Ultrastructure and morphogenesis of ceroid pigment. II. Late changes of lysosomes in Kupffer cells of rat liver after phagocytosis of unsaturated lipids (author's transl)]

Wistar rats were injected intravenously with cod liver oil emulsion. The lipid droplets ere phagocytized by Kupffer cells and stored in lysosomes. The transformation of these lipid-containing lysosomes into ceroid pigment granules was studied electron-microscopically and cytochemically for a period of 12 weeks after the injection. The lipid droplets enclosed in lysosomes show an increasing and continous condensation from the periphery towards the center due to oxidation and polymerization of unsaturated fatty acids. During the first week almost the total amount of the stored lipids is transformed into an amorphous, highly electron-dense material which disintegrates into cloddy and globular fragments during the following time. The fragments are embedded in a fine granular, slightly electron-dense matrix showing a marked activity of acid phosphatase. The lysosomal structures which contain remnants of condensed oxidized and polymerized lipids are the electron-microscopic equivalent of the granules as seen by light microscopy. These lipids, which have been changed in their molecular structure, cannot be hydrolized by lysosomal enzymes. They remain as an indigestible material, as a waste product in lysosomal residual bodies. Both lipofuscin and ceroid are lysosomal structures containing oxidized and polymerized lipids. The differences between these lipogenous pigments are due to their different formal and causal genesis. Lipofuscin develops in parenchymal and muscle cells by autophagocytosis and by subsequent oxidation and polymerization of segregated membrane lipids. Ceroid is formed in macrophages by heterophagocytosis of unsaturated lipid material which is also oxidized and polymerized.     

An Ultrastructural Study of the Hemocytes of the Pericardial Body in the Ascidian Ciona intestinalis (L.)

Abstract The hemocytes of the pericardial body of Ciona intestinalis were studied by electron microscopy. Our findings showed that stem cells, clear vesicular granulocytes, microgranulocytes, unilocular granulocytes and globular granulocytes are present at the periphery of the smaller-sized pericardial bodies. The stem cells are small round cells with a large nucleus, with or without nucleolus, and homogeneous cytoplasm containing numerous ribosomes. The clear vesicular granulocytes are characterized by an ameboid shape and cytoplasm containing several large electron-lucent vacuoles and small electron-dense granules. The microgranulocytes are variable in shape and contain numerous large electron-dense granules. The unilocular granulocytes show a single large vacuole with an electron-dense or electron-lucent content and a thin layer of peripheral cytoplasm that contains the flattened nucleus. The globular granulocytes are characterized by the presence of large vacuoles containing either fibrogranular material or electron-dense aggregates.     

Oöcytes degeneration in the queen honey bee after infection by Nosema apis

Terminal o?cytes containing yolk in both healthy and nosema infected queen honey bees were studied. In the healthy queens the terminal o?cytes exhibited a layer of follicular cells which were covered by a smooth-surfaced ovariole sheath. In the o?plasm were numerous electron-dense yolk granules and lipid yolk droplets. The elecron-dense yolk granules exhibited a crystalline structure. Stacks of endoplasmic reticulum were observed in the yolk granules throughout the o?plasm. Numerous mitochondria possessing well defined cristae were also observed. O?cytes in the ovary of queen honey bees appeared degenerated after 7 days of infection by Nosema apis. The ovariole sheath was wrinkled. In the o?plasm, yolk granules were broken down into small spheres and granular substances. Numerous ribosomes without stacks of endoplasmic reticulate were observed. Lysosomes were abundant and numerous electron-dense materials surrounded by a membrane were detected. The o?cytes appeared to be extensively autolysed. The significance of these observations is discussed.     

Neuronal types in the striatum of man

Summary Nerve cells of the human striatum were investigated with the use of a newly developed technique that reveals the pattern of pigmentation of individual nerve cells by means of transparent Golgi impregnations of their cell bodies and processes. Five types of neurons are distinguished:Type I is a medium-sized spine-laden neuron with an axon giving off a great number of collateral branches. The vast majority of the cells in the striatum belong to this type. Numerous intensely stained lipofuscin granules are contained in one pole of the cell body and may also extend into adjacent portions of a dendrite.Type II is a medium-sized to large neuron with long intertwining dendrites decorated with spines of uncommon shape. A distinguishing feature of this cell type is the presence of somal spines. This cell type is devoid of pigment or contains only a few tiny lipofuscin granules.Type III is a large multipolar neuron. The cell body generates a few rather extended dendrites that are very sparsely spined. The finely granulated pigment is evenly dispersed within a large portion of the cytoplasm.Type IV is a large aspiny neuron with rounded cell body and richly branching tortuous dendrites. The axon branches frequently in the vicinity of the parent soma. Large pigment granules are concentrated within a circumscribed part of the cell body close to the cell membrane.Type V is a small to medium-sized aspiny neuron. The dendrites break up into a swirling mass of thin branches. More than one axon may be given off from the soma. The axons branch close to the soma into terminal twigs. Cells of this type contain numerous large and well-stained lipofuscin granules.Each of the cell types has a characteristic pattern of pigmentation. The different varieties of nerve cells in the striatum can therefore be distinguished not only in Golgi impregnations but also in pigment-Nissl preparations.     

Microscopic anatomy of the orbital Harderian gland in the common tree shrew (Tupaia glis)

The orbital Harderian gland of the common tree shrew (Tupaia glis) was investigated at the macroscopic and microscopic levels. In the glands of both sexes only one acinar cell type was found. The cell is characterized by the presence of numerous lipid vacuoles of variable size and by a small number of PAS-positive, electron-dense granules distributed throughout the cytoplasm, which are predominant at the basal portion of each acinar cell. The duct system is well developed within the gland. The content of lipid vacuoles within the acinar cells is secreted from the apical portions by exocytosis, indicating the exocrine function of the organ. Apart from the lipid vacuoles, both acinar and ductal luminal contents of the Harderian gland also contain accretion of electron-dense materials. The vascularization within the Harderian gland is unique in that two capillary types (small fenestrated and irregular sinusoidal capillaries) could be demonstrated. The presence of fenestrated capillaries together with other morphological features (such as accumulation of the small electron-dense granules at the basal pole and the presence of basolateral microvilli) near the basal portion of the acinar cells suggest that the Harderian gland in T. glis might also be involved in an endocrine function.     

Neuronal types in the lateral geniculate nucleus of man

Summary Nerve cell types of the lateral geniculate body of man were investigated with the use of a transparent Golgi technique that allows study of not only the cell processes but also the pigment deposits. Three types of neurons have been distinguished:Type-I neurons are medium-to large-sized multipolar nerve cells with radiating dendrites. Dendritic excrescences can often be encountered close to the main branching points. Type-I neurons comprise a variety of forms and have a wide range of dendritic features. Since all intermediate forms can be encountered as well, it appears inadequate to subdivide this neuronal type. One pole of the cell body contains numerous large vacuolated lipofuscin granules, which stain weakly with aldehyde fuchsin.Type-II and type-III neurons are small cells with few, sparsely branching and extended dendrites devoid of spines. In Golgi preparations they cannot be distinguished from each other. Pigment preparations reveal that the majority of these cells contains small and intensely stained lipofuscin granules within their cell bodies (type II), whereas a small number of them remains devoid of any pigment (type III). Intermediate forms do not occur.     

Ultrastructural studies of vagal paraganglia in Syrian hamsters

Summary Typical vagal paraganglia of Syrian hamsters are encapsulated in connective tissue and consist of groups of epithelial cells. Ganglion cells, a few fenestrated capillaries, and bundles of unmyelinated nerve fibers are intermingled among the parenchymal cells. The parenchymal cells are of two types: chief or paraganglion and sustentacular or supporting cells. The processes of the supporting cells partly or completely surround the paraganglion cells. In addition to the nucleus, Golgi complex, mitochondria, parallel-arrayed granular endoplasmic reticulum, and lipofuscin pigment, the chief cells are characterized by the presence of numerous membrane-bound, electron opaque granules. After an injection of ³H-dopa, labelings were concentrated over the chief cells and were associated predominantly with the granules. Following glutaraldehyde-dichromate treatment the granules gave a positive reaction for unsubstituted amines. These results suggest that the chief cells contain catecholamines in the electron opaque granules.Research supported by USPHS Grants NS 05665, 00690 and HE 12751. A preliminary report of this research was presented before the American Society for Cell Biology, 1969.Sponsored by National Council on Science Development, Republic of China.Recipient of Career Research Development Award 1 K3 GM 28064.     

Coelomic Cells in the Connective Tissue Spaces of the Clitellar Epithelium of Lumbricus friendi Cognetti, 1904 (Oligochaeta): an Ultrastructural Study

The ultrastructure of the connective tissue spaces in the clitellar epithelium has been studied in the earthworm Lumbricus friendi. Four morphological types of coelomic cells are described: amoebocytes, mucocyte-like cells, pigment cells and crystal-containing cells. The amoebocytes are characterized by the presence of spherical to oval electron-dense granules, phagocytic vacuoles and numerous microtubules located in the Golgi areas. The mucocyte-like cells show the features of the mucocytes reported in enchytraeid worms (globular inclusions with filamentous and homogeneous, moderately electron-dense material, as well as a filopodous process). The pigment cells contain typical spindle-shaped osmiophilic granules, microtubules (not reported before) and glycogen particles. The crystal-containing cells show inclusions which are polygonal in section with a striated substructure (periodicity of about 4.5 nm). Apart from the mucocyte-like cells, the coelomocytes showed cytoplasmic processes attached to the basement membrane of the spaces. The possible functions of these cells are discussed and a common peritoneal origin is postulated on the base of their morphological and cytological features.     

Neuronal types and their percent distribution within the magnocellular nuclei of the human basal forebrain

Three neuronal types constituting the magnocellular nuclei of the human basal forebrain have been differentiated with the aid of preparations stained for both Nissl material and pigment deposits: type I = large multipolar neurons contain loosely packed and faintly stained lipofuscin granules occupying a large portion of the cell body; type II = large spindle-shaped neurons reveal a densely packed accumulation of coarse and intensely stained lipofuscin granules, and type III = small nerve cells, scattered among these large neuronal components, with only a small number of faintly stained lipofuscin granules. The determination of the projection areas of the somata of the three neuronal types has led to a distribution pattern with three peaks. The ratio of the nerve cell types has been evaluated: 73.6% type I; 8.6% type II, and 17.8% type III neurons.     

Biogenesis of melanosomes in Kupffer cells of Proteus anguinus (Urodela,Amphibia)

The ultrastructural characteristics of melanosomes and premelanosomes observed during the biogenesis of melanosomes in liver pigment cells of the neotenic cave salamander Proteus anguinus (Proteidae) are described. It is well known that amphibian liver pigment cells, also known as Kupffer cells (KC), contain melanosomes and are able to synthesize melanin. Liver pigment cells of P. anguinus contain numerous siderosomes and melanosomes. The melanosomes are grouped together within single-membrane-bounded bodies, named as 'clusters of melanosomes' or 'melanosomogenesis centers'. Inside such clusters, different structures are present: (1) filament-like structures, characteristic of the initial stage of melanosome biogenesis, (2) medium electron-dense melanosomes in different stages of melanization, (3) melanosomes with an electron-dense cortical area and a less electron-dense medullar area, and (4) uniformly highly electron-dense mature melanosomes or melanin granules. Histochemical and cytochemical dihydroxyphenylalanine (DOPA) oxidase reactions in pigment cells were positive. Our results confirm the ability of amphibian KC to synthesize melanin and contribute to this little known subject.     

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.     

Ultrastructure of the cells of the ovarian interstitial gland in hypophysectomized rats

Summary The fine structure of the interstitial gland of the ovary was studied in hypophysectomized rats and in hypophysectomized rats after denervation of the ovary or stimulation of the ovarian plexus. Hypophysectomized rats were used to eliminate gonadotropic influences on interstitial cells. In hypophysectomized rats, there was a large amount of intercellular space and cells had irregularly shaped nuclei and a large nuclear-cytoplasmic ratio. Prominent cytoplasmic features included small mitochondria with an electron-dense matrix, rough and smooth endoplasmic reticulum, polysomes and large osmiophilic lipid droplets. Interstitial cells from stimulated ovaries had reduced intercellular space and a reduced nuclear-cytoplasmic ratio. Mitochondria had tubular cristae; smooth endoplasmic reticulum-surrounded lipid droplets, and large polysomes were present. After section of the ovarian plexus, intercellular space was increased and filopodia were numerous. Cytoplasmic features included mitochondria with a dense matrix and indistinct cristae, large electronlucent lipid droplets, and variously sized multivesicular structures. These observations suggest that stimulation of the ovarian plexus in hypophysectomized rats causes regressed cells to assume the fine structural features of active steroidogenic cells. In contrast, interruption of the ovarian nerve supply causes a qualitative and quantitative increase in ultrastructural features characteristic of regressed steroidogenic cells. These responses of interstitial gland cells to denervation and stimulation provide morphological evidence for a functional role for the adrenergic nerves to this ovarian compartment.     

Efferent fibers and daily rhabdomal changes in the anteromedial eye of the liphistiid spider,Heptathela kimurai

The presence of efferent fibers in the retina of liphistiid spiders, kept in natural daily cycles of illuminance, was examined by electron microscopy. The efferent fibers were observed to extend their processes through the ocellar nerve to the retina. They contained characteristic large electron-dense granules and branched repeatedly within the retina with varicosities, to provide synaptoid contacts with the receptor cells. They ran mostly among receptor cells and glial cells but sometimes protruded into receptor cells to establish invaginated synaptoid contacts. The synaptoid structures were characterized by spherical clear vesicles located at the presynaptic region, with electron-dense material adhering to the plasma membranes of the receptor cell and the efferent fiber, and a cleft about 10 nm wide formed by the two opposed parallel membranes. The clear vesicles and the electron-dense granules were secreted by exocytosis. The efferent fiber was characteristically presynaptic in relation to the receptor cell. In addition, the rhabdoms differed in size from day to night.     

Opposite effect of lipofuscin granules and melanosomes from human retinal pigment epithelium of eye on photooxidation of cardiolipin

The influence of lipofuscin granules and melanosomes from human retinal pigment epithelium on the light-induced photooxidation of cardiolipin liposomes and the generation of superoxide radicals was studied. Lipofuscin granules were able to stimulate, while melanosomes inhibited, the cardiolipin photooxidation. The visible light irradiation of both melanosomes and lipofuscin granules generated superoxide radicals with mean rates of 1.5 nmole/min/10(7) and 38 nmole/min/10(7) granules, accordingly. However, melanosomes but not lipofuscin granules reacted readily with superoxide radicals. Moreover, the rate constant of degradation of superoxide radicals in the presence of melanosomes was about five orders of magnitude higher than the rate constant of its photogeneration. Therefore, we propose that melanosomes in retinal pigment epithelium cells have a photoprotective role whereas lipofuscin granules may stimulate photodestructive reactions.     

The effect of aging on the rat submandibular gland: an ultrastructural, cytochemical and biochemical study

The effect of aging on the rat submandibular gland was studied by using ultrastructural, ultrastructural cytochemical and biochemical techniques. There was an age-related clumping of the nucleolar-associated and peripheral chromatin in many of the acinar cells and a decrease in the number of cisternae of rough endoplasmic reticulum. Many aged acinar cells were binucleated. There was also an age-related increase in pigment granules throughout the gland. These membrane bound granules consisted of a lipid droplet and an associated dense cap which had a granular matrix and pigment droplets. The lead capture method for acid phosphatase activity demonstrated that activity was associated with the granular matrix of the dense cap. These results were correlated with the age-related increase in acid phosphatase activity as determined by colorimetric procedures. There was an age-related increase in the number of cells characterized by small secretory granules. These cells were found as part of the intercalated ducts or at the junction of the duct with the acini. Oncocytes were also found as part of the parenchyma of the aged submandibular gland. These cells were characterized by the pleomorphic mitochondria that fill their cytoplasm. Occasionally, cells that possessed extraordinary numbers of mitochondria and small secretory granules were also observed. The determinations of total DNA and RNA revealed and age-related decrease in RNA while there was no significant change in the concentration of DNA.     

Lipofuscin granule dynamics during development of age-related macular degeneration

The pigment epithelium cell structure and therapeutic effect of antioxidant SkQ1, selectively penetrating into mitochondria from eye drops, were studied upon development in OXYS rats of age-related retinopathy as a model of macular degeneration. The characteristic dynamics and ultrastructural peculiarities of the layer of electron-dense cytoplasmic structures of the pigment epithelium apex part and incorporated lipofuscin granules were revealed. The therapy of OXYS animals for 68 days using 250 nM SkQ1 drops decreased the extent of development of age-related macular degeneration. Electron-microscopic investigation showed that SkQ1 prevented development of ultrastructural changes in the pigment epithelium characteristic of macular degeneration, the condition of which after therapy with SkQ1 drops corresponded to ultrastructure of pigment epithelium in Wistar rats of the same age having no symptoms of retinal damage. It is supposed that ultrastructural changes in the electron-dense layer upon development of age-related macular degeneration are indicative of disturbances in the optical cycle functioning, especially of disturbances in functioning of photoreceptor membranes.     

An ultrastructural stereologic study of aldosterone secreting adrenal adenomas and of adjacent zona glomerulosa

The ultrastructure of four aldosterone secreting adenomas and of the adjacent zona glomerulosa has been described by the use of stereological techniques. Adenomatous cells (about 2800 microns 3 in volume) invariably displayed a striking abundance of lipid droplets, which occupied about 30% of the cytoplasm. Mitochondria prevalently contained tubulo-lamellar or lamellar cristae, but some cells exhibited organelles with vesicular cristae. Smooth endoplasmic reticulum (SER) was not very abundant. Small lipofuscin-pigment granules were frequently seen and in a few cells they were exceedingly numerous. Zona glomerulosa cells were smaller (about 950 microns 3 in volume) and possessed mitochondria with typical tubulo-lamellar cristae, a plentiful SER and few lipid droplets. They showed the ultrastructural features of elements actively engaged in steroid synthesis. The possible origin of aldosteronoma cells from the zona glomerulosa is discussed.     

Somatostatin-like immunoreactivity in non-pyramidal neurons of the human entorhinal region

Summary The distribution of somatostatin-immunoreactive cells and processes throughout the human entorhinal region and subjacent white matter was examined either by the unlabelled antibody-enzyme method or by the avidin-biotin method. The brain slices were obtained at autopsy with a short post-mortem delay. The majority of somatostatin immunoreactive nerve cells was found in the inner principal layer and subjacent white matter. In addition, individually scattered immunoreactive neurons were observed in both the outer principal layer and lamina dissecans. The immunoreactive perikarya varied in shape and ranged in size from 10 to 30 m. Without exception the neurons could be classified as belonging to the group of non-pyramidal neurons. Each neuron gave rise to a few thick dendrites and a thin axon with a beaded appearance. In the adult human brain, the pattern formed by lipofuscin granules deposited in the nerve cells can be considered characteristic for the type of the neuron. Therefore, immunoreactive perikarya were documented, destained of chromogen and restained to demonstrate lipofuscin pigment and basophilic substance. It became evident from these studies that the previously immunoreactive cells were characterized by a large rounded and eccentrically located nucleus, sparse basophilic substance and, in most cases, a lack of lipofuscin granules. A few of the immunoreactive cells were laden with coarse pigment granules. The findings permit classification of entorhinal somatostatin-immunoreactive neurons as either non-pigmented or pigment-laden non-pyramidal neurons.Dedicated to Prof. Dr. J. Lang, Würzburg, on the occasion of his 65th birthdayA portion of the results has been presented at the annual meeting of the European Neuroscience Association 1986 in Marseille, France (Friederich-Ecsy et al. 1986)     

Ultrastructural studies on the morula in cattle

Embryos at morula stage were ultrastructurally examined. Blastomeres of normal embryos are characterized by the presence of ribosomes, short single and branched cisterns of endoplasmic reticulum, small spherical mitochondria and vacuolar-granular structures. An enhanced autophagy and the presence of numerous and large lipid droplets are signs of the beginning degeneration.     

On the fine structure of the small,heavily pigmented non-pyramidal cells in lamina II and upper lamina III of the human isocortex

Summary With the aid of a newly developed technique for the successive examination of both the Golgi and pigment picture of individual neurons (Braak, 1974a) Braak (1974b) demonstrated that within lamina II and upper lamina III of the human isocortex, heavily pigmented non-pyramidal cells are distributed irregularly and sparsely. The lipofuscin pigment granules serve as excellent internal markers to identify these non-pyramidal cells in ultrathin sections. This favourable circumstance facilitates the study of these interneurons in the electron microscope.The heavily pigmented non-pyramidal cells are small, spherical to ovoid with diameters of about 12–15 m. One pole of the cell comprising a large cytoplasmic area gives rise to a few dendrites, while the other pole is occupied by the nucleus and in some cases is in close apposition to another nerve cell body. The nucleus is deeply invaginated by the large cytoplasmic area and occasionally displays nuclear inclusions. Among the usual organelles distributed within the large cytoplasmic area the mitochondria with a moderately electron dense matrix are abundant and the coarse lipofuscin pigment granules are the most striking elements. The latter contain densely packed filamentous or tubular material and a single vacuole. The perikaryon rarely receives more than 3 type I and type II synapses per section per cell, whereas the dendrites receive numerous synapses of both type I and type II. Within the apposition zone to another nerve cell body (which in no case is a heavily pigmented non-pyramidal cell) puncta adhaerentia occur and also contacts in which the cleft of 8 nm is intersected by a dense stratum.Some of the ultrastructural findings are summarized in the schematic drawing of Figure 15.