Correlative Fine Structural,Behavioral, and Histochemical Analysis of Ascidian Blood Cells

Abstract The blood cells of a solitary ascidian, Halocynthia roretzi, were examined by electron microscopy (EM) with reference to their appearance by light microscopy (LM). In addition, their movement and stainability by vital dyes was observed by phase-contrast microscopy, and their stainability by Giemsa was also examined. Nine cell types were recognized: vacuolated cells, hyaline amoebocytes, small amoebocytes, granular amoebocytes, macrogranular cells, globular cells, lymphocyte-like cells, large basophilic cells and large granular cells. Vacuolated cells were found to possess various numbers of vacuoles containing strongly electron-dense materials and could be divided into at least three subgroups. Granular amoebocytes contained microfilaments and many granules of uniform size. Hyaline amoebocytes and small amoebocytes seemed to be specialized as phagocytes. Macrogranular cells and globular cells were not well characterized. In the blood of adult individuals, hemoblasts were rarely found, although lymphocyte-like cells were present. Each of two large cells, large basophilic cells and large granular cells, possessed novel granules or vacuoles, whose functions remain to be elucidated. The possible functions and relationships of these cells among various ascidian species are discussed.     

The lipofuscin in neuroglial cells of the optic nerve of Formosan rock-monkey (Macaca cyclopis)

The ultrastructure of lipofuscin granules in neuroglial cells of the optic nerve of the Formosan Rock-Monkey was investigated by electron microscopy. In the cytoplasm of astroglial cells, numerous irregular lipofuscin granules were characterized by the presence of large lipid droplets, small electron-dense pigment granules, and some lamellar structures. The lipofuscin granules of the oligodendroglial cells were composed largely of dense, coarse pigment granules, multilinear structures, and a few small lipid droplets. The lipofuscin granules in microglial cells were characterized by numerous lipid droplets in various sizes, small electron-dense pigment granules, and prominent lamellar structures. It was reported that the lipofuscin granules are wear-and-tear materials and products from the cells in lower functional activity. However, our observations suggest that the presence of lipofuscin granules in the neuroglial cells of the optic nerve is likely a characteristic product of active phagocytosis.     

The formation of glandular secretion in larval Austramphilina elongata (Amphilinidea)

The ultrastructure of three types of gland cells of embryos and free-swimming larvae of Austramphilina elongata is described. Type I gland cells contain large, more or less round electron-dense granules which are formed by numerous Golgi complexes. Type II gland cells contain thread-like, membrane-bound secretory granules with longitudinally arranged microtubules inside the granules; secretory droplets are produced by Golgi complexes and the microtubules apparently condense in the cytoplasm or in the droplets. Type III gland cells contain irregular-ovoid membrane-bound granules with coiled up microtubules which have an electron-dense core; the granules are formed by secretionderived from Golgi complexes and the microtubules aggregate around and migrate into the secretion; microtubules are at first hollow and the early secretory granules have a central electron-dense region.     

A light and electron microscopic study of the pineal gland of the ground squirrel, Citellus tridecemlineatus.

The pineal gland of the 13-lined ground squirrel (Citellus tridecemlineatus) has been examined at the light and electron microscopic level. This gland is composed of low-density parenchymal cells interspersed among which are occasional glial, vascular and neural elements. Punctuating the glandular parenchymal mass are prominent perivascular and intercellular spaces. The parenchymal cells possess numerous mitochondria and less prominent profiles of rough and smooth-surfaced endoplasmic reticulum. Golgi apparatus, microtubules and lipid droplets of varying size and electron density constitute regular cytoplasmic features, with dense-core vesicles being present occasionally. The parenchymal cells have numberous processes. One among these in each cell extends for several micra to terminate in a bulbous expansion containing both clear and dense-core vesicles and occasional electron-dense inclusions. These bulbous terminals are found within the perivascular and intercellular spaces where they course in close proximity to both other parenchymal elements and axon terminals. Glial cells and their processes invest the pineal periphery and incompletely separate the parenchymal cells.     

Morphological Characterization of Three Phenotypes of the Isopod Armadillidium vulgare

The morphological characteristics and ommochrome quantity in the integument of red, white, and wild type (black-grey) Armadillidium vulgare were studied. The red phenotype was found to possess two kinds of immature ommochrome pigment granules within its pigment cells, in addition to mature pigment granules. The immature granules seemed to contain uniformly distributed fibrilles, or to have an electron-dense central region surrounded by an electron-lucent outer edge. Since these immature pigment granules were typically observed to be distributed along with the mature ones, and were also more easily extractable than the wild type's, it is hypothesized that ommochrome granule maturation in the red phenotype may occur slowly due to a defect in the pigment granule internal process which combines pigments with matrix proteins. Regarding the white phenotype, although its pigment cells were undeveloped, several large-sized vesicles containing a small amount of electron-dense material appeared in the pigment cell cytoplasm. The wild and red type males of A. vulgare were found to have an ommochrome content twice as large as that of the corresponding females, with no ommochrome pigment being detected in the white phenotype. The genetic relationship between the white and red phenotypes was discussed using as a basis the observed pigment granule structure.     

Differences between blood cells of juvenile and adult specimens of the pond snail Lymnaea stagnalis

Summary Blood cells (amoebocytes) of juvenile and adult specimens of the pond snail Lymnaea stagnalis were compared. Juvenile snails contain fewer circulating amoebocytes per l haemolymph. However, a higher percentage of these cells shows mitotic activity, as determined by incorporation of ³H-thymidine in vitro. Relatively more amoebocytes of juvenile snails have the characteristics of less differentiated cells: they are small and round with few inclusions, a high nucleus-to-cytoplasm ratio, and a high pyronin stainability. Enzyme cytochemical studies showed that acid phosphatase (AcP), non-specific esterase (NSE), and alkaline phosphatase (AlP) are present in all amoebocytes of juvenile and adult snails. AcP activity is relatively weak. NSE activity is dispersed throughout the cytoplasm and occasionally found in granules, whereas AlP is clearly localized in granules. Differences between the two age groups were found only for the enzyme peroxidase (PO). In juvenile snails a lower percentage of the cells is positive and the granules that contain the activity are less abundant than in amoebocytes of adults. It is suggested that, due to the above-mentioned characteristics of the amoebocytes, the activity of the internal defence system in juvenile L. stagnalis is on a lower level than that in adult snails. This might be an explanation for the fact that juvenile L. stagnalis are highly susceptible to infection by the schistosome Trichobilharzia ocellata, whereas adult snails are less susceptible.     

Occurrence of ommochrome-containing pigment granules in the central nervous system of the silkworm, Bombyx mori

Dark-red pigment granules were found in the brain and ganglion of the normal strain of the silkworm, Bombyx mori, by light microscopy. No other pigmentation was seen in the brain or ganglia. Electron microscopy showed that the granules were electron-dense. The granules were similar to the ommochrome-containing pigment granules that are present in the epidermal cells of the quail mutant, as previously reported. The pigment in the larval central nervous system (CNS) of the normal silkworm was identical to the ommin standard with respect to the absorption spectrum, the infrared spectrum, and the Rf value in thin-layer chromatography (TLC). After acid hydrolysis of the pigment, 3-hydroxykynurenine was detected by TLC. The pigment granules in the CNS contained mainly ommin. An ommochrome-binding protein was also detected in the CNS by in vitro binding studies and Western blotting. The ommochrome granules may have an important function in the CNS of the silkworm.     

Ultrastructural observations on the granular leucocytes of the tuatara Sphenodon punctatus (gray)

The granular leucocytes of an active, mature female tuatara, Sphenodon punctatus (Gray) were examined in the electron microscope. Eosinophils contained a lobulated nucleus, homogeneous, dense, irregularly shaped granules, assorted smaller granular inclusions, mitochondria and beta-glycogen. Endoplasmic reticulum, Golgi complexes and ribosomes were scanty. Immature neutrophils (myelocytes) were regular in outline and contained a compact nucleus. In the adjacent centrosomal region were paired centrioles with connected microtubules, and Golgi complexes. Ovoid electron-dense granules, mitochondria, lipid droplets and numerous microfilaments arranged randomly or in bundles, lay in the cytoplasm. Mature neutrophils were often highly irregular in outline, had a segmented nucleus and contained possibly a second type of granular inclusion. The basophils were regular in outline with a compact nucleus. Numerous ovoid homogeneous, electron-dense granules, mitochondria, beta-glycogen particles and some microfilaments were seen in the cytoplasm. The granules in many basophils appeared 'altered' or degenerate and most of these contained microtubules. The cytology of the granulocytes of the tuatara is compared with that in other vertebrates.     

Ultrastructure of secretion in the atrial gland of a mollusc (Aplysia)

Extracts of the atrial gland of the sea hare Aplysia californiea (Mollusca) induce egg laying when injected into mature individuals. Since egg laying is controlled endogenously by a peptide secreted by neuroendocrine cells in the central nervous system, the relationship between the atrial gland and these central neurons has become an issue of interest. With the particular objective of examining secretory structures we undertook an ultrastructural study of the atrial gland and adjacent tissues. This study revealed that the atrial gland epithelium is composed of two major cell types: ‘goblet-like’ exocrine cells containing large electron-dense granules, and ciliated ‘capping cells’. A non-secretory, and possibly post-secretory, cell containing electron-lucent granules was noted. A region of the large hermaphroditic duct contiguous to the atrial gland, known as the red hemiduct, also displayed capping cells and secretory cells with large granules. The content of these granules is organized into crista-like condensations. The cell also contains iron-rich pigment inclusions.     

Ultrastructure of the eyes of the grass shrimp,Palaemonetes pugio

Summary The cone cells and corneagenous cells possess extensive networks of smooth tubular endoplasmic reticulum that may be involved in optical reflectance and light-adaptational responses, respectively. The extracellular basal lamina of the basement membrane is confluent with glial cell capillary walls and may prove to be a viaduct for the transmission of hemolymph-borne substances to the retina or of retinal degradation products to the hemolymph. In addition to dense pigment granules, the distal pigment cells are shown for the first time to contain migratory reflecting platelets that are usually polymorphic in light-adapted eyes but are rectangular in dark-adapted eyes. In the latter these plates become aligned against the crystalline cones and presumably contribute to the reflection superposition optics of the grass shrimp. Dark-adapted retinular cells possess well-developed perirhabdomal cisternae, oblong or ovoid mitochondria, generally vesicular rough endoplasmic reticulum, and occasional, spherical, calcium-like intrarhabdomal inclusions. Light-adapted retinular cells possess poorly developed perirhabdomal cisternae, lamelliform rough endoplasmic reticulum, and condensed mitochondria frequently associated with lipid droplets and pigment granules. The cytoplasmic boundaries of the reflecting pigment cells expand into the extracellular spaces between individual ommatidial retinular cells during dark adaptation and recede to the interommatidial extracellular spaces during light adaptation. Cytoplasmic microfilament bundles found only at the bases of partially light-adapted rhabdomeric microvilli may be involved in microvillar shortening.     

THE ROLE OF MICROTUBULES IN THE MOVEMENT OF PIGMENT GRANULES IN TELEOST MELANOPHORES

When microtubules in teleost melanophores are disrupted with antimitotic agents, colchicine, high hydrostatic pressure, low temperature, and vinblastine, the alignment and movement of the pigment granules in these cells disappear; during recovery, the return of alignment and movement corresponds in both time and space with the repolymerization of microtubules. Furthermore, analysis of nearest neighbor distances in untreated melanophores reveals that pigment granules are closely associated with microtubules. Other structures such as microfilaments, the endoplasmic reticulum, and the cytoplasmic matrix do not appear to be involved. Thus we conclude that microtubules determine the alignment and are essential for the selective movements of the pigment granules in these cells. Investigations of the mechanism of movement show that microtubules are required for both centrifugal and centripetal migrations and that they do not change in number or location during redistribution of pigment. Our results further indicate that microtubules in melanophores behave as semistable organelles as determined by investigation with colchicine and hydrostatic pressure. These observations and others rule out a push-pull mechanism based on the polymerization and depolymerization of microtubules or one which distinguishes two operationally different sets of microtubules. We propose instead that particles move by sliding along a fixed array of microtubules.     

Fine structure and taxonomic position of the giant amoeboid flagellate Pelomyxa palustris

Specimens of Pelomyxa palustris from five collecting sites had numerous nonmotile flagella. The structures are called flagella because of morphological similarities to flagella and because P. palustris has affinities with amoeboid flagellates. Flagella were photographed on living cells and studied by transmission and scanning electron microscopy. From 64 to 742 flagella per cell were estimated from scanning electron microscopy of ten cells 204 to 1269 micron in length. The nonmotile flagella arise from basal granules which were, in one strain, surrounded by radiating electron-dense microtubules. This strain also had excess axonemal microtubules. Abundant cytoplasmic microtubules were arranged in several different patterns. In about half of the P. palustris cells in which nuclei were studied, microtubules were either apposed to the nuclear membrane in a parallel alignment (with some also radiating) or radiating from the nuclear membrane (with none parallel). Bacteria associated with nuclei were of three characteristic types: Gram-negative rods, Gram-positive rods, and large rods. All nuclei within a given trophozoite had similar perinuclear features. Recent proposals for separation of Pelomyxa to its own phylum (based on its proposed primitive, unique nature) can not be justified. Pelomyxa is a complex, highly specialized organism adapted to live in a specific fresh-water environment. Mastigamoebid amoeboid flagellates of the genera Mastigamoeba, Mastigella, Mastigina, and possibly Dinamoeba are placed with Pelomyxa within the order Pelobiontida Page, 1976, emend., containing two families. Pelomyxidae Schulze, 1877, and Mastigamoebidae Goldschmidt, 1907.     

Ultrastructural changes in the pars distalis of the maturing male rat

Anterior pituitary glands of male rats (2, 3, 5, 8, 12, 25, 36, 52, 56, and 62 days of age) were processed for electron microscopy. During early postnatal stages secretory cells are found in various stages of differentiation and comparatively few secretory granules are seen. Nuclei are mostly irregular, and the nucleo-cytoplasmic ratio is large. Many free ribosomes are present; the endoplasmic reticulum is generally sparse and the Golgi complex small or invisible. Cells are of variable shape, and numerous cytoplasmic processes project into large intercellular spaces. Many electron-dense cells which often contain myelinlike figures are seen. Lysosomes and lysosomal precursors are frequently found in secretory cells, predominantly in somatotrophs, of all immature glands. Mitotic figures are numerous in early stages after brith and decrease in number as the gland grows in size. A gradual increase in cytoplasmic volume with concomitant differentiation of cytoplasmic components as well as accumulation of secretory granules, accompanied by loss of myelin-like figures and decrease in the number of electron-dense cells, is observed as the animal reaches the prepuberal stage. Few lysosomes are seen in cells of mature glands. At 36 days of age all secretory cells seem to have differentiated, and morphological features as well as granule content show little change until puberty is reached. Gonadotrophs attain their characteristic morphology later than other cells. Cilia are observed in all developmental stages but are relatively infrequent in the mature gland. The described ultrastructural characteristics reflect the degree of maturation as well as the functional capacities of secretory cells at particular stages of development.     

Identification of two types of melanocyte within the stria vascularis of the mouse inner ear

We have distinguished two types of melanocyte within the intermediate layer of the stria vascularis in the cochlea of normally pigmented mice: light and dark intermediate cells. The light intermediate cells are present in the stria from birth and have the typical appearance of a melanocyte. They are large and dendritic with electron-lucent cytoplasm containing numerous vesicles that show tyrosinase activity, and pigment granules in various stages of development. These granules have the ultrastructural and histochemical characteristics of premelanosomes and melanosomes. The light intermediate cells persist throughout life, but less frequently contain pigment in older animals. The dark intermediate cells, present only in adult mice, vary considerably in number and distribution between animals. Pigment granules, bound within an electron-dense acid phosphatase-rich matrix, form the main component of the dark intermediate cells. The intermediate cells may comprise either two distinct cell populations or different developmental stages of the same cell type; ultrastructural observations suggest the latter. In young mice, light intermediate cells contain the electron-dense matrices, which at later stages of development are found almost exclusively in dark cells. The dark intermediate cells contain few cell organelles other than pigment granules accumulated within lysosomal bodies and they often have pycnotic nuclei. These observations suggest that the dark intermediate cells are a degenerate form of the light intermediate cells. Clusters of melanosomes also occur in the basal cells, and to a much lesser extent in the marginal cells. These cells do not stain after incubation in DOPA, suggesting that they are not capable of melanin synthesis, and therefore probably acquire melanin by donation from adjacent melanocytes. Pigment clusters are also found within the spiral ligament at all stages of development.     

Rat intestinal galactoside-binding lectin L-36 functions as a structural protein in the superficial squamous cells of the esophageal epithelium

Using an affinity purified antibody raised against the RI-H fragment of rat intestinal lectin L-36, the latter protein has been identified within the esophageal epithelium by means of ultracryotomy followed by immunogold labeling. The epithelium consists of 4 morphologically distinct cell-types, namely, the basal, spiny, granular and squamous cells, and each of these exhibits a different immunolabeling pattern. The basal cells form a layer on the basal lamina, and in these a diffuse cytoplasmic staining is observed. This basal cell layer is overlaid by spiny cells that extend many cell processes into wide intercellular spaces. In these cells, immunogold particles are found only on small granular inclusions consisting of an electron-lucent homogeneous substance. The granular cells from a third layer over the spiny cells, and are characterized by a number of large granular inclusions with an electron-dense core rimmed by a less electron-dense substance. Immunogold labeling is found on these granules, both on the core and peripheral region. Squamous cell-types constitute the most superficial layer of the epithelium. They are without granular inclusions, and immunogold labeling is confined to the cytoplasmic surface of the thickened plasma membrane. These findings suggest that L-36 is produced in the basal cells as free cytosolic protein, then becomes progressively aggregated into the granular inclusions of the spiny and granular cells, and is eventually transferred onto the cytoplasmic surface of the squamous cell plasma membrane where it may interact with complementary glycoconjugate(s) located at this site. The membrane lining substance thus formed may play a role in stabilizing the squamous cell membranes, thereby maintaining the structural integrity of the epithelium against mechanical stress coming from the esophageal lumen.     

Firm structural associations between migratory pigment granules and microtubules in crayfish retinula cells

The morphology of associations between mobile pigment granules and microtubules of the crayfish retinula cells was examined with transmission electron microscopy. Many pigment granules were found associated with microtubules through linkages of fuzzy appearance in thin sections. The linkages were revealed as discrete strands of variable shape in rotary-shadowed replicas of freeze-fractured and deep- etched specimens. The only feature of constant morphology among these connections consisted of 2-4-nm filaments projecting laterally from the microtubules. The firmness of the pigment granule-microtubule associations was judged by their ability to hold up during cell disruption procedures of increasing disaggregation effects in a low- Ca++ stabilization buffer. The results of these tests were inspected with scanning electron microscopy and with transmission electron microscopy of negatively stained preparations. Numerous pigment granules remained associated with a stable microtubule framework after the plasma membrane had been stripped away. Moreover, granule- microtubule attachments survived breakdown of this framework into free fascicles of microtubules. The pigment granules were associated with the free microtubules either individually or as clusters entangled in a fibrous material interwoven with 10-nm filaments. These findings attest that many pigment granules are bound to microtubules through linkages that constitute effective attachments. Further, it is demonstrated that a highly cohesive substance associates the pigment granules with one another. These conclusions are discussed in terms of a pigment transport mechanism in which a network of interconnected granules would establish firm transient interactions with a supporting skeleton of microtubules.     

The compound eye in the opaque-eye phenotype of Drosophilia melanogaster.

The compound eye of the opaque-eye mutant of Drosophila melanogaster was investigated by means of electron microscopy to determine the morphological and physical properties of ommatidial elements. These elements in the mutant were found to differ from those of the wild-type flies in the following ways: (1) The cuticular lens was thinner than that of the control and lacked the typical lamellar construction. (2) The Semper cells were irregular in shape and contained many membranous inclusions similar to those found in degenerating cells; also their nuclei contained virus-like particles. (3) The primary pigment cells contained an abundance of drosopterin-containing granules which were lacking in those of wild-type flies. (4) The superior and inferior central photoreceptor cells were misplaced and their rhabdomeres evidenced some degeneration. (5) The secondary pigment cells had only one type of pigment granules instead of the three types found in the control. These morphological changes in ommatidial elements induced physical abnormalities such as the apparent opaqueness of the eye, the lack of a pseudopupil, the probable disability of the photoreceptor cells to respond to light and the inability of the dioptric system to produce utilizable geometric images.     

Morphometric and densitometric study of the biogenesis of electron-dense granules in Fonsecaea pedrosoi

Fonsecaea pedrosoi is a polymorphic pathogenic fungus, etiological agent of chromoblastomycosis, that synthesizes a melanin-like pigment. Although this pigment has been described as a component of the outer layers of the cell wall, electron-dense cytoplasmic bodies have also been visualized. In this work, we have correlated the appearance of intracellular electron-dense granules with the melanization process in F. pedrosoi. For this, conidial forms were grown under conditions where melanin was not synthesized. Afterwards, cells were incubated in Hank's medium supplemented with bovine fetal serum, at 37 degrees C, to stimulate the pigment production. The genesis of cytoplasmic bodies, with different stages of electron density, was demonstrated by transmission electron microscopy. The appearance of fungal acidic compartments, visualized by confocal laser scanning microscopy in cells stained with acridine orange, was time coincident with the formation of electron-dense granules observed by transmission electron microscopy. The quantification of granule numbers as well as morphometric and densitometric studies were performed.     

Transformations in the structure of the cytoplasmic ground substance in erythrophores during pigment aggregation and dispersion. I. A study using whole-cell preparations in stereo high voltage electron microscopy

Pigment migration in cultured erythrophores of the squirrel fish Holocentrus ascensionis, after manipulation with K+, epinephrine, 3',5'- dibutyryl cyclic adenosine monophosphate, theophylline, and caffeine, is essentially identical to that observed in this chromatophore in situ. For such observations, the erythrophores are dissociated from the scales with hyaluronidase and collagenase, and allowed to spread on an amorphous collagen substrate, where they resemble the discoid erythrophore in situ. In this state, they are readily fixed by glutaraldehyde and osmium tetroxide, and are then critical-point dried for whole-cell viewing in the high voltage electron microscope. The organization and fine structure of the erythrophore cytoplast was stereoscopically examined after fixation of the pigment granules in four experimental states: pigment dispersed, pigment aggregated, pigment aggregating, and pigment dispersing. In the dispersed cell, granules are contained in an extensive three-dimensional lattice composed of radially oriented microtubules and a network of fine filaments 3-6 nm in diameter (microtrabeculae), whereas in the aggregated cell, the microtrabecular system is absent, and the majority of the microtubules appear displaced into the cortices on the cytoplasmic surface of the plasma membrane. In cells fixed while aggregating, few microtrabeculae are observed, although formless thickenings are observed in the cortices, on granules, and between clumped granules. In dispersing cells, the microtrabecular system is reformed from material stored in the cortices and with the granules in the centrosphere. These observations suggest that the granules are suspended in a dynamic microtrabecular system that withdraws during pigment aggregation and is restructured during pigment dispersion. The microtubules guide linear granule motion not by defining physical channels, but by a recognizable affinity of microtubules, microtrabeculae, and granules for one another.     

中国鲎变形细胞超微结构的进一步观察

中国鲎的变形细胞可在血液中发育成熟,根据其超微结构,可分为原始变形细胞、幼变形细胞和变形细胞三个阶段。 变形细胞受到外环境或内毒素的刺激后,第一型颗粒出现一系列变化,最终把内容物释放到细胞外。与此同时,细胞表面形成若干胞质性突起,边缘带分散断裂,质膜下和胞质性突起中出现微丝。第二型颗粒无变化,提示它们和血液凝胶化过程无直接关系。