Ultrastructural and autoradiographic studies of mouse cleavage stages

Studies of cleavage stage mouse embryos are reported, with particular emphasis upon nucleolar fine structural and functional changes. Multiple fibrillar primary nucleoli are present in the early 2-cell embryo. In late 2-cell embryos, some of these nucleoli acquire a peripheral zone of granules, while others reticulate, forming nucleoli composed of fibrillo-granular cortices and fibrillar cores. The nucleoli of early 4-cell embryos are composed only of fibrils. In the middle of the 4-cell stage, some of the nucleoli acquire a peripheral granular zone, while others reticulate. The reticulated nucleoli of both the late 2-cell and 4-cell embryos can be considered, on the basis of their fine structure, to be definitive nucleoli. Early 8-cell and morula embryos usually contain only two definitive nucleoli per nucleus. ³H-5-uridine-pulsed embryos contain label localized in the nucleus, particularly over definitive nucleoli. Nucleolar labeling increases at each successive developmental stage. Beginning at the 8-cell stage, re-incubation in nonradioactive medium results in a significant decrease in nucleolar labeling and an increase in cytoplasmic labeling suggesting that more ribosomal RNA is transferred from the nucleus to the cytoplasm at the later cleavage stages.     

A cytochemical study of the nucleolus and nucleolus-related structures during human spermatogenesis

Besides the components of typical nucleoli, three other nuclear structures were identified in spermatogonia by positive silver staining: dense centers, fibrillar regions, and dense granules. There was a close relationship between the dense centers and fibrillar regions in spermatogonia and spermatocytes, whereas the dense granules, which appeared dispersed throughout the nucleoplasm of spermatogonia, became localized at the periphery of chromosomes in spermatocytes. From the beginning of spermiogenesis, these three structures then appeared in direct relationship with the dense fibrillar component of the nucleolus. At the Golgi phase, a new nuclear structure also appeared in close relationship with the dense fibrillar component. As it was morphologically similar to the fibrillar region, but not silver stained, it was designated fibrillar structure. With further spermatid development, as the dense fibrillar component gradually disappeared, a sharp increase in the number of the associated dense granules was observed; these granules then disappeared as well. In spermatids at cap phase, each fibrillar region appeared intimately associated with several fibrillar structures. In maturing spermatids, silver staining became confined to the fibrils that appeared located inside the nuclear vacuole. The nuclear vacuole also contained a dense fibrillar structure in intimate relationship with these fibrils and the peripheral condensed chromatin. Ethidium bromide-PTA, Na-tungstate and EDTA regressive staining suggest the presence of RNP in the fibrillar regions, dense granules and intravacuolar fibrils, and the presence of DNP in the fibrillar structures.     

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.

     

Coiled bodies in supraoptic nuclei of the rat hypothalamus during the postnatal period

In electron microscopic studies of the supraoptic nuclei of the rat hypothalamus, structures identified as “coiled bodies” were found in magnocellular neurons. Although they could be seen elsewhere in mature neurosecretory cells, coiled bodies were commonly encountered in developing neurons during the postnatal period in both sexes. They appeared as distinctive nuclear inclusions consisting of round-to-oval networks of short electron-dense strands embedded in a less dense, fibrillar matrix, and lacking a limiting membrane. In fine structure and stain-affinity, they bore a resemblance to the fibrillar component of the nucleolus. Coiled bodies were located either in close association with the nucleolus or free within the nucleoplasm, showing no specific relationships with the perinucleolar chromatin or with the nuclear envelope. Their origin and functional meaning is discussed in the light of recent ultrastructural and biochemical data on cellular differentiation and nucleolar behavior.     

Subnuclear localization and phosphorylation of Epstein-Barr virus latent infection nuclear proteins

Functions of the six Epstein-Barr virus latent infection nuclear proteins (EBNA-1, -2, -3A, -3B, -3C, or -LP) in maintaining latent infection or cell growth transformation are only partially understood. Using antibodies specific for each EBNA in immunofluorescence microscopy, EBNA-2, -3A, and -3C localized to subnuclear granules which fill much of the nucleus, excluding nucleoli. EBNA-LP localized to a small number of discrete subnuclear particles, also excluding nucleoli. Only EBNA-1 associated with metaphase chromosomes. Concordantly, in biochemical nuclear fractionation studies, EBNA-1 was the major chromatin-associated EBNA. EBNA-1 also differed from the other EBNAs in the extent of its association with the nucleoplasm and in its lack of nuclear matrix association. EBNA-LP, -2, -3A, and -3C were associated with the nuclear matrix, although they were also found in the nucleoplasm and to a lesser extent in the chromatin fractions. Metabolic 32Pi-labeling of cells followed by two-dimensional gel electrophoresis showed that EBNA-LP could be resolved into multiple phosphorylated isoforms. EBNA-2 was also phosphorylated and many isoforms were detected by isoelectric focusing.     

Nuclear inclusions in immature glial cells of the rat hypothalamus during the postnatal period

Ultrastructural study of the hypothalamic ventromedial nucleus during the postnatal period showed the presence of nuclear inclusions in immature glial cells. These inclusions, identified as "coiled bodies", consist of round-to-oval formations of coiled electron-dense strands embedded in a less dense fibrillar matrix. Coiled bodies are located free within the nucleoplasm, showing no specific relationships with the nucleolus or the nuclear membrane. The cells containing coiled bodies were typified as oligodendrocyte precursors, mainly oligodendroblasts. The coiled bodies were not found in mature glial cells nor in other types of immature glial elements. The nature and possible functional role of coiled bodies are suggested in the light of recent morphological and biochemical data.     

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.     

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.     

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.      

Ultrastructure of preimplantation human embryos co-cultured with human ampullary cells

Ova with two pronuclei were co-cultured with established human ampullary cell lines and various stages of preimplantation embryonic development were monitored by Nomarski optics and then assessed by transmission electron microscopy (TEM). Fifteen embryos ranging from the 2-cell stage to blastocyst hatching were examined for normal and abnormal features. Their ultrastructure was similar to that of embryos cultured in Whittingham's T6 medium, reported previously. Seven embryos were evidently morphologically normal and showed good organization of fine structure. Most cellular organelles underwent progressive changes during early development. There was evidence of enhanced embryonic genome activation at the 8-cell stage. Invariably, all embryos had few too many fragments, some internalized, which were later segregated into the blastocoele or found outside the trophoblast of the late morula and blastocysts. Six grossly 'normal' embryos assessed by Nomarski had multiple nuclei of various dimensions, which highlights the subjectivity of embryo assessment in the IVF laboratory. Incomplete incorporation of chromatin into nuclei and formation of micronuclei were evident in some blastomeres. The results are discussed in relation to early embryonic loss, prevalent in IVF. Significant events reported include the detection of centrioles at the 8-cell stage, cavitation of the early blastocyst and the initiation of blastocyst hatching visualized by TEM.     

Number of nucleoli and coiled bodies and distribution of fibrillar centres in differentiating Purkinje neurons of chick and rat cerebellum

We used differentiating chick and rat Purkinje cells to investigate in homologous neurons the influence of the number of nucleolar organizer regions (two in the chick and six in the rat) on the behaviour of the nucleolus and coiled bodies. We employed specific silver-staining methods on smear preparations and on semithin and ultrathin sections. In chick Purkinje cells the number of nucleolar silver-staining granules increased from 15.7±3 (mean±SD) at embryonic day 13 to 23.8±3 at post-hatching day 7. These nucleolar granules were unevenly distributed between the two nucleoli of binucleolated cells. Electron-microscopic cytochemistry showed that nucleolar granules are equivalent to the fibrillar centres with their associated shell of dense fibrillar component. A reduction in the number of nucleoli was found during the differentiation of both chick and rat Purkinje cells, although in mature cells the average number of nucleoli per cell was higher in the chick (1.60) than in the rat (1.07). The number of coiled bodies decreased from 1.33 in newborn rats to 0.47 at postnatal day 90 in the rat. Coiled bodies were not observed in homologous chick Purkinje cells. The dynamic behaviour of nucleoli and coiled bodies during neuronal differentiation and the relationship of these two nuclear organelles with the number of nucleolar organizer regions is discussed.     

Comparison of the effects of controlled-rate cryopreservation and vitrification on 2-cell mouse embryos and their subsequent development.

Effects of two cryopreservation procedures (conventional slow controlled-rate freezing using a programmable freezer and vitrification by direct plunging into liquid nitrogen) were compared on 2-cell embryos and their subsequent development to blastocysts, fresh or cryopreserved 2-cell mouse embryos were developed into blastocysts in vitro. The percentage of vitrified embryos which developed into blastocysts was significantly lower than that of fresh and slow controlled-rate frozen embryos. Although blastocysts from each cryopreservation procedure appeared morphologically normal and neither number of cells in the blastocysts nor in-vitro trophoblast spreading differed significantly, there were significant differences in their functional viability. First, the glucose incorporation activity in terms of [(3)H]2-deoxyglucose (2-DG) uptake in vitrified and thawed 2-cell embryos significantly decreased compared with fresh or slow controlled-rate frozen and thawed 2-cell embryos. Second, 2-DG uptake by blastocysts developed in vitro from fresh 2-cell embryos and from slow controlled-rate frozen or vitrified 2-cell embryos was 105 +/- 75, 43.0 +/- 28.3 and 22.0 +/- 11.4 fmol/embryo/h respectively. Third, the implantation rate of blastocysts developed in vitro from vitrified 2-cell embryos (10.2%) was significantly lower than that from fresh 2-cell embryos (30.8%) or slow controlled-rate frozen 2-cell embryos (22.1%). Since these data suggest that cryopreservation may have ulterior consequences on the functional development of embryos and that vitrification may exert a more harmful effect than slow controlled-rate freezing, more attention should be paid to its safety before vitrification is used routinely in a clinical programme.     

Adenosinetriphosphatase (Mg-ATPase) activity in the plasma membrane of preimplantation mouse embryo as revealed by electron microscopy

The ultrastructural localization of Mg2+-ATPase activity was investigated in unfertilized eggs and in preimplantation mouse embryos. Enzyme activity was detected on the entire surface of unfertilized eggs and in one-cell embryos. From the two-cell stage up to the 12-cell embryo the reaction product was localized both at the exposed surface of the embryo and between blastomeres. At the late morula stage, activity was only present on the free surface of the peripheral cells outside the apical tight junctions. In the early blastocyst the membranar reaction was observed both on the outside membrane of the trophoblastic cells and on the inner surface facing the blastocoele, whereas in the late blastocyst the reaction remained unchanged on the outer surface of the embryo but was absent from the membranes bordering the blastocoele. These results are discussed with reference to the modification of the cell surface membrane during preimplantation development.     

Expression and localization of Werner syndrome protein is modulated by SIRT1 and PML

Mutations in genes for WRN and BLM RecQ family helicases cause cancer prone syndromes. Werner syndrome, resulting from WRN mutation, is a segmental progeria. Endogenous WRN and BLM proteins localize in nucleoli and in nuclear PML bodies defined by isoforms of the PML protein, which is a key regulator of cellular senescence. We further characterized WRN and BLM localization using labeling with monomeric red fluorescence protein (mRFP). When ectopically expressed, mRFP-WRN (or untagged WRN) forms nuclear bodies, which are donut-shaped in some cells. We identified PML isoforms associating with the nuclear bodies. Interestingly, mRFP-WRN relocalizes from nucleoli to the nucleoplasm, frequently showing conspicuous nucleolar exclusion as well as a decrease in frequency of mRFP-WRN nuclear bodies in response to overexpression of wild-type and deacetylase mutant (H363Y) SIRT1 proteins. Similar nucleolar relocalization in response to wild-type SIRT1 was detected for mRFP-labeled BLM. Moreover, increased SIRT1 expression was associated with the downregulation of endogenous WRN and a decreased frequency of cells with BRCA1 foci. Our data indicate for the first time that SIRT1 protein may be functionally associated with WRN and BLM helicases and that some major SIRT1 functions may not require its deacetylase activity.