Ultrastructural and autoradiographic studies of non-generative cells in the antheridium of Chara vulgaris. I. Manubria

During the development of an antheridium DNA content in manubria gradually increases to 8C-16C level. 3H-thymidine incorporation into the nuclei of the manubria lasts till the stage of quantitative predominance of the 16 celled antheridial filaments. The nucleus of the manubria is characterized by the low content of the condensed chromatin and the presence of nucleoli with the nucleolonema-like structure the number of which increases from 6-8 to 32-38 along with the increase of DNA content in a nucleus. In the cytoplasm of the manubria there are numerous secretive vesicles filled with fine-granular substance discharged outside plasmalemma, active Golgi apparatus, well-developed rough ER, numerous polysomes, mitochondria with the condensed structure and plastids with granar and inter-granar thylakoids as well as plastoglobules which increase in number and size along with the development of the antheridium. During spermiogenesis the cells are vacuolated, the number of the secretive vesicles decreases whereas the electron density of their content increases, smooth ER appears while rough ER is reduced. The manubria actively incorporate 3H-uridine, 3H-tryptophane and 3H-leucine. The increase of the incorporation activity is gradual in the period of increasing polyploidy of the manubria and rapid during the initiation of the spermatozoid differentiation. It has been suggested that the manubria should play an important role in the process of spermatogenesis and the induction of spermatozoid differentiation.     

In vivo imaging of nuclear-cytoplasmic deformation and partition during cancer cell death due to immune rejection

In this report, we investigated the in vivo cell biology of cancer cells during immune rejection. The use of nestin-driven green fluorescent protein (ND-GFP) transgenic mice as hosts, in which nascent blood vessels express GFP, and implanted dual-color mouse mammary tumor 060562 (MMT) cells, in which the cytoplasm expresses red fluorescent protein (RFP) and the nuclei express GFP, allowed very important novel observations of angiogenesis and subcellular death pathways during immune rejection of a tumor. Nascent blood vessels did not form in the initially-growing mouse mammary tumor in ND-GFP immunocompetent mice. In contrast, in ND-GFP immunodeficient nude mice, numerous GFP-expressing nascent blood vessels grew into the tumor. The results suggest that insufficient nascent tumor angiogenesis was important in tumor rejection. During immune rejection, the cancer cells deformed their cytoplasm and nuclei, which were readily imaged by RFP and GFP, respectively. The nuclear membrane of the cancer cells ruptured, and chromatin extruded during partition of cytoplasm and nuclei. T lymphocytes infiltrated into the initially-growing tumor in the nestin-GFP transgenic immunocompetent mice. The cytotoxic role of the sensitized T lymphocytes was confirmed in vitro when they were co-cultured with MMT cells. The CD8a-positive lymphocytes attached to the cancer cells and caused nuclear condensation, deformation, and partition from their cytoplasm, similar to what occurred in vivo. The color-coded subcellular fluorescence-imaging model of immune rejection of cancer cells can provide a comprehensive system for further testing of immune-based treatment for cancer.     

Cytology of the human seminiferous epithelium

The appearances in cytologic specimens of the principal cell types in the normal human seminiferous epithelium are described and illustrated. Sertoli cells, which are larger than spermatogenic cells, are characterized by a slightly basophilic, ill-defined cytoplasm of triangular, elongated or columnar shape; the cytoplasm may be vacuolated and may contain spermatozoa. The nuclei of Sertoli cells are round, with a uniformly finely granulated chromatin and a single nucleolus. Spermatogenic cells are round or oval and show scanty cytoplasm with deeper basophilia and well-defined cytoplasmic borders. Multinucleation is common in spermatogenic cells. The Sertoli cells constitute a very homogeneous cell population as compared to the spermatogenic cells, which show several distinct cell types (spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa) whose nuclear structures depend on the stage of meiosis. Both cell types may occur as naked nuclei. Some problems of cell classification are discussed.     

Villous trophoblast of human placenta: a coherent view of its turnover, repair and contributions to villous development and maturation

A coherent view of human villous trophoblast as a continuously renewing epithelium is presented. Epithelia undergoing continuous renewal (e.g. intestinal mucosa, epidermis) display clonogenic cells which pass through several transit divisions before migrating out of proliferation zones and into zones of maturation/differentiation. Quantitative relations (e.g. relative numbers of cells) between proliferation and differentiation zones help to define the steady state and this may vary in response to physiological and pathological circumstances. From the differentiation compartment, cells or cell fragments are eventually extruded by mechanisms which may involve apoptosis. All these features are seen in trophoblastic epithelium. Cytotrophoblast cells (CT, proliferation zone) divide continuously throughout gestation and post-mitotic cells are recruited into syncytiotrophoblast (ST, differentiation zone) after membrane fusion. Evidence of fusion events includes localised confluence of CT and ST cytoplasms, and intrasyncytial plasma membrane segments bearing desmosomal remnants. During differentiation, nuclei undergo changes in shape, chromatin condensation and packing density. Densely-clustered nuclei are associated with cytokeratin intermediate filaments and annulate lamellae. Both clustered and non-clustered nuclei show ultrastructural features of pre-apoptosis and apoptosis. Normally, apoptosis is triggered only when nuclei are in the syncytium. Some (pre-)apoptotic nuclear aggregates are sequestered in syncytial knots, extruded as trophoblast fragments into the intervillous space and then deported into the maternal circulation to be phagocytosed at extraplacental sites. During gestation, there is some constancy in the numerical ratios between CT and ST nuclei pointing to a normal steady state. The steady state may be perturbed when the epithelium is damaged locally. Where the epithelium is denuded, fibrin-type fibrinoid from the intervillous space plugs the discontinuity and, with CT proliferation, facilitates reepithelialisation. Features of normal villous development (e.g. sprouting, intervillous bridge formation, bridge abruption, syncytial knot formation) are explicable in the context of trophoblast turnover with early CT proliferation being mainly for growth and later proliferation for renewal and repair. Adaptive re-settings of the epithelial steady state may also occur in abnormal pregnancies.     

Interactions between endoplasmic reticulum and nuclear membranes of different types of cells during repair of damaged Amoeba nuclei

Repair of amoeba nuclear envelopes that have been damaged microsurgically involves the association of pieces of endoplasmic reticulum with the damaged nuclear membranes. The capacity of endoplasmic reticulum of one type of cell to interact with the nuclear membranes of a different type was tested by placing the damaged nucleus of one kind of amoeba into the cytoplasm of another. Damaged nuclei from Amoeba proteus underwent repair in the cytoplasm of A. discoides or A. indica, as was the case in the reciprocal combinations of these nuclei and cytoplasms. In samples prepared 30 min after operation, heterologous endoplasmic reticulum was associated with holes in the nuclear membranes and appeared to fuse with the nuclear membranes at the margins of the holes. By 5 h after operation, almost all of the cells survived, and the nuclear membranes were largely intact, indicating that repair had occurred. In contrast, when an Amoeba dubia nucleus was damaged and placed in A. proteus cytoplasm there was no evidence of repair and many cells died within a few hours. The results indicate that endoplasmic reticulum and nuclear membranes from different types of cells can interact during repair of damaged nuclear membranes. There appears to be a specificity to this interaction, however, since in a combination of relatively dissimilar cells no association of endoplasmic reticulum with damaged nuclear envelopes was observed and repair did not occur.     

Enzyme synthesis and potential during induction synchrony in the fission yeast Schizosaccharomyces pombe

The fine structure of L cells is described at 30 min and 24 h after enucleation by centrifugation in cytochalasin B. The morphology of the 30-min enucleates is the same as that of the cytoplasm of nucleated cells. Centrioles, a normal Golgi apparatus, endoplasmic reticulum, mitochondria, microtubules, and some microfilaments, are present in enucleates. At 24 h after enucleation, the enucleates are extensively vacuolated. The cisternae of the Golgi apparatus are extremely dilated, and the granular ER is sometimes dilated. Microtubules, and, in particular, microfilaments, are still abundant. Nuclei removed from cells by enucleation in cytochalasin B are surrounded by a thin shell of cytoplasm containing numerous ribosomes, an occasional mitochondrion, a few pieces of endoplasmic reticulum, and an enclosing plasma membrane. Continuities between the nuclear envelope and the ER are particularly frequent. These nuclei possesses a normal fine structure.     

Ultrastructure of cell fusion and premature chromosome condensation (PCC) of thymocyte nuclei in metaphase II mouse oocytes

Following PEG (polyethylene glycol) treatment of ovulated metaphase II mouse oocytes aggregated with thymocytes, fusion of cell membranes occurs. Prerequisites for cell fusion are: close apposition of lectin-agglutinated (phytohemagglutinin-treated) membranes of both cells, formation of firm punctual adhesion sites, and expansion of adhesion sites over a certain area. Establishment of the firm cell-cell contact is associated with development of actin-like filaments along both of the adhering plasma membranes. Membrane fusion occurs at single or multiple sites, and is followed by internalization of thymocyte-oocyte membrane complexes decorated with actin filaments into the hybrid cell cytoplasm. A filamentous actin layer forms also along the inner surface of newly formed hybrid oocyte-thymocyte plasma membrane. Thymocyte nuclei incorporated into oocyte cytoplasm undergo nuclear envelope breakdown and premature chromosome condensation (PCC) leading, eventually, to formation of single chromatids complete with kinetochores. Concomitantly with chromatin condensation an extensive polymerization of microtubules starts in the center of the chromatin mass which leads to the formation of an apparently non-functional spindle-like structure.     

The effect of cooling rate and of dimethyl sulfoxide concentration on the ultrastructure of neonatal rat heart cells after freezing and thawing

The ultrastructure of neonatal rat heart cells in suspension and in tissue culture after freezing at optimal, suboptimal, and supraoptimal cooling rates with 2.5, 5, 7.5, and 10% DMSO was investigated. The effect of DMSO treatment only on the structure of the cells was also studied. A comparison was made with the survival in culture.Without freezing, increasing DMSO concentrations caused an increase of morphological damage, correlating with a decrease of the survival in culture. With 2.5% DMSO there was no difference with untreated cells. At higher DMSO concentrations, the ultrastructural damage increased from spaces between cell membrane and cytoplasm at 5% DMSO to interrupted cell membranes, swollen or destroyed mitochondria, and nuclei with clumped chromatin at 10% DMSO.After freezing at optimal or nonoptimal cooling rates with 5 or 7.5% DMSO, the ultrastructure correlated well with the survival. After freezing with 2.5 or 10% DMSO at optimal or nonoptimal cooling rates, differences in survival were found, which were not reflected in the ultrastructure of the cell. After 8 days of culturing, cells which were frozen at all the different cooling rates and DMSO concentrations appeared to have a normal structure.     

Cytological changes in the testes of vitamin-A-deficient rats. II. Ultrastructural study of the seminiferous tubules.

Ultrastructural study confirmed that, in rats, vitamin A deficiency initially caused the sloughing of some spermatids and spermatocytes into the lumina of the seminiferous tubules around day 3 following the initial decrease of body weight. From days 5 to 10, a considerable number of spermatocytes and spermatids, which still remained in the epithelium, underwent necrosis. Several stages of dying spermatocytes and abnormal spermatids were observed. The latter were distinguished by the presence of chromatin aggregating along the nuclear envelopes and highly vacuolated mitochondria. These cells range from single to multinucleate forms. They were incapable of differentiating further into spermatozoa and ultimately degenerated. Within the same period, Sertoli cells exhibited numerous darkly stained lysosome-like inclusions, and the upper part of their cytoplasm appeared as irregular processes, some of which were broken off and resulted in the thinning of the epithelium. From days 10 to 20, the remaining germ cells comprised mainly spermatogonia and few abnormal spermatocytes. The latter appeared enlarged and were very lightly stained. Their nuclei exhibited unusual blocks of heavily condensed chromatin amidst very highly dispersed chromatin fibers. Though their number was reduced, most of the spermatogonia appeared unaltered. Processes of Sertoli cells became even more irregular and were interrupted at certain sites by large empty spaces. Darkly stained inclusions in their cytoplasm were fewer than observed earlier.     

Catheterized urine cytology of mucinous carcinoma arising in the renal pelvis. A case report

BACKGROUND: Primary mucinous carcinoma of the renal pelvis is a rare tumor; therefore, criteria for cytologic diagnosis of this tumor have not been established. CASE: An 81-year-old woman suffered from macrohematuria for six months and was found to have a tumor in the right kidney by radiographic examination. Catheterized urine obtained from the right renal ureter was viscous and contained spherical clusters of cells with occasionally vacuolated, lacy and basophilic cytoplasm. In the small to medium-sized nuclei, chromatin was coarse and granular, and the nuclear membrane was thin and nearly smooth. Large nucleoli were evident in some of the nuclei. These findings were consistent with adenocarcinoma possibly of mucinous type. CONCLUSION: Preoperative diagnosis of mucinous carcinoma is possible by cytologic findings of catheterized urine together with clinical data.     

Ultrastructural aspects and programmed cell death in the tapetal cells of Lathyrus undulatus Boiss

Programmed cell death (PCD) in the tapetum of Lathyrus undulatus L. was analyzed based on light, fluorescence and electron microscopy to characterize its spatial and temporal occurrence. Development and processes of PCD in secretory tapetal cells of Lathyrus undulatus L. were correlated with the sporogenous cells and pollen grains. At early stages of development the tapetal cells appeared similar to pollen mother cells, structurally. Concurrent with meiosis, tapetum expanded both tangentially and radially as vacuoles increased in size. Tapetal cells most fully developed at young microspore stage. However, tapetum underwent substantial changes in cell organization including nucleus morphology monitored by DAPI. The TUNEL staining confirmed the occurrence of intra-nucleosomal DNA cleavage. In addition to nuclear degeneration which is the first hallmark of PCD other diagnostic features were observed at vacuolated microspore stage intensely; such as chromatin condensation at the periphery of the nucleus, nuclear membrane degeneration, chromatin release to the cytoplasm, vacuole collapse according to tonoplast rupture, shrinkage of the cytoplasm, the increase and enlargement of the endoplasmic reticulum cisternae and disruption of the plasma membrane. After vacuole collapse due to possible release of hydrolytic enzymes the cell components degraded. Tapetal cells completely degenerated at bicellular pollen stage.     

Preblastoderm Nuclear Division in the Embryo of the Large Milkweed Bug, Oncopeltus fasciatus (Dallas)

The preblastoderm nuclei of the large milkweed bug, Oncopeltus fasciatus (Dallas), divide in the absence of centrioles and a spindle fibre apparatus. The preblastoderm nuclei occur in cytoplasmic islands that are not bound by cytoplasmic membranes. The chromatin condenses in association with the nuclear envelope and intranuclear lamellae into two compact masses. The chromatin of the sister nuclei disperse as the nuclei increase in volume and move away from each other in a common cytoplasm. A model is proposed for the orderly separation of the chromatin of the indicated eukaryotic system that is patterned after the site and site territory concept for the control of prokaryotic DNA replication by the membrane. In the case of the milkweed bug preblastoderm nuclei, the nuclear envelope would play a distributive role by providing only one site and site territory in each half of the nucleus for the attachment of a homologue. The presence of a site on the membrane for the attachment of a given chromosome would prevent the formation of a new site for the chromosome homologue within a certain distance.     

FERTILIZATION IN OEDOGONIUM. III. KARYOGAMY

Karyogamy is described in Oedogonium cardiacum from ultrastructural studies. Close proximity of the two gamete nuclei in the fusion cell is established by plasmogamy, whereas karyogamy appears to be initiated by multiple contacts formed between the outer membranes of the adjoining nuclear envelopes. Blebs of endoplasmic reticulum (ER) originate from the outer membrane of each nuclear envelope; these ER blebs presumably contact and fuse with the outer membrane of the nuclear envelope of the opposing nucleus. This is followed by the fusion of the inner membranes of the opposing nuclear envelopes, thereby resulting in a series of small connective bridges between the two gamete nuclei. It is estimated that in this manner 30–50 bridges are formed, perhaps many more. Several of these bridges enlarge relative to the others; one presumably becomes the major connection between the fusing nuclei. As it continues to enlarge, any organelles positioned between the fusing nuclei are pushed aside. There is also evidence, particularly in later stages of karyogamy, that the smaller connective bridges fuse to form larger ones. Temporary cytoplasmic channels often result at the juncture of fusion. In other instances, isolated inclusions of cytoplasm may be delimited by remnants of nuclear envelope deep within the developing zygote nucleus; these inclusions disappear with subsequent development. Throughout karyogamy the contribution of the male gamete nucleus is readily recognized by the characteristic appearance of its highly condensed chromatin. Ultimately, however, this distinction is lost and the content of the mature zygote nucleus assumes a more uniform appearance very similar to that of an egg nucleus. The complete process of fertilization in Oedogonium may occur within 15 min of mixing the spermatozoids with eggs.     

Flow cytometric and ultrastructural aspects of the synergistic antitumor activity of vitamin C-vitamin K3 combinations against human prostatic carcinoma cells

Transmission and scanning electron microscopy and flow cytometry were employed to characterize the cytotoxic effects of vitamin C(VC), vitamin K₃ (VK₃), or VC-VK₃ combinations on a human prostate carcinoma cell line (DU145) following a 1-h vitamin treatment and a 24-h incubation in culture medium. Cells exposed to VC exhibited membranous blebs, aberrant microvillar morphology, mitochondria with swollen cristae and intramitochondrial deposits, as well as nucleoli with segregated components. VK₃-treated cells displayed a damaged cytoskeleton and membranes, a cytoplasm which contained large lumen, condensed polysomes, and severely damaged mitochondria with residual bodies, and nuclei which exhibited chromatic condensation, pyknosis, and karyolysis. VC-VK₃-treated cells exhibited characteristics consistent with necrosis, i.e. swollen mitochondria and swollen, achromatic nuclei with marginated chromatin and intact envelopes, while other cells displayed characteristics consistent with apoptosis, i.e. expulsion of organelle-containing blebs, margination of nuclear chromatin, and segregation of nucleolar components. Vitamin treatment also decreased DNA synthesis, induced a S/G₂ block in the cell cycle, and resulted in the accumulation of fragmented DNA. These results suggested that increased oxidative stress, subsequent membrane damage, and DNA fragmentation were responsible for enhanced cytotoxicity of the vitamin combination.     

Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs

We have studied the pathway of nuclear assembly from demembranated sperm chromatin by fractionating a cell-free system from Xenopus eggs (Lohka, M. J., and Y. Masui. 1983. Science (Wash. DC). 220:719-721). Both the soluble fraction and a washed vesicular fraction are required for formation of normal nuclei that initiate replication in vitro. The soluble fraction alone decondenses chromatin and the vesicular fraction alone surrounds chromatin with membranes. Both fractions are required for formation of nuclear pore complexes. Recombining these two fractions recovers approximately 100% of the nuclear assembly and DNA replication activities. Restricting the proportion of the vesicular fraction slows acquisition of the nuclear membrane and allows observation of immature nuclear pores ("prepores"). These form as arrays around and within the chromatin mass before membranes form. Subsequently membrane vesicles bind to these prepores, linking them by a single membrane throughout the chromatin mass. At the periphery this single membrane is surrounded by an outer membrane. In mature nuclei all membranes are at the periphery, the two membranes are linked by pores, and no prepores are seen. Nuclear assembly and replication are inhibited by preincubating the chromatin with the vesicular fraction. However nuclear assembly is accelerated by preincubating the condensed chromatin with the soluble fraction. This also decreases the lag before DNA replication. Initiation of DNA replication is only observed after normal nuclei have fully reassembled, increasing the evidence that replication depends on nuclear structure. The pathway of nuclear assembly and its relationship to DNA replication are discussed.     

Cytologic findings of atypical adenosis of the breast. A case report

BACKGROUND: Atypical apocrine adenosis, a well-described histopathologic entity, can sometimes be misdiagnosed as carcinoma. Apocrine cells can also appear atypical in cytopathology and be mistaken for carcinoma. Occasional case reports describe false positive cases due to the presence of apocrine cells in a few cases of radial scars and atypical apocrine metaplasia and in a degenerated cyst. CASE: A 37-year-old female underwent ultrasound-guided fine needle aspiration of an ill-defined breast nodule. The aspirate showed clusters and single cells containing abundant granular to focally vacuolated cytoplasm; enlarged, pleomorphic nuclei with irregular nuclear membranes; granular chromatin; and prominent nucleoli. These cells were distinct from and larger than the surrounding ductal and myoepithelial cells. Excision showed a nodular area of atypical apocrine adenosis adjacent to previous biopsy changes, correlating with the cytologic findings. CONCLUSION: Atypical apocrine adenosis can mimic carcinoma in histopathology and cytopathology. One should be cautious when reviewing apocrine cells in cytology, given their atypical features, especially their single, dispersed nature. However, the presence of accompanying benign cellular elements supports a benign diagnosis. Surgical biopsy should be recommended based on the cytologic findings.     

Programmed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx mori

In the present study, we describe the features of programmed cell death of the ovarian nurse cells occurring during vitellogenesis of the silkmoth Bombyx mori. At developmental stage 5, the nurse cells occupy one-half of the follicular volume and obtain a rather spherical shape, while the nurse cell nuclei appear large and elongated, forming impressive projections. At the following stage, stage 6, the nurse cells decrease in size and their shape becomes elliptic. The nuclei remain elongated, being also characterized by large lobes. The lobes of the ramified nurse cell nuclei seem to retain the nucleus in the center of the cell during the dumping of the nurse cell cytoplasm into the growing oocyte. At stage 7, membrane enclosed vacuoles can be easily detected into the nurse cells cytoplasm. Ultrastructural analysis and fluorescent microscopy using mono-dansyl-cadaverine staining of these vacuoles also reveal that they represent autolysosomes. Caspase activity is detected during stage 7, as it is demonstrated by using the Red-VAD-FMK staining reagent. At developmental stages 8 and 9, the nurse cells exhibit chromatin condensation, DNA fragmentation and caspase activity. Finally, during the following stage 10, the nuclear remnants are assembled into apoptotic vesicles, which, after being phagocytosed, are observed in the cytoplasm of adjacent follicle cells. We propose that apoptosis and autophagy operate synergistically during vitellogenesis of B. mori, in order to achieve an efficient and rapid clearance of the degenerated nurse cell cluster.     

ULTRASTRUCTURAL STUDIES PLASMA MEMBRANE RELATED SECONDARY VACUOLES IN CULTURED CELLS

The plasma membrane of cultured cells of several plant species was observed to possess invaginations, or secondary vacuoles, of variable size in the adjacent cytoplasm. These structures, which occurred in cells at different phases in vacuolation, were very numerous in thin sections of some cells but fewer in others. In vacuolated cells enlarged secondary vacuoles protrude into the primary vacuole but are delimited from the tonoplast by an intermembrane zone of variable width. The plasma membrane at the orifice of an invagination may fuse and detach the secondary vacuole from the membrane to form in the cytoplasm a structure bounded by a single membrane. Complex accumulations of membranes consisting of spherical, tubular, and laminar structures, possibly containing cytoplasm, may develop within secondary vacuoles. Contents of many of these vacuoles arise from folds along its limiting membrane which pinch off into the interior of the secondary vacuole. A fibrous substance, possibly derived from the wall, is present in some secondary vacuoles. Observed folding of the plasma membrane and measurements of membrane width of various organelles and cytomembranes support an interpretation that endocytosis occurs in cultured cells.