Disordered development of macrophages in non-Hodgkin's lymphoma

Macrophage development in 20 untreated patients with non-Hodgkin's lymphoma (NHL) has been studied and compared with that in 20 normal subjects. Morphometric measurements were carried out on ultrastructural features of cell, nucleus and mitochondria during 6 days suspension culture of blood monocytes in the presence of autologous serum and lymphocytes. The results were subjected to multivariate and univariate analysis of variance. Statistically significant differences were found between the subject groups with respect to the volumes and surface areas of cell, nucleus and mitochondria, to the excess surface membrane of cell and nucleus (as compared with equivalent spheres) and to the number of mitochondrial profiles per section. It would appear that the patients' cell grew less, showed less elaboration of surface features and had reduced nuclear and mitochondrial development, the latter affecting mitochondrial numbers rather than individual size. The findings provide further evidence that mononuclear phagocytes are deranged in NHL.     

Quantitative ultrastructural analysis of barley sperm

Summary Complete serial ultrathin sections of seven sperm pairs, computer-assisted measurements of cell, nuclear and organelle surface areas and volumes, and three-dimensional imagery were used to demonstrate that a process of cytoplasm and organelle elimination occurs during sperm maturation in barley. The number of mitochondria per sperm cell is reduced by 50%; sperm cell surface area and volume are reduced by 30% and 51% respectively. Mean volume and surface area per mitochondrion are significantly less in mature sperms. No examples of mitochondrial fusion or degeneration were observed within sperm cells. These data, along with observations of plasma membrane apposition and vesiculation within cytoplasmic extensions containing mitochondria, support the proposition that cytoplasm and organelle loss results primarily from the formation of cytoplasmic projections that are subsequently discarded from the sperm cell body. Comparisons of the quantitative data, including the number of mitochondria, indicate that differences between sperm cells of a pair are absent to very slight. Spatial organization within the pollen grain is such that the mature sperms, as well as the sperms and vegetative nucleus, are not in close proximity.     

Disordered development of macrophages in non-Hodgkin’s lymphoma

Macrophage development in 20 untreated patients with non-Hodgkin’s lymphoma (NHL) has been studied and compared with that in 20 normal subjects. Morphometric measurements were carried out on ultrastructural features of cell, nucleus and mitochondria during 6 days suspension culture of blood monocytes in the presence of autologous serum and lymphocytes. The results were subjected to multivariate and univariate analysis of variance. Statistically significant differences were found between the subject groups with respect to the volumes and surface areas of cell, nucleus and mitochondria, to the excess surface membrane of cell and nucleus (as compared with equivalent spheres) and to the number of mitochondrial profiles per section. It would appear that the patients’ cell grew less, showed less elaboration of surface features and had reduced nuclear and mitochondrial development, the latter affecting mitochondrial numbers rather than individual size. The findings provide further evidence that mononuclear phagocytes are deranged in NHL.     

Effect of water stress on cotton leaves : I. An electron microscopic stereological study of the palisade cells

Palisade cells from fully expanded leaves from irrigated and nonirrigated, field grown cotton (Gossypium hirsutum L. cv. Paymaster 266) were subjected to a microscopic examination to evaluate the effect of water stress on subcellular structures. The water potential difference between the two treatments was 13 bars at the time of sampling. The dimensions of the palisade cells and their density per unit leaf area were determined by light microscopy. Palisade cells from stressed plants had the same diameter, but were taller than their counterparts in irrigated plants. The density of the palisade cells was the same in both treatments as was the fractional volume of the intercellular space. It was concluded that the reduced leaf area observed in the stressed plants resulted primarily from a mitotic sensitivity to water stress. Further, expansion of palisade cells was not inhibited by the stress imposed in this study.

Morphometric analysis of electron micrographs was used to evaluate the subcellular structure of palisade cells from nonstressed and stressed plants. The fractional volumes of cell walls, total cytoplasm, chloroplasts, starch granules, intrachloroplast bodies, mitochondria, peroxisomes, and central vacuoles were determined. The surface densities of grana and stroma lamellae, outer chloroplast membranes, mitochondrial cristae, endoplasmic reticulum and Golgi cisternae were also measured. The number of chloroplasts, mitochondria, and peroxisomes were determined. These data were expressed as actual volumes, areas, and numbers per palisade cell for each treatment. Palisade cells from stressed plants had thinner cell walls, larger central vacuoles and approximately the same amount of cytoplasm compared to cells from nonstressed plants. Within the cytoplasm, stressed plants had more but smaller chloroplasts with increased grana and stroma lamellae surfaces, larger mithchondria with reduced cristae surfaces, smaller peroxisomes and reduced membrane surfaces of endoplasmic reticulum and Golgi cisternae.

     

The nuclear dry weight of liver cells from patients with virus hepatitis and from controls.

Interferometric investigations were performed at liver cell nuclei isolated in 70 per cent glycerol. In 11 patients with virus hepatitis and seven patients without liver disease the nuclear dry weight of liver cells obtained by liver biopsy was determined by interferometry. The average nuclear dry weight of diploid liver cells from controls was 39.9 pg while an average value of 45.4 pg was found for patients with hepatitis. The corresponding nuclear volumes were 241 and 274mu3 respectively. The dry mass and volume of tetraploid nuclei was twice as big as that of diploid nuclei in both materials. The nuclear water content was neither significantly different between diploid and tetraploid nuclei nor significantly different between nuclei from controls and patients with hepatitis.     

Morphology of the differentiation and maturation of LLC-PK1 epithelia

In the present study, a stereologic approach was utilized to quantitatively assess morphological changes during the differentiation of LLC-PK1 cells into an epithelial membrane. This renal epithelial cell line has been described to undergo morphological changes during differentiation and maturation from subconfluent culture to a confluent epithelial layer. An increase in the number of apical microvilli, interpreted as an areal increase in this membrane domain was reported. This morphological differentiation was found to be accompanied by an increase in the expression of apical Na(+)-dependent hexose transport and the activities of certain brush border enzymes. Since no data are available that quantify the morphologic changes during LLC-PK1 differentiation, a quantitative morphologic-stereologic-investigation was performed for an early (6 days) and a late (12 days) state of confluence of LLC-PK1 monolayer cultures. The following morphological parameters were determined by light and electron microscopic morphometry: volume fractions (Vv) of nuclei, mitochondria, and lysosomes, and surface densities (Sv) of the apical and basolateral cell membrane domains. For the apical membrane surface, the microvillous fraction has been measured separately. Since the stereologic approach used in the present study allows the determination of absolute cell volumes, the absolute measures of organelle volumes (V) and membrane surfaces (S) per average cell can be calculated from volume and surface densities. Although no changes in cell density were found for 6 and 12 day old LLC-PK1 monolayers, indicating ceased cell proliferation due to contact inhibition, remarkable changes were found concerning the absolute cell volume and apical membrane surface. The observed increase in the apical cell surface was exclusively due to the enlarged microvillous surface fraction. This finding is in good agreement with the increased number of Na(+)-dependent hexose transporters as well as with the increased expression of apical membrane marker enzymes observed during the differentiation of LLC-PK1 monolayers.     

Stereological analysis of the guinea pig pancreas. I. Analytical model and quantitative description of nonstimulated pancreatic exocrine cells

A stereological model which provides detailed quantitative information on the structure of the fasted, nonstimulated gland has been developed for the guinea pig pancreas. The model consists of morphologically defined space and membrane compartments which were used to describe the general composition of the tissue and the specific components of exocrine cells. The results are presented, where appropriate, relative to a cubic centimeter of pancreas, a cubic centimeter of exocrine cell cytoplasm, and to the volume of an average exocrine cell. The exocrine cells, accounting for 82% of the pancreas volume, consisted of 54% cytoplasmic matrix, 22% rough-surfaced endoplasmic reticulum (RER), 8.3% nuclei, 8.1% mitochondria, 6.4% zymogen granules, and 0.7% condensing vacuoles. Their total membrane surface area was distributed as follows: 60% RER, 21% mitochondria, 9.9% Golgi apparatus, 4.8% plasma membranes, 2.6% zymogen granules, 1.8% plasma membrane vesicles, and 0.4% condensing vacuoles. The application of this model to the study of membrane movements associated with the secretory process is discussed within the framework of an analytical approach.     

Photoreceptor number and outer segment disk membrane surface area in the retina of the rat: stereological data for whole organ and average photoreceptor cell

A random sampling scheme is employed to obtain stereological estimates of disk membrane surface area in the entire retina and in the average photoreceptor cell. The scheme involves the use of vertical sections with combined light and electron microscopy at several magnification levels. Left and right retinas from six albino animals were analysed. There were no significant lateral differences. On average, the retina had a volume of 16 mm3, thickness of 200 μm and surface area of 80 mm2 (representing about 56% of the external surface of the eyeball). Photoreceptor disk membranes within outer segments amplified total retinal surface by almost 1000-fold (final surface 770 cm2 per retina). The retina contained 3×107 photoreceptors (packing density 374 000 mm-2) with an average disk membrane surface area of 2600 μm2. Mean nuclear volume in photoreceptor cells was 59 μm3 and the coefficient of variation for the distribution of nuclear volumes was 57%. The data are consistent with an average of 700 disks per photoreceptor cell, a membrane area of 4 μm2 per disk and a convergence ratio of ～260 photoreceptors per optic nerve fibre. The basic scheme could be modified for other species and for direct cell counts conducted on rods and cones separately.     

Ultrastructural morphometry of blastogenesis I: Transformation of small lymphocytes stimulated in vivo with dinitrochlorobenzene

Summary Changes in the volumes and surfaces of subcellular compartments of unstimulated small lymphocytes and immunoblasts in mouse axillary lymph nodes have been established using stereological techniques. Blast transformation was induced in vivo with dinitrochlorobenzene (DNCB). Cell samples were obtained by random sampling regimes applied at light and electron microscopic levels.From electron micrographs the volume densities of euchromatin, heterochromatin, nucleoli, mitochondria, Golgi apparatus and rough endoplasmic reticulum were determined. Cell surface/volume ratios were also computed. By estimating mean nuclear volumes using light microscopy, it was possible to calculate absolute compartmental volumes and to evaluate the plasma membrane surface areas of average cells.Transformation in this model was characterized by a considerable cellular hypertrophy and a substantial increase in plasmalemma surface. Hypertrophy was the consequence of increases in the volumes of all measured intracellular compartments, notably euchromatin and residual cytoplasm (including ground cytoplasm and free ribosomes). These changes are discussed in the context of the altered metabolic status of cells.M.M. Al-Hamdani was supported by a grant from the Ministry of Higher Education, Republic of Iraq     

Nuclear and Cell Sizes in Different Regions of Root Meristems of Zea mays L.

Nuclear volumes and cell areas were determined for seven regionsof the meristem of roots of Zea mays. Roots were fixed in 10per cent neutral buffered formalin, in 3 per cent glutaraldehydeor in acetic acid/alcohol; they were prepared as sections oralls were teased apart. Mean volumes of interphase nuclei weresimilar in all regions of the root except the vascular tissueof the stele. Mean nuclear volumes and the overall range ofvolumes were similar in sub-populations of cells with differentproportions of G¹, S and G² cells, e.g. in row I of root capinitials, whose cells lack a G¹ phase, and in quiescent centrecells, which are mainly in G¹. Nuclear volume does not appearto be closely correlated with DNA content. Nuclear volumes covereda 6 to 12-fold range within a meristem and even within specificregions, in which cells are part of the same cell lineages,there was a 4- to 9-fold range. Nuclear volumes were comparedin sister cells in rows I and II of the root cap initials. In10 per cent of the pairs, sister nuclei had identical volumes;the other pain had different volumes and mean difference was68 µm³. Mechanisms by which this variability could begenerated are discussed, particularly asymmetry, at mitoses,of factors that regulate nuclear growth. Zea mays L., nuclear volume, cell size, root mcristem, DNA content, mitosis     

A rapid procedure for isolating hemopoietic cell nuclei

A new method for isolating cell nuclei is described which involves freezing and thawing cells in 2% Tween 40, then gentle homogenization to release nuclei, followed by immediate microcentrifugation through 50% sucrose. Purified nuclei were obtained in 3 min and yields of 78-95% were obtained from a variety of human hemopoietic cells. Electron microscope analysis of nuclei obtained from HL60 cells showed that 89% of the nuclei were intact and have an appropriate morphology. A low level of contamination with other organelles was revealed by electron microscopy and by using specific assays for plasma membrane, mitochondria, lysosomes, Golgi membrane, and endoplasmic reticulum (0.5-5.5%). The value of the technique is that nuclear proteins and small metabolites which might be lost by rapid leakage from isolated nuclei and the possibility of biochemical modification of cellular constituents are minimized by using a rapid isolation procedure.     

Mitochondrial growth and division during the cell cycle in HeLa cells

The growth and division of mitochondria during the cell cycle was investigated by a morphometric analysis of electron micrographs of synchronized HeLa cells. The ratio of total outer membrane contour length to cytoplasmic area did not vary significantly during the cell cycle, implying a continuous growth of the mitochondrial outer membrane. The mean fraction of cytoplasmic area occupied by mitochondrial profiles was likewise found to remain constant, indicating that the increase in total mitochondrial volume per cell occurs continuously during interphase, in such a way that the mitochondrial complement occupies a constant fraction( approximately 10-11(percent)) of the volume of the cytoplasm. The mean area, outer membrane contour length, and axis ratio of the mitochondrial profiles also did not vary appreciably during the cell cycle; furthermore, the close similarity of the frequency distributions of these parameters for the six experimental time-points suggested a stable mitochondrial shape distribution. The constancy of both the mean mitochondrial profile area and the number of mitochondrial profiles per unit of cytoplasmic area was interpreted to indicate the continuous division of mitochondria at the level of the cell population. Furthermore, no evidence was found for the occurrence of synchronous mitochondrial growth and division within individual cells. Thus, it appears that, in HeLa cells, there is no fixed temporal relationship between the growth and division of mitochondria and the events of the cell cycle. A number of statistical methods were developed for the purpose of making numerical estimates of certain three-dimensional cellular and mitochondrial parameters. Mean cellular and cytoplasmic volumes were calculated for the six time-points; both exhibited a nonlinear, approx. twofold increase. A comparison of the axis ratio distributions of the mitochondrial profiles with theoretical distributions expected from random sectioning of bodies of various three-dimensional shapes allowed the derivation of an "average" mitochondrial shape. This, in turn, permitted calculations to be made which expressed the two-dimensional results in three-dimensional terms. Thus, the estimated values for the number of mitochondria per unit of cytoplasmic volume and for the mean mitochondrial volume were found to remain constant during the cell cycle, while the estimated number of mitochondria per cell increase approx. twofold in an essentially continuous manner.     

A QUANTITATIVE STEREOLOGICAL DESCRIPTION OF THE ULTRASTRUCTURE OF NORMAL RAT LIVER PARENCHYMAL CELLS

The principles of stereology have been applied to a morphometric analysis of parenchymal cells from the peripheral, midzonal, and central regions of normal rat liver lobules. The fractional volumes of cytoplasm occupied by mitochondria, peroxisomes, lysosomes, lipid, and glycogen have been determined. The surface densities of smooth- and rough-surfaced endoplasmic reticulum and of mitochondrial envelope and cristae have also been measured. The average number and dimensions of mitochondria and peroxisomes have been evaluated. By the use of an independent measurement of the average cytoplasmic volume, these data have been expressed as the actual volumes, areas, and numbers per cell in the different parts of the hepatic lobule. Similarly, the volumes of the envelope, cristae, and matrix compartments and the area of cristae membranes have been calculated for the average-sized mitochondrion in each lobular zone. Structural homogeneity is found in over 80% of normal rat liver parenchymal cells, with most of the significant differences being confined to those cells immediately surrounding the central veins.     

Ultrastructural changes in the rat exocrine pancreas after jejunoileal bypass

The influence of a 90% jejunoileal bypass on the rat exocrine pancreas was studied by morphometrical procedures. In sham-operated animals exocrine acinar cells accounted for 80.3% of the pancreas volume. These cells are composed of 9.9% nuclei, 8.4% mitochondria, 12.2% zymogen granules, 0.3% lipid droplets and 69.2% of a compartment ("ERGLS") composed of endoplasmic reticulum, ribosomes, Golgi areas, lysosomes and the cytoplasmic ground substance. Intestinal bypass did not change the volume density of exocrine cells nor that of nuclei in the cells during the three postoperative months. The means nuclear diameter was approximately the same in both groups. However, the volume density of secretory granules diminished by 50%. This was accompanied by a decrease in mean granular diameter, but not in their numerical density. The volume density of lipid droplets increased 10 fold, that of mitochondria increased slightly from the 15th postoperative day but significantly from the 45th day. The remaining cellular compartment composed of "ERGLS" was not modified by intestinal bypass. These findings suggest that a 90% jejunoileal bypass induces major changes in the composition of pancreatic acinar cells but not in their size.     

Characterization of the ultrastructure of gingival mast cells in alloxan-induced diabetic rats

The morphological changes of gingival mast cells of alloxan-induced diabetic rats were studied by electron microscopy. The following observations were made. The cell nucleus and cytoplasm degenerated. The electron density of the granules in the cell cytoplasm clearly decreased. Some granules had dense irregular threads and the granules were surrounded by a thin vacuole. A ghost vacuole formed in some mast cells and disorganized materials accumulated in the cytoplasm. The mast cell nuclei were generally irregular and degenerating mast cells had pyknotic nuclei. General destruction of the cell membrane and granule shedding in some samples was noted and mitochondria with atypical cristae in the cytoplasm of the mast cells were seen. We conclude that the characteristics of the ultrastructure of gingival mast cells in diabetics are distinctive and should be used as criteria for pathogenesis of gingival inflammation.     

Freeze-fracture features of epithelioid cells,multinucleated giant cells,and phagocytic macrophages

The freeze-fracture morphology of epithelioid cells, multinucleated giant cells (Langhans' type), and phagocytic macrophages was investigated. The intensely folded and interdigitating surface membranes of epithelioid cells and multinucleated giant cells displayed no specialized areas of cell contact. The size of the intramembranous particles (IMP) and the fact that the area density of IMPs was higher in the cytoplasmic (P) faces than in the external (E) faces of the cell membranes agreed with observations in other eukaryotic cells. The area densities of the IMPs suggest lower transport rates of molecules across the cell membranes of granuloma cells than of certain epithelial cells. Small pits were detected in the surface membranes of the granuloma cells but an extrusion of granules was not observed. The cytoplasmic granules displayed very different sizes and shapes ranging from spherical to rod-shaped. The latter type of granules (probably primary lysosomes) dominated in multinucleated giant cells. The granule membranes were studded with IMPs whose area densities increased with the granule size. Multilamellar bodies with smooth (lipid) fracture faces were found only in phagocytic macrophages. The nuclear pores of the granuloma cells were distributed over the entire surfaces of the nuclei and displayed moderate clustering. The values of the area densities of the nuclear pores were in keeping with the values observed in mammalian and human epithelial or mesenchymal cells, indicating similar exchange rates of molecules between the nucleoplasm and the cytoplasm in these different cell types. In a single phagocytic macrophage the E-face of the inner membrane of the nuclear envelope displayed a network of fine filaments whose nature is at present unknown.     

A quantitative morphological study of osmotically induced swelling and shrinkage in nervous tissue

The rabbit retina was used to study, in vitro, the responses of central nervous tissue to changes in extracellular osmolarity. After isolation, retinas were incubated in either hypertonic or hypotonic medium containing 80 milliosmols more or 80 milliosmols less sodium chloride than the isotonic control medium. After fixation and embedding, comparable areas of each retina were sectioned and studied with the phase and electron microscopes. The diameters of receptor cell inner segments, synapses, nuclei, and mitochondria were measured on micrographs; mean nuclear areas and volumes were calculated. Cutouts from micrographs also provided areas and volumes of the receptor cell nucleus and its 'surround' of axons, dendrites, glial processes, and extracellular space. In general, hypertonic incubation produced decreases in the linear dimensions, areas, and volumes of the receptor cell, its nucleus, and its mitochondria that were consistent with their behaviour as osmometers. After hypotonic incubation, the increases in the diameters of inner segments, synapses, and mitochondria were in the predicted range. The increases for the nuclei themselves, and the nuclei and their 'surround' were less than expected. This may have been due to the failure of the preparative techniques to maintain the swollen state of these larger structures.     

Occurrence of Mitochondria in the Nuclei of Tobacco Sperm Cells

Tobacco sperm cells contain intact mitochondria within their nuclei with a frequency of 0.35 [plusmn] 0.13 per cell. These inclusions appear to originate from mitochondria found among chromatids in the highly elongated metaphase plate of the dividing generative cell. These organelles are apparently captured during the reconstitution of the nuclear envelope. Only sperm cells were observed to contain these nuclear mitochondria; generative cells, vegetative pollen cells, transmitting tissue cells, unfertilized egg cells, and central cells lacked them. Nuclear mitochondria were also seen in the nuclei of the egg and central cell after fusion with sperm nuclei, suggesting that nuclear mitochondria are transmitted into the zygote and primary endosperm cells during double fertilization. Organellar inclusions in the sperm nucleus provide a potential mechanism for transmitting organellar DNA into the next generation and could potentially facilitate the transfer of genetic material between the nucleus and other organelles.     

Bimodal variations in subcellular structures of rat thyroid follicular cells during 24 hours: fine structural and morphometric studies

To clarify 24-hr variations in rat thyroid follicular cells under physiological conditions, their subcellular structures were examined at six evenly spaced times during 24 hr by using a morphometric technique. The volume, surface, and numerical densities of subcellular structures varied distinctly over each 24-hr period, with a bimodal pattern. The cellular and nuclear volumes varied also bimodally over 24 hr. A decrease in the surface density of the apical plasmalemma at 1200 and 0000 hr coincided with an increase in volume density of cytoplasmic granules representing colloid droplets and dense bodies. Most granules (colloid droplets) appearing at these times were reduced in electron density. At other times, especially at 1600 and 0400 hr, morphometric parameters of rough endoplasmic reticulum (rER), Golgi complex, and subapical vesicles were prominently increased, although values for rER did not peak at 1600 hr. At these times, the volume densities of cytoplasmic granules, most of which were heterogeneous and of homogeneous electron density, were decreased. These findings coincided with immediate and subsequent reactions of follicular cells after injection of thyroid-stimulating hormone (TSH). From the evidence, it seems likely that variations in follicular cells over a 24-hr period reflect variations in blood TSH concentration. The total membrane areas of membrane components in follicular cells were calculated from the morphometric measurements. These areas fluctuated unimodally during 24 hr over a 65% range. This suggests that the membranes in follicular cells are subjected to cyclic degradation and regeneration during each 24-hr period.     

The differentiation of monocytes into macrophages,epithelioid cells,and multinucleated giant cells in subcutaneous granulomas

The morphological changes occurring in monocytes during their differentiation into macrophages, epithelioid cells, Langhans-type giant cells, and foreign-body-type giant cells were investigated in foreign-body granulomas induced by subcutaneous implantation of pieces of Melinex plastic. Analysis based on Adams's (1974) criteria for discrimination between the several types of cell of the monocyte line, showed that each type has a characteristic type of granule. Primary and secondary granules, numerous in the Golgi area of monocytes were generally found close to the cell membrane and decreased in number in maturing macrophages. This was accompanied by an increase in the number of microtubules. Mature macrophages show numerous characteristic macrophage granules, which are round (average diameter: 280 nm) and have a halo between the limiting membrane and granular matrix. Mature epithelioid cells have characteristic epithelioid cell granules, and multinucleated giant cells a heterogenous population of granules. Fusing macrophages generally have their Golgi areas facing each other, and also show a reduced thickness of the cell coat. The morphology of the multinucleated giant cell is closely related to the number of nuclei present. In Langhans-type giant cells, which generally have two to ten nuclei, a giant centrosphere with numerous aggregated centrioles is found. In transition forms between Langhans-type and foreign-body-type giant cells, which generally contain 10--30 nuclei, the centrioles show less aggregation. In the foreign-body-type giant cells, which generally have more than 30 nuclei, centrioles are virtually absent and never aggregated. These differences between the Langhans-type giant cells, the foreign-body-type giant cells, and the transition forms, support our previous finding that Langhans-type giant cells are the precursors of foreign-body-type giant cells.