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.     

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

During early embryogenesis of the nematode Parascaris univalens (2n=2) the processes of chromatin diminution and segregation of the germ and somatic cell lineages take place simultaneously. In this study we analyzed the nucleolar cycle in early embryos, both in germinal and somatic blastomeres, by means of silver staining and antibodies against the nucleolar protein fibrillarin. We observed an identical nucleolar cycle in both types of blastomeres, hence, the chromatin diminution process has no effect on the nucleolar cycle of somatic blastomeres. We report the existence of outstanding differences between this cycle and those previously reported during early embryogenesis of other species. There is a true nucleolar cycle in early embryos that shows a peculiar nucleolar disorganization at prophase, and a preferential localization of prenucleolar bodies only on the euchromatic regions during nucleologenesis. Moreover, fibrillarin does not form a perichromosomal sheath in metaphase or anaphase holocentric chromosomes, probably owing to their special centromeric organization. The number and location of nucleolus organizer regions (NORs) in the chromosomal complement have been determined using silver impregnation, chromomycin A₃/distamycin A staining, and fluorescent in situ hybridization using an rDNA probe. There are only two NORs, one per chromosome, and these are lost in blastomeres after chromatin diminution. Moreover, the constant presence of two nucleoli in somatic blastomeres suggests that NORs are not affected during the fragmentation of euchromatic regions when this process occurs.     

Patterns of activity of nucleolar organizer regions during spermatogenesis and oogenesis in Kalotermes flavicollis Fabr. (Insecta: Isoptera) analyzed by silver staining

The distribution and the behaviour of the nucleolus organizer regions (NORs) were analysed during the spermatogenesis and oogenesis of K. flavicollis with the silver staining method. The Ag-stainability of the NORs increases in growing spermatocytes up to pachytene and is absent during the remainder of the meiotic prophase. During female meiosis the nucleolar material undergoes a more complex transformation. It is active until pachytene; in early diplotene the mass of silver stainable material progressively increases as an effect of rDNA amplification. By the end of meiotic prophase the nucleolar strands disappear and a large nucleolus is rebuilt in the mature oocyte.     

An InCytes from MBC Selection: Nucleolar Separation from Chromosomes during Aspergillus nidulans Mitosis Can Occur Without Spindle Forces

How the nucleolus is segregated during mitosis is poorly understood and occurs by very different mechanisms during closed and open mitosis. Here we report a new mechanism of nucleolar segregation involving removal of the nucleolar-organizing regions (NORs) from nucleoli during Aspergillus nidulans mitosis. This involves a double nuclear envelope (NE) restriction which generates three NE-associated structures, two daughter nuclei (containing the NORs), and the nucleolus. Therefore, a remnant nucleolar structure can exist in the cytoplasm without NORs. In G1, this parental cytoplasmic nucleolus undergoes sequential disassembly releasing nucleolar proteins to the cytoplasm as nucleoli concomitantly reform in daughter nuclei. By depolymerizing microtubules and mutating spindle assembly checkpoint function, we demonstrate that a cycle of nucleolar “segregation” can occur without a spindle in a process termed spindle-independent mitosis (SIM). During SIM physical separation of the NOR from the nucleolus occurs, and NE modifications promote expulsion of the nucleolus to the cytoplasm. Subsequently, the cytoplasmic nucleolus is disassembled and rebuilt at a new site around the nuclear NOR. The data demonstrate the existence of a mitotic machinery for nucleolar segregation that is normally integrated with mitotic spindle formation but that can function without it.     

Divergent location of ribosomal genes in chromosomes of fish thorny-headed worms, <Emphasis Type="Italic">Pomphorhynchus laevis</Emphasis> and <Emphasis Type="Italic">Pomphorhynchus tereticollis</Emphasis> (Acanthocephala)

We studied distribution of ribosomal DNA (rDNA) sequences along with chromosomal location of the nucleolar organizer regions (NORs) in males of two fish parasites, Pomphorhynchus laevis and Pomphorhynchus tereticollis (Acanthocephala). Fluorescence in situ hybridization with 18S rDNA probe identified two clusters of rDNA in each species, but revealed a remarkable difference in their location on chromosomes. In P. laevis, the rDNA-FISH signals were found in long arms of the first chromosome pair and in short arms of the second pair. Whereas in P. tereticollis, rDNA clusters were located in long arms of both the first and second chromosome pairs. The divergent location of rDNA clusters in the chromosome No. 2 supports current classification of P. tereticollis, previously considered a synonym of P. laevis, as a separate species. A possible scenario of the second chromosome rearrangement during karyotype evolution of the two species involves two successive pericentric inversions. In both species, one or two prominent nucleoli were apparent within interphase nuclei stained with either silver nitrate or a fluorescent dye YOYO-1. However, a single large nucleolus was observed in early stages of mitosis and meiosis I regardless the number of rDNA clusters. Nevertheless, two bivalents with silver-stained NORs in diakinesis and two silver-stained sites in early prophase II nuclei indicated that all NORs are active. This means that each Pomphorhynchus NOR generates a nucleolus, but the resulting nucleoli have a strong tendency to associate in a large body.     

Nucleolar assembly of the rRNA processing machinery in living cells

To understand how nuclear machineries are targeted to accurate locations during nuclear assembly, we investigated the pathway of the ribosomal RNA (rRNA) processing machinery towards ribosomal genes (nucleolar organizer regions [NORs]) at exit of mitosis. To follow in living cells two permanently transfected green fluorescence protein-tagged nucleolar proteins, fibrillarin and Nop52, from metaphase to G1, 4-D time-lapse microscopy was used. In early telophase, fibrillarin is concentrated simultaneously in prenucleolar bodies (PNBs) and NORs, whereas PNB-containing Nop52 forms later. These distinct PNBs assemble at the chromosome surface. Analysis of PNB movement does not reveal the migration of PNBs towards the nucleolus, but rather a directional flow between PNBs and between PNBs and the nucleolus, ensuring progressive delivery of proteins into nucleoli. This delivery appeared organized in morphologically distinct structures visible by electron microscopy, suggesting transfer of large complexes. We propose that the temporal order of PNB assembly and disassembly controls nucleolar delivery of these proteins, and that accumulation of processing complexes in the nucleolus is driven by pre-rRNA concentration. Initial nucleolar formation around competent NORs appears to be followed by regroupment of the NORs into a single nucleolus 1 h later to complete the nucleolar assembly. This demonstrates the formation of one functional domain by cooperative interactions between different chromosome territories.     

蚕豆染色体周边RNP形成过程的电镜研究

本文运用Bernhard染色方法研究了蚕豆根端分生组织细胞中染色体周边RNP的超微结构以及这种周边RNP在有丝分裂前期到中期的形成过程。我们观察到,在前期核仁解体过程中,来自核仁的RNP物质结合于染色体表面,形成染色体周边RNP。前期末时,大量核仁RNP颗粒向周围扩散并进一步结合于染色体表面,使染色体周边RNP有所增加。中期染色体的周边RNP明显多于前期,由直径15-20 nm的RNP颗粒构成。RNP物质在染色体周边的分布是不均匀的。姊妹染色单体之间往往有较多的RNP物质存在。本文观察结果表明染色体周边RNP来源于核仁RNP。     

Nucleolar organizing chromosomes ofRicinus

Summary Pachytene chromosome morphology was compared in nine races ofRicinus communis L. (2n = 20), using pollen mother cells (PMCs) and light microscopy. Of the ten bivalents, only the two possessing nucleolar organizing regions (NORs), chromosomes 2 and 7, exhibit structural variations among the races. The NORs are located in the short arms of these two chromosomes. Most of the observed structural variations affect these short arms, which are similar morphologically and consist largely of heterochromatic segments. The PMCs contain a single nucleolus and this is associated with the NOR of each of the two chromosomes at a particular frequency in each race. In eight races, a nucleolar constriction (NC) is present in either chromosome 2 or chromosome 7. In these races, the nucleolus is associated with the chromosome possessing an NC at a frequency of 100% and with the chromosome lacking an NC at a frequency ranging between 5.6 and 100%, depending upon the race. No microscopically visible NC is present in the ninth race. In this race, the nucleolus is associated with both chromosomes 2 and 7 at a frequency of 100%. The association of the nucleolus with a chromosome possessing an NC is at the NC and with a chromosome lacking an NC is at the terminal heterochromatic segment of the short arm. Several interpretations are offered to account for the variations in frequency of association between the nucleolus and each of the nucleolar organizing chromosomes. It is suggested that the two non-linked NORs have evolved through some intragenomic changes rather than polyploidy, that this species is highly intolerant to structural variations other than those occurring in or near the NORs, and that structural variations in the nucleolar organizing chromosomes are not associated with racial variations in plant phenotype.Paper of the Journal Series, New Jersey Agricultural Experiment Station     

Localization of rRNA genes in Phyllostomidae bats reveals silent NORs in Artibeus cinereus

We analyzed the nucleolus organizer regions (NORs) of thirteen bats from genera Phyllostomus, Phylloderma, Trachops, Tonatia, Sturnira, Platyrrhinus, Artibeus and Glossophaga. We used silver staining and FISH with rDNA probe. Nine species had only one Ag-NOR-bearing chromosome pair. Artibeus lituratus, A. jamaicensis and A. fimbriatus presented multiple Ag-NORs located in the short arms of pairs 5, 6 and 7, and an additional mark in the long arm of one chromosome 5 in A. fimbriatus. Artibeus cinereus showed Ag-NORs in the chromosome pairs 10 and 13. The chromosomal location of rRNA genes using FISH agreed with the number and position of NORs in all but one species. In A. cinereus the hybridization signals were seen in three chromosome pairs 9, 10 and 13. This suggests the occurrence of silent NORs in pair 9. Differences in the size and intensity of the hybridization signals were also observed in the pair 9 of A. cinereus.     

‘Nucleolar dominance’ as observed in barley translocation lines with specifically reconstructed SAT chromosomes

Summary Diploid homo- and heterokaryotypes of barley translocation lines with only one satellite chromosome pair containing two nucleolus organizer regions (NORs) in opposite arms were found to show repressed nucleolus formation by the transposed NOR as evident from the formation of only micronucleoli. The same was true for auto-tetraploid homokaryotypes and for translocation lines with all NORs tandemly arranged into the same chromosome arm. When NORs were transposed to chromosomes without NOR in the standard karyotype, the normal pattern of nucleolus formation remained unaffected. The modified mode of nucleolus formation after the combination of all NORs in one chromosome pair is interpreted to be due to intrachromosomal nucleolar dominance analogous to interchromosomal nucleolar dominance observed in certain interspecific hybrids.     

High Ag-NOR-site variation associated to a secondary contact in brown trout from the Iberian Peninsula

The analysis of nucleolar organizer regions (NORs) using silver (Ag-) staining and in situ hybridization (ISH) in brown trout (Salmo trutta) from various river basins in the Iberian Peninsula revealed high variation in the number and location of NORs. A total of 17 different Ag-NOR sites were revealed in 10 different chromosome pairs. Three different Ag-NOR patterns clustered by river basins and strongly associated to the internal transcribed spacer 1 (ITS1) variation were detected. The main variability in NOR-sites was found in a secondary contact between two divergent lineages of brown trouts at Duero basin. Our results confirmed the abrupt break in the spatial distribution of genetic variation of brown trout populations previously reported at Duero basin. We hypothesize that NOR-site variation might be a consequence of hybridization between divergent lineages of brown trouts and that NORs could play a major role in the maintenance of a hybrid zone in Duero basin via post-zygotic isolation mechanisms.     

Structure and variation of the Nucleolus Organizer Region in turtles

Differential staining techniques were used to study the structure and variation of the NORs of 27 species of cryptodiran turtles. No species or individuals had more than a single pair of NORs. Extensive variation in NOR structure and chromosomal location was found among higher taxa and individual variation in NOR size was common. Thirty eight percent of all individuals studied were heterozygous for the size of the NOR. However, interspecific variation in chromosomal location and structure of the NOR within major taxa was relatively rare. It is concluded that ( I ) the pattern of variation of NORs is consistent with patterns of chromosomal evolution in turtles; (2) Turtles have only a single pair of NORs whereas other animals, such as some mammals, possess numerous NORs; (3) The heterochromatin associated with the NOR is involved in the structure of the nucleolus.     

NOR and nucleolus in the spermatogenesis of acridoid grasshoppers

By means of silver staining procedures of light microscopy the characteristics of the nucleolus and the NORs have been investigated in meiocytes of different grasshopper species. Our results show that: (1) Two is the most common number of chromosomes per haploid genome carrying active NORs although this number may vary from one up to five; (2) NOR activity is preferentially located on medium and short chromosomes but the X and the megameric chromosome are involved in nucleolar organization in a high proportion of the species studied; (3) The NOR location is normally restricted to one end in acro-telocentrics and to the short arm, near the centromere region, in metacentrics; (4) A marked correlation is observed between the number of nucleoli present in the spermatogonial cells and in the first meiotic prophase of a given species; (5) In some cases, the nucleoli are associated to chromosomes during spermatogonial premetaphases.     

Conservation of nucleolus organizer regions during evolution in sheep, goat, cattle and aoudad.

There are ten nucleolus organizer regions (NORs) in domestic sheep (Ovis aries L.). cattle (Bos taurus L.), goat (Capra hircus L.) and aoudad (Ammotragus lervia Blyth) and these are located terminally on chromosomes with homologous (G-banding patterns. The similarity in number of nucleolus organizer regions in these species may indicate that their ribosomal DNA regions are infrequently involved in exchange events which could lead to different numbers of active nucleolus organizer regions. Other possible explanations of the conservation of number of nucleolus organizer regions in these species are discussed. The homology of NOR location in these species supports the idea that the Bovidae karyotype tends to be fairly stable apart from changes due to centric fusion events.     

Silver staining of the nucleoli of pig embryonic kidney cells during the hyperactivation and inhibition of the synthesis of nucleolar RNA by UV microirradiation

Silver staining of the nucleoli in pig embryo kidney cells (PK) was studied during the cell cycle and also upon mature nucleoli modifications induced by UV microirradiation. During anaphase only four silver-stained granules were revealed in each daughter set of chromosomes in the four nucleolus-organizing regions (NORs). In the following 1-2 hours, the number of granules in the NORs rapidly increased up to 25-30 per nucleus. During the next 20-25 hours of the cell cycle, the number of silver-stained granules was slowly doubling as the nucleoli grew in size. UV microirradiation of one nucleolus in the nucleus with two nucleoli induced a profound degradation of the injured nucleolus and a compensatory hypertrophy of the intact one. Such nucleolar modifications were accompanied by redistribution of the silver-stained granules between the injured and non-injured nucleoli and by alterations in the levels of nucleolar RNA synthesis in the NORs. These data support a hypothesis that silver-stained proteins may be involved in the regulation of the nucleolar activity.