found per cell; two “rod” bivalents, with single chiasmata, two “ring” bivalents, each with two or three chiasmata, or one “rod” and one “ring” bivalent (Fig. 1a, b and c). The nature of this interlocking and the metaphase orientational and congressional properties of interlocked bivalents are analysed in detail elsewhere¹.     

The reaction of Lupinus angustifolius L. root meristematic cell nucleoli to lead

The effect of 2–48 h treatment of Lupinus angustifolius L. roots with lead nitrate at the concentration of 10⁻⁴ M on the nucleoli in meristematic cells was investigated. In the lead presence the number of ring-shaped as well as segregated nucleoli increased especially after 12–48 h of treatment, while spindle-shaped nucleoli appeared after 24 h and 48 h. Lead presence also increased the frequency of cells with silver-stained particles in the nucleus and the number of these particles especially from the 12th hour of treatment. It was accompanied by significant decline of nucleolar area. Analysis of these cells in transmission electron microscope confirmed the presence of ring-shaped and segregated nucleoli. Moreover, electron microscopy revealed compact structure nucleoli without granular component. Additionally, one to three oval-shaped fibrillar structures attached to nucleolus or lying free in the nucleoplasm were visible. The possible mechanism of lead toxicity to the nucleolus is briefly discussed.     

The nucleolar organizer of Plethodon cinereus cinereus (Green)

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