The ultrastructure and argentophilic properties of the nucleolus organizer and centromeric regions of the chromosomes in early mouse embryogenesis

The Ag-staining of metaphase chromosomes in one-cell mouse embryos shows that the nucleolus organizer regions (NORs) are Ag-negative, whereas centromeric regions (CRs) are Ag-positive. Starting from 8-16-cell embryos, NORs stained by AgNO3 constantly, CRs remaining argentophobic. On the ultrathin sections of multicell embryos, Ag(+)-NORs differ from the chromosomal arms: they consist of loosely filaments about 6-8 nm in diameter, characterized by a low electron density. On the contrary, at one-cell stage Ag(-)-NORs are not morphologically identified: chromosomal bodies consist of uniform DNP-fibrils about 20 nm in diameter. These data permit to suppose that extended rDNA may form supranucleosomal and nucleosomal DNP-fibrils in the absence of Ag-proteins. The Ag(+)- and Ag(-)-CRs contain 10-20 nm DNP-fibrils mainly, although their density at multicell stages is higher than in one-cell mouse embryos.     

Evolution of the cytoplasmic organelles during female meiosis in Pisum sativum L.

In this paper we have traced the evolution of the cytoplasmic organelles in the female germinal cell of Pisum sativum L., from the beginning of meiosis to the early stages of the maturing megaspore, in order to correlate the morphological changes with the physiological aspects of megasporogenesis.A process of intense cytoplasmic vacuolation takes place in the megaspore mother cell (MMC) during prophase I, probably proceeding from the smooth endoplasmic reticulum and dictyosomes; it results in the formation of big vacuoles, which play a role in MMC polarization. By means of this polarization most plastids and mitochondria are incorporated into the functional megaspore at the end of meiosis.There are plastid and mitochondria cycles which consist of dedifferentiation followed by redifferentiation, During these cycles a transient morphology appears, called a cup-shaped form, which we interpret as an expression of low organelle activity.The wall of the MMC thickens throughout megasporogenesis and loses its plasmodesmata during middle prophase I. The ribosome population is reduced during prophase I and then restored during the early stages of the megaspore maturing process, as shown by the quantitative study that we have carried out. The nucleolar cytoplasmic bodies play a part in this restoring process. These bodies have a special morphology and appear to be originated from the activity of the nucleolar organizing region (NOR) during nucleolar disorganization in prophase I.We think that this cytoplasmic evolution is a response to nuclear genic recombination, in order to provide the most adequate expression of the zygote genome.Abbreviations EDTA ethylene-diamine-tetracetic acid - ER endoplasmic reticulum (SER: smooth ER) - MMC megaspore mother cell - NOR nucleolar organizing region - RNP ribonucleoproteins This work has been partially supported by the Comisión Asesora para la investigación Cientifica by Técnica Projects n^o 613/02 and 613/10     

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.     

Onset of nucleolus organizer activity in early mouse embryogenesis and evidence for its regulation

Ovulated mouse oocytes and preimplantation embryos were examined for NOR activity by means of selective silver staining. Evidence of the first staining activity appeared in two cell embryos, which was later followed by an increase in nucleolar activity, whereas the ovulated oocytes and pronuclei showed no such activity whatsoever. The staining of chromosomes was restricted to the nucleolus organizing region. Our results agree with earlier observations that genes for ribosomal RNA (rRNA) are transcribed as early as in the 2-cell stage in mouse embryogenesis. In addition to the nuclear staining we also observed some silver staining within the cytoplasm, at least from 4-cell stages onwards. Cytoplasmic staining was resistant to incubation with cycloheximide and actinomycin D. Nuclear staining was depressed, or even totally blocked, after actinomycin D incubation but was not blocked by cycloheximide. The onset of silver staining depends not on a specific embryonic stage but on the time interval following ovulation. This appears to indicate that the initiation of ribosomal cistrons is regulated by molecules which are activated or synthesized within the oocyte soon after ovulation.     

Appearance and heterochromatin localization of HP1α in early mouse embryos depends on cytoplasmic clock and H3S10 phosphorylation

Pericentric constitutive heterochromatin surrounds centromeric regions and is important for centromere function and chromatid cohesion. HP1 (heterochromatin protein 1), a homolog of yeast Swi6, has been shown to be indispensible for proper heterochromatin structure and function. In mammalian somatic cells, two HP1 isoforms, HP1α and HP1β, are constitutively present in pericentric heterochromatin until late G₂, when they dissociate from heterochromatin. Subsequently, they re-associate with heterochromatin at late anaphase. In one-cell mouse embryos, pericentric heterochromatin has a unique configuration and features. It does not form heterochromatin clusters observed in somatic cells and known as chromocenters. Instead, in both pronuclei, it surrounds nucleolar precursor bodies (NBPs), forming ring-like structures. These regions contain HP1β but lack HP1α in both pronuclei. In subsequent interphases, HP1β is constitutively found in heterochromatin until the blastocyst stage. It is not known when HP1α appears and what is its function in early mouse embryos. Here, we show that HP1α appears for the first time at late S phase of two-cell stage, at the time when pericentric heterochromatin is replicated. Its appearance is regulated at the level of translation. In two-cell embryos, the amount of HP1α that can bind to these regions is regulated by phosphorylation of serine 10 of histone H3 (H3S10Ph). Elimination of HP1α by siRNA interfered with centromere relocation from heterochromatin surrounding NPBs to pro-chromocenters at the two-cell stage but did not affect preimplantation develoment to the blastocyst stage.     

Nucleolus,nucleolar chromosomes,and nucleolus-associated chromatin from early diplotene to dictyotene in the human oocyte

Summary 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 of³H-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 of³H-uridine uptake showed that the oocyte was not at a resting stage. The possible cytogenetic consequences of these observations are discussed.     

Nucleolar rDNA folds into condensed foci with a specific combination of epigenetic marks

Arabidopsis thaliana 45S ribosomal genes (rDNA) are located in tandem arrays called nucleolus organizing regions on the termini of chromosomes 2 and 4 (NOR2 and NOR4) and encode rRNA, a crucial structural element of the ribosome. The current model of rDNA organization suggests that inactive rRNA genes accumulate in the condensed chromocenters in the nucleus and at the nucleolar periphery, while the nucleolus delineates active genes. We challenge the perspective that all intranucleolar rDNA is active by showing that a subset of nucleolar rDNA assembles into condensed foci marked by H3.1 and H3.3 histones that also contain the repressive H3K9me2 histone mark. By using plant lines containing a low number of rDNA copies, we further found that the condensed foci relate to the folding of rDNA, which appears to be a common mechanism of rDNA regulation inside the nucleolus. The H3K9me2 histone mark found in condensed foci represents a typical modification of bulk inactive rDNA, as we show by genome-wide approaches, similar to the H2A.W histone variant. The euchromatin histone marks H3K27me3 and H3K4me3, in contrast, do not colocalize with nucleolar foci and their overall levels in the nucleolus are very low. We further demonstrate that the rDNA promoter is an important regulatory region of the rDNA, where the distribution of histone variants and histone modifications are modulated in response to rDNA activity.     

rRNA genes are not fully activated in mouse somatic cell nuclear transfer embryos

The well known and most important function of nucleoli is ribosome biogenesis. However, the nucleolus showed delayed development and malfunction in somatic cell nuclear transfer (NT) embryos. Previous studies indicated that nearly half rRNA genes (rDNA) in somatic cells were inactive and not transcribed. We compared the rDNA methylation level, active nucleolar organizer region (NORs) numbers, nucleolar proteins (upstream binding factor (UBF), nucleophosmin (B23)) distribution, and nucleolar-related gene expression in three different donor cells and NT embryos. The results showed embryonic stem cells (ESCs) had the most active NORs and lowest rDNA methylation level (7.66 and 6.76%), whereas mouse embryonic fibroblasts (MEFs) were the opposite (4.70 and 22.57%). After the donor cells were injected into enucleated MII oocytes, cumulus cells and MEFs nuclei lost B23 and UBF signals in 20 min, whereas in ESC-NT embryos, B23 and UBF signals could still be detected at 60 min post-NT. The embryos derived from ESCs, cumulus cells, and MEFs showed the same trend in active NORs numbers (7.19 versus 6.68 versus 5.77, p < 0.05) and rDNA methylation levels (6.36 versus 9.67% versus 15.52%) at the 4-cell stage as that in donor cells. However, the MEF-NT embryos displayed low rRNA synthesis/processing potential at morula stage and had an obvious decrease in blastocyst developmental rate. The results presented clear evidences that the rDNA reprogramming efficiency in NT embryos was determined by the rDNA activity in donor cells from which they derived.     

Variation in the activity of nucleolar organizers and their ribosomal gene content

In metaphase preparations from leucocytes of the toad, Bufo marinus, conspicuous secondary constrictions are present in the number 7 pair of chromosomes. These constrictions were considered to be the nucleolar organizers since they were associated with nucleoli during prophase. In 35 out of 60 individuals taken from natural populations, the homologous nucleolar organizers produced two equal-sized nucleoli and secondary constrictions (Group I animals). Pour animals (Group II) had only one very large secondary constriction in the majority of their metaphase preparations and an abnormally high frequency of cells containing one nucleolus. The remaining 21 animals (Group III) had unequal-sized constrictions in most of their metaphases but were more variable than the individuals of Groups I or II since they also had metaphases with two equal constrictions or only one constriction.The DNA from individuals of each group was hybridized with radioactive ribosomal RNA in order to correlate the size of nucleoli and constrictions with the amount of DNA (rDNA) homologous to ribosomal RNA. The two animals of Group II which were studied contained 0.056% of their genome homologous to ribosomal RNA a value considerably higher than those found for any of the animals of Groups I or III. These high values for rDNA coupled with the morphological appearance of the nucleolar homologues suggested a duplication of the nucleolar organizer in the homologue with the long constriction. The amount of rDNA in animals of Group I and III varied between 0.025 and 0.048% of the genome. Although the animals with unequal-sized constrictions (Group III) had generally lower contents of rDNA than those with equal-sized constrictions (Group I), the values overlapped between the two groups. Further evidence which correlates the size of nucleoli with the number of ribosomal RNA genes comes from studies with a small nucleolar mutant of the Mexican axolotl (Ambystoma mexicanum). Animals homozygous for this deletion were found to contain only 55% of the complement of rDNA present in the wild type. It is concluded that partial deletions and duplications of the nucleolar organizer as well as highly variable contents of rDNA are common in the genome of these amphibians.Presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Biology Department, University of Rochester.     

Localization by high resolution in situ hybridization of the ribosomal minichromosomes during the nucleolar cycle of Physarum polycephalum.

We have used biotinylated rDNA probes to localize by in situ hybridization the extrachromosomal genes for ribosomal RNA in the slime mold Physarum polycephalum. We established conditions that allow for highly specific hybridization at the ultrastructural level and determined that the 60-kb palindromic rDNA molecules are confined to the nucleolus in interphase. Our study definitively locates these extrachromosomal genes in mitosis in the form of thin DNA fibers contained within nucleolar remnants. We further show that these rDNA minichromosomes do not condense and that they segregate as entities independent of the condensed chromosomal DNA. In telophase, these minichromosomes migrate from the poles toward the equatorial region of the nucleus in a direction opposite that of the chromosomes. Our results illustrate the discontinuous nature of the nucleolar organizing region in Physarum.     

Ribonucleic acid transfer from nucleus to cytoplasm during interphase and mitosis in mouse somatic cells cultured in vitro

Summary Kidney cells from primary cultures of 15-day old mouse embryos were incubated for 2, 5 or 10 min with H³-uridine, then either fixed immediately or incubated again for various periods in a chase medium containing an excess of unlabeled uridine and cytidine. The number of grains over the non-nucleolar part of the nucleus (chromatin), the nucleolus and the cytoplasm were counted on the autoradiograms.The grain count showed that both chromatin and nucleolus incorporate very rapidly H³-uridine from the medium, whereas a time lag elapses before any H³-radioactivity above background is detected in the cytoplasm. Incorporation of H³-uridine into the RNA of the nucleus and the nucleolus is not immediately blocked after chase, suggesting that the labeled precursor pool is not completely washed out from the living cell, or diluted by the excess of unlabeled uridine present in the medium. The grain count over the nucleus and the nucleolus rises for a certain time after chase and then gradually declines; H³-radioactivity appears in the cytoplasm 10 min after chase and keeps rising through a 110-min interval. The experiment, then — even though it suggests that the bulk of cellular RNA is synthesized in the chromatin and the nucleolus and then continuously released into the cytoplasm — does not rule out the possibility that some RNA fraction, characterized by a low turnover rate, is synthesized independently in the cytoplasm.Synthesis of RNA is a continuous process throughout the cell cycle, except during metaphase and anaphase. It ceases at prometaphase after the disappearance of the nucleolus and disintegration of the nuclear membrane, and resumes in early telophase. Part of the chromosomal RNA does not remain associated with the chromosomes through division, but is suddenly released into the cytoplasm when the cell enters metaphase.