Noncanonical structural-functional organization of nucleoli in maturing oocytes of sea urchin Paracentrotus lividus

The dynamics of structural and functional organization of the nucleolus in the oocytes of P. lividus is described. At the late stages of oogenesis the nucleolus is composed of two main components, namely the peripheral zone (PZ) and the central zone (CZ) which are spatially separated. This two-component structure of the nucleolus is formed, at early stages of oogenesis, by stepwise segregation of the fibro-granular component and by its migration to the nucleolar periphery. Absence of morphologically distinct fibrillar centers and dense fibrillar component in nucleoli of both somatic cells and oocytes makes it possible to classify the nucleoli of P. lividus as 'noncanonical' type. Based on detailed morphological and cytochemical analysis the following molecular interpretation of nucleolar ultrastructure in oocytes of P. lividus is proposed: 1) the PZ, containing RNP-positive granules 15 nm in size, but lacking Ag-NOR proteins and BrU incorporation, can be considered a structural equivalent of the granular component of 'typical' nucleoli; 2) the CZ, which is the site of incorporation of RNA precursors, contains intranuclear DNA, RNP-fibers and accumulates Ag-NOR proteins, corresponds to both FC and DFC of 'typical' nucleoli; 3) nucleolar growth during oogenesis, leading to the 1000-fold increase of nucleolar volume, seems to be correlated with the stockpiling of nonfunctioning mature preribosomal particles which will be utilized during embryogenesis.     

Structure of the blood vessels of the microcirculatory bed in different organs of albino rats in neonatal period of ontogenesis

In newborn albino rats the walls of some of the blood vessels of microcirculatory bed are not completely formed in the myocardium of left ventricle, testis, adrenal cortex and liver. Arterioles and venules are found to be few. Unformed areas of these blood vessels contain immature endotheliocytes with nucleolonemal nucleoli (3-4 (min diameter) and numerous ribosomes in the cytoplasm. In these nucleoli the Ag-NOR-proteins are forming a continuous network that unites fibrillar centers and a dense fibrillar component. In rats on postnatal day 8 the number of such endotheliocytes is significantly reduced. In animals aged 15 days the walls of all the vessels of microcirculatory bed acquire definitive structure and only mature endotheliocytes are revealed, the majority of which have annular nucleoli with corresponding one Ag-positive zone.     

Differentiating characteristics of myocardial nuclei in cardiomyopathy

Myocardial tissue obtained by endomyocardial biopsy was semiquantitatively evaluated for nuclear and nucleolar characteristics in six groups of patients: patients with normal cardiac function (group 1), doxorubicin-induced cardiomyopathy (group 2), idiopathic cardiomyopathy (group 3), alcoholic cardiomyopathy (group 4), post-viral cardiomyopathy (group 5), and chronic valvular heart disease (group 6). From each patient, ten nuclei containing nucleoli were examined and rated on the basis of the following characteristics: chromatin clumping, number of fibrillar centers per nucleolus, nucleolonemal structure, size of nucleolus, number of nucleoli per nucleus, and stage of nucleolar change. Mean values for the nuclear characteristics in each group were compared with normal values using the unpaired t test. In the doxorubicin treated group there were significantly increased chromatin clumping, decreased fibrillar centers, decreased nucleolonemal structure, and increased numbers of contracted nucleoli (indicating later stage). These changes may be linked to lowered nuclear and nucleolar activity. In the idiopathic and post-viral groups, characteristics were consistent with increased nuclear and nucleolar activity. There were no significant changes from normal in the nucleolar features of either the alcoholic or valvular groups. Further comparisons between groups using one-way analysis of variance and multivariate statistical analysis support the conclusion that there are significant differences in the nuclear and nucleolar characteristics of these groups.     

Nucleoli in embryos: a central structural platform for embryonic chromatin remodeling?

Nucleoli are the site of ribosomal RNA production and subunit assembly. In contrast to active nucleoli in somatic cells, where three basic sub-compartments can be observed, mammalian oocytes and early embryos contain atypical nucleoli termed “nucleolus-like bodies” or “nucleolus precursor bodies”, respectively. Unlike their somatic counterparts, these structures are composed of dense homogenous fibrillar material and exhibit no polymerase activity. Irrespective of these unusual properties, they have been shown to be absolutely essential for embryonic development, as their microsurgical removal results in developmental arrest. Historically, nucleolus-like and nucleolus precursor bodies have been perceived as passive storage sites of nucleolar material, which is gradually utilized by embryos to construct fully functional nucleoli once they have activated their genome and have started to produce ribosomes. For decades, researchers have been trying to elucidate the composition of these organelles and provide the evidence for their repository role. However, only recently has it become clear that the function of these atypical nucleoli is altogether different, and rather than being involved in ribosome biogenesis, they participate in parental chromatin remodeling, and strikingly, the artificial introduction of a single NPB component is sufficient to rescue the developmental arrest elicited by the NPB removal. In this review, we will describe and summarize the experiments that led to the change in our understanding of these unique structures.

     

Development of nucleolar apparatus in the chick primitive erythroid cells

The primitive erythroid line cells of chick embryos were studied during embryonic days 2–14 by means of a cytochemical method to investigate the appearance and frequency of the main nucleolar types. The populations of erythroblasts and erythrocytes were classified according to the presence of functionally dominant nucleoli in their nuclei. In the course of primitive erythroid cell differentiation and maturation, compact nucleoli and nucleoli with nucleolonemas (both supposed to be RNA biosynthetically active) were gradually replaced by ring-shaped nucleoli and finally by micronucleoli reflecting the reversible and irreversible inhibition of RNA synthesis, respectively. The occurence of the main nucleolar types and their values in primitive erythroid cells of the developing chick depend not only on the maturation stage of the blood cells, but also on the developmental stage of the chick embryo. In comparison with the definitive erythroid line of the post-hatching chick and hen, the cells of the chick embryonic primitive erythroid line possess relatively high values of active nucleolar types. These are still present in advanced maturation stages, and occur also as definitive erythroid lines of lower vertebrates.     

The silver reaction of nucleolar proteins in the main structural compartments of ring-shaped nucleoli in smear preparations.

The present study was undertaken to provide more information on the conditions which result in preferential silver staining of the main nucleolar structural compartments using silver stainable proteins as their markers at the light microscopic level. For this study the mostly used method in cytology and pathology in which the nucleolar silver-positive structures are "developed" with the colloidal developer (Howell and Black, 1980; Ploton et al., 1986) was selected as silver reaction. Ring-shaped nucleoli of mature human lymphocytes represent a convenient model for such a study because they consist of one large fibrillar center, adjacent nucleolar regions with dense fibrillar components and the nucleolar peripheral shell with dense granular components. All these nucleolar compartments are known to possess characteristic silver stainable proteins. The results demonstrated that proteins of the fibrillar center and possibly adjacent nucleolar regions reacted preferentially with silver after a relatively long fixation with formaldehyde or methanol in unwashed specimens before the silver reaction. In contrast, the preferential staining of proteins in the nucleolar peripheral shell with silver was achieved after the fixation with acidified methanol or ethanol as well as after short fixation with formaldehyde vapors. In addition, the commonly used fixation before the silver reaction are not necessary and may be omitted for the visualization of all silver stainable proteins present in the fibrillar center as well as in the adjacent nucleolar regions and the nucleolar peripheral shell. In addition, similar results were achieved for the simultaneous visualization of proteins in the fibrillar center and nucleolar peripheral shell after fixation with ethanol.     

Effect of phosphate on the second cleavage division of the rat embryo

Development of the rat embryo is arrested at the 2-cell stage in vitro in the presence of inorganic phosphate (Pi). Rat embryos were affected by exposure to 1.19 mM KH2PO4 in modified hamster embryo culture medium- 1 at the late 2-cell stage only. When exposure durations were 6 h, embryos whose exposure timings were prior to cleavage had a reduced rate of development to the blastocyst stage (2-8%) when compared with embryos with no exposure to Pi (97%, P < 0.05). When exposure durations were 18 h, all embryos were arrested at the 2- to 4-cell stage. These timings would correspond to the G2 to M phase of the second cell cycle. Maturation-promoting factor (MPF), which is regulated by a phosphorylation cascade, controls cell division, and its kinase activity is necessary in order for the cell to enter the M phase. However, the histone H1 kinase activity levels and the patterns of the state of phosphorylation of cdc2 were the same in blocked and non- blocked embryos. Because MPF was active in blocked embryos, the developmental block in rat 2-cell embryos caused by phosphate was not due to MPF activity or its phosphorylation cascade.      

p66shc, but not p53, is involved in early arrest of in vitro-produced bovine embryos

High embryo loss occurs in the first week of bovine embryo development, with a high percentage of embryonic arrest. We hypothesized that arrested embryos enter a 'senescence-like state' and that both the cell cycle regulatory protein p53 and the stress-related protein p66(shc), which are involved in the onset of senescence in somatic cells, are responsible for this early embryonic arrest. In our in vitro production system, 13.5 +/- 0.5% of embryos arrest at the 2-4-cell stage. First cleavage occurs between 26 and 48 h post insemination (hpi), with early cleaving embryos showing only 0.6 +/- 0.3% arrest, with later cleaving embryos exhibiting up to 14.2 +/- 0.9% arrest. We compared 2-4-cell embryos collected at 28 hpi with those arrested at the 2-4-cell stage collected at day 8 post insemination. Quantification by real-time PCR and by semi-quantitative immunofluorescence showed significantly higher p66(shc) mRNA and protein levels in both arrested and late cleaving embryos versus 28 hpi embryos. By comparison, no significant changes in p53 mRNA, protein and phosphorylation levels were detected. Taken together, these results demonstrate that embryonic developmental potential is related to the time of first cleavage and that p66(shc), but not p53, is up-regulated in early arrested in vitro-produced bovine embryos.     

Nerve cell nuclear and nucleolar abnormalities in the human oedematous cerebral cortex. An electron microscopic study using cortical biopsies

Cerebral cortical biopsies of 17 patients with clinical diagnosis of congenital hydrocephalus, complicated brain trauma, cerebellar syndrome and vascular anomaly were examined with the transmission electron microscope to study the nuclear and nucleolar abnormalities induced by moderate and severe brain oedema, and the associated anoxic-ischemic conditions of brain tissue. In infant patients with congenital hydrocephalus and Arnold-Chiari malformation two different structural patterns of immature chromatin organization were found: the clear type characterized by a clear granular and fibrillar structure of euchromatin, scarce heterochromatin masses and few perichromatin granules, and a dense granular and fibrillar euchromatin with abundant and scattered heterochromatin masses, and increased number of perichromatin granules. The lobulated nuclei exhibited an irregularly dilated and fragmented perinuclear cistern, and areas of apparently intact nuclear pore complexes alternating with regions of nuclear pore complex disassembly. In moderate traumatic brain injuries some nucleoli exhibit apparent intact nucleolar substructures, and in severe brain oedema some nucleoli appeared shrunken and irregularly outlined with one or two fibrillar centers, and others were disintegrated. The nuclear and nucleolar morphological alterations are discussed in relation with oxidative stress, peroxidative damage, hemoglobin-induced cytotoxicity, calcium overload, glutamate excitotoxicity, and caspase activation.     

Nucleolar grading of renal cancer. Correlation of frequency and localization of nucleoli to histologic and cytologic grading and stage of renal cell carcinomas

Cytologic-nucleolar grading of carcinomas includes mitotic frequency, mean nuclear size, nuclear variability, degree of chromasia and nucleolar frequency, and the occurrence of singular or multiple, small or large nucleoli in the central or peripheral location. A total of 186 renal cell carcinomas were analyzed cytologically in this respect. The results were correlated to the histologic pattern, tumor stage, and grading. Renal cell carcinomas of low malignancy and stage possess a low nucleolar frequency and small solitary nucleoli in the central location. Highly malignant carcinomas (T3 or T4) are characterized by high nucleolar frequency and multiple prominent nucleoli in the eccentric position. There is no correlation to the histologic pattern. Therefore, the nucleolar status is helpful in grading and determination of prognosis of renal cell carcinomas.     

Embryo morphology or cleavage stage: how to select the best embryos for transfer after in-vitro fertilization

This retrospective study of 1001 in-vitro fertilization (IVF) cycles included a consecutive series of single transfers (n = 341), dual transfers (n = 410) and triple transfers (n = 250) where all the transferred embryos in each cycle were of identical quality score and identical cleavage stage. In our 2 day culture system, transfer of 4- cell embryos resulted in a significantly higher implantation rate and pregnancy rate (23 and 49%) compared with 2-cell embryos (12 and 22%) and 3-cell embryos (7 and 15%). Furthermore, the transfer of 4-cell embryos resulted in a significantly higher pregnancy rate compared with embryos that had cleaved beyond the 4-cell stage (28%). The implantation rate (21%) and pregnancy rate (43%) after transfer of embryos of score 1.0 were significantly higher than after transfer of embryos of score 2.0 (14 and 32% respectively). Transferring embryos of score 2.1 resulted in significantly higher implantation rates (26%) and similar pregnancy rates compared with score 1.0. Transferring embryos of score 2.2-3.0 resulted in a significantly lower implantation rate (5%) and pregnancy rate (15%). A striking finding was that embryos of quality score 2.0 had a significantly lower implantation rate compared with embryos of quality score 1.0 and 2.1 and a significantly lower pregnancy rate compared to embryos of quality score 1.0. We also found a lower implantation rate and pregnancy rate when transferring 3-cell embryos. These findings may indicate periods of increased sensitivity to damage during the cell cycle. In conclusion, these results substantiate the idea of the superiority of 4-cell embryos and demonstrate that minor amounts of fragments in the embryo may not be of any importance. These findings may call for a shift when weighing the two main morphological components (quality score and cleavage stage) in the sense that reaching a 4-cell cleavage stage even with the presence of a minor amount of fragments should be preferred to a 2-cell embryo with no fragments.      

Conceptus polarity and cell-zona adhesion during early cleavage (fertilized tubal egg to 8-cell stage) in the marsupial Sminthopsis macroura

This study outlines the ultrastructural changes that occur in Sminthopsis macroura tubal zygotes to the 8-cell stage in relation to observations of development in vitro, oocyte polarity and cell-zona adhesion. The extremely polarized mature oocytes and zygotes have nuclear material at one pole and accumulated vesicular bodies at the other. The first division is associated with extrusion of vesicular bodies and some cytoplasm as a membrane-bound yolk mass into the perivitelline space. Early cleavage is accompanied by the appearance of an extensive, highly structured extracellular matrix (ECM) comprised of amorphous substance, granules and filaments. At the 2- and 4-cell stage the decrease in density of the ECM in the vicinity of the blastomeres may facilitate cell-zona contact. At the 8-cell stage, discharge of vesicular bodies, which mostly appear to be empty, may contribute to the ECM by increasing the area of plasma membrane for synthesis of a hyaluronan-like ECM. As in other marsupials, the precedence of cell-zona adhesion over cell-cell contacts prevents morula formation. The earliest cell-zona contacts appear when microvilli contact the zona in the uterine zygote 12-16 h after uterine entry and continue at later stages. This early contact is possible because of the absence of a dense subzonal ECM in this species. Between late zygote and late 4-cell stage the cytoplasm also contains, beside a large amount of vesicular bodies, demarcated areas where smooth endoplasmic reticulum encloses mitochondria, vesicles, granular material and fibrillar arrays. The latter develop in the late zygote stage and are found outside demarcated areas as well, often closely surrounding large vesicles, probably helping vesicle extrusion. A putative germ plasm was identified at the 4-cell stage.     

Sinistral equal-size spiral cleavage of the indirectly developing polychaete Hydroides elegans.

Two major variants of the stereotypic spiral cleavage correlate with distinct developmental modes in polychaetes. Indirect development through a feeding trochophore larva correlates with development from four equal-sized blastomeres, whereas direct development correlates with unequal cleavage characterized by a large dorsal blastomere precursor maternally predetermined. The equal-size spiral cleavage of the indirectly developing serpulid Hydroides elegans has been reconstructed from serial sections of nuclei-stained embryos. The order of cell divisions has been determined from the 2-cell stage to the 80-cell stage, when gastrulation cell movements start to overlap with late spiral-cleavage divisions. In contrast to related species, the third cleavage in Hydroides elegans is invariably sinistral. The four quadrants remain indistinct until the 60-cell stage, when the small 2d22 and large 2d21 cells are generated. The developmental significance of the invariant spiral cleavage relates to the spatial distribution of gene functions that it partitions and their relation to blastomere fate commitments. The conservation and divergence of the cleavage pattern among spiralians is well suited to study the developmental control of the cell-cleavage machinery and its evolution.