Morphology of the epithelium of the extratesticular rete testis, ductuli efferentes and ductus epididymidis of the adult male rabbit

The fine structure of the epithelium lining the extratesticular rete testis, ductuli efferentes and ductus epididymidis of the rabbit has been investigated. In the ductuli efferentes the epithelium is composed of two cell types, principal cells and ciliated cells. The latter type is distinguished from principal cells by the presence of cilia projecting into the lumen and the position of the nucleus in the apical half of the cell. Principal cells in this segment are characterized by micropinocytotic vesicles on the surface plasma membrane and a variety of small dense bodies scattered throughout the cytoplasm. In the ductus epididymidis basal cells replace ciliated cells as the second cell type, but differences between various segments of the epididymis are related to the fine structure of the principal cells. In the proximal caput epididymidis (Nicander's region 1) the principal cells are tall with long microvilli. They typically contain a small Golgi apparatus and a cluster of dense bodies adjacent to the nucleus. In the distal caput epididymidis (Nicander's regions 2-5) the apical cytoplasm of principal cells is filled with numerous micropinocytotic vesicles and large multivesicular bodies; these features are interpreted as signs of absorptive activity. The multivesicular bodies are absent from the cytoplasm of principal cells in the corpus epididymidis (Nicander's region 6) and, instead, numerous elements of smooth endoplasmic reticulum, a large Golgi apparatus, lipid droplets and dense bodies characterize principal cells in this segment. Towards the proximal cauda epididymidis (Nicander's region 7), the number of dense bodies (lysosomes) in the cytoplasm increases considerably. In the globose cauda (Nicander's region 8), the principal cells are reduced in height, and in addition to the features described in region 7, are characterized by a concentric array of rough endoplasmic reticulum in the basal cytoplasm. These observations are discussed in relation to the role of the epididymis in promoting the maturation and survival of spermatozoa.     

In situ hybridization at the electron microscope level: an improved method for precise localization of ribosomal DNA and RNA

In situ hybridization using biotinylated rDNA probes and secondary antibody coupled to gold particles was developed on ultrathin sections of Lowicryl-embedded Ehrlich tumor cells for precise localization of ribosomal RNA (rRNA) and ribosomal DNA (rDNA). For the detection of rDNA, an immunocytochemical approach involving an antibody against single-stranded DNA was used in order to determine the more efficient denaturation procedure. Using this technique, rDNA can be visualized in the fibrillar centers of nucleoli, especially in their peripheral regions at the proximity of both the dense fibrils and the nucleolar interstices as well as within the latter. rDNA was occasionally detected in some clumps of dense nucleolus-associated chromatin. Besides the presence of rRNA in the ribosome-rich cytoplasmic areas and in the dense fibrillar component and the granular component of the nucleolus, rRNA was also found in the fibrillar center areas close to the boundary region to the dense fibrillar component. These results are discussed in the light of the present knowledge on the functional organization of the nucleolus.     

Ultrastructural study of the shell-repair membrane in the snail,Helix pomatia L.

Summary The shell-repair membrane of the snail, Helix pomatia, has been studied with the transmission electron microscope (TEM). The ultrastructure of the repair membrane, in the initial stages of calcification, revealed the presence of a fibrillar protein, proteoglycan granules, osmiophilic vesicles, and cytoplasmic dense bodies of different size and structure. The involvement of the cell constituents in the formation of calcifying centra and initial crystal formation is discussed. The amoebocytes present within the repair membrane appeared to be involved in three different functions: (1) phagocytosis, (2) release of granules, vesicles and dense bodies, and (3) secretion of a fibrillar protein. The possible lytic function of the amoebocytes is mentioned. The common features in the mineralizing process of the shell-repair membrane of the snail and the epiphyseal cartilage of the mammals were noted.This investigation was supported by a grant from the Swedish Natural Science Research Council, which is gratefully acknowledged. I am indebted to Miss Ch. Stensjö and Mrs. E. Hellmén for their technical assistance     

Ultrastructure and fluorescence histochemistry of endocrine (APUD-type) cells in tracheal mucosa of human and various animal species

This study describes distrinctive cells with ultrastructural and histochemical features of APUD-type endocrine cells within the tracheal epithelium of human fetuses, newborns and children as well as different animal species. These cells referred to as Kultschitzky cells (K cells) were found to be argyrophilic, but not argentaffin, and are considered analogous to the same type of cells in lung and gastro-intestinal tract. Fluorescence histochemistry demonstrated the presence of intracellular amine within tracheal K cells, but only after in-vitro or in-vivo administration of amine precursor (L-DOPA). Ultrastructurally, these cells are characterized by the presence of numerous cytoplasmic granules (dense core vesicles) which show species related morphologic variations. Two different types of K cells were found in trachea of lamb and armadillo, each type possessing morphologically different dense core vesicles. In human and rabbit tracheas, only one type of K cell was identified. K cells in the trachea are distributed as single cells between other epithelial cells; neuroepithelial bodies such as those found in bronchial mucosa were not identified. Well differentiated K cells were found in tracheas of early human fetuses and throughout gestation, infancy, and childhood. Preservation of K cells in human autopsy material and widespread occurence of these cells in various laboratory animals will permit further studies into the nature and function of tracheobronchial endocrine cells.     

A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus.

Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.     

Density-dependent expression of keratins in transformed rat liver cell lines

Immunomorphological examination of the distribution of three keratins in cultured rat liver-derived epithelial cell lines of the IAR series was performed in order to find out the effects of neoplastic evolution on the expression of these epithelium-specific markers. Specific monoclonal antibodies were used to reveal various intermediate filament proteins: keratins with molecular masses of 55, 49 or 40 kD (K55, K49 or K40), and vimentin. The expression of keratins was negligible in sparse and dense cultures of non-transformed lines, which had typical epithelial morphology. The examined keratins were also absent in the sparse cultures of transformed lines, which have lost partially or completely the morphological features of epithelia. However, cells in dense cultures of most transformed lines contained numerous keratin filaments. It is suggested that the paradoxical increase of keratin expression after transformation is due to increased saturation density of transformed cultures; this high density favours the expression. As shown by the experiments with culture wounding, the effects of density are local and reversible. While K55 was present in all the cells of dense cultures, the expression of the other two keratins was dependent on the cell position within these cultures. It is suggested that the expression of the latter two keratins, besides high cell density, also requires the presence (K40) or the absence (K49) of cell-substratum contacts. Possible mechanisms of the effects of cell density on the expression of keratins are discussed.     

Conservative segregation of tetrameric units of H3 and H4 histones during nucleosome replication

We have specifically investigated the behavior of H3 and H4 histones during the replication cycle of MH-134SC cells. Mononucleosomes obtained from cells density-labeled with IdU or dense amino acids in the presence of appropriate radiolabeled precursors were applied to sucrose gradients containing 0.3 M NaCl and 4 M urea for rate zonal centrifugation. This allowed the resolution of dense and normal subnucleosome particles composed of DNA and two molecules each of H3 and H4 without any measurable interparticle histone exchange. On labeling with dense amino acids and radiolabeled lysine, a distinct peak of radiolabeled dense particles was obtained. In contrast, pre-radiolabeled H3 and H4 remained in the normal subnucleosome peak region even after one generation time of culturing with dense amino acids. These data indicate the formation of (H3-H4)2 tetramers composed entirely of new H3 and H4 molecules as well as the conservation of pre-existing tetramers. Density labeling for 1 h with IdU in the presence of radiolabeled lysine yielded a distinct peak of radiolabeled dense particles, indicating the deposition of new tetramers on newly replicated DNA. Similar rate zonal analysis of subnucleosome particles obtained from cells prelabeled for 1 h with radiolabeled lysine followed by various IdU-labeling schedules in nonisotopic media yielded data suggesting that tetramers once deposited do not move about randomly during the replication cycle. A possible mode of nucleosome replication is discussed in the light of the present data.     

Evidence for distinct populations of human Merkel cells

Merkel cells (MCs) are neuroendocrine cells of unknown origin located in the skin. They are identified at electron microscopic level by electron dense granules, at light microscopic level by the presence of cytokeratins 8, 18, 19 and 20. Contradictory reports concerning the presence of other molecules of epithelial as well as neural origin prompted us to investigate whether there are distinct populations of human MCs. Here, we show the heterogeneous expression of villin, N-CAM, NGF-R, and neurofilaments in MCs. Synaptophysin is found in all MCs but with different intensity, nestin is absent. Expression patterns vary between interfollicular epidermis, hair follicles and glabrous epidermis. We conclude that there are distinct populations of MCs, but all populations contain markers for epithelial as well as neural cells. Putative functions of the distinct populations are discussed. A.-C. Eispert and F. Fuchs have contributed equally to the paper.     

Evidence for basal secretion in the subcommissural organ of the adult rabbit

Summary Morphological evidence is presented supporting the possibility of basal secretion into hypendymal capillaries of the adult rabbit subcommissural organ (SCO). The synthetic apparatus of the SCO cell is described as well as the heterogeneous granules and vesicles which are concentrated in the basal processes bordering a widened perivascular space. The origin of the electron dense granules, of which two fairly distinct subgroups are found, is discussed.A binding of secretory sacs to the lateral plasma membrane is seen. The possibility of a lateral secretion is supported by the presence of a system of extracellular channels between SCO cells which are filled with a flocculent material resembling that of the secretory sacs.Nerve perikarya which are separated from the SCO by only a few glial fibers are demonstrated. Synapses are described in nerve fascicles bordering on the hypendymal capillaries. The possibility of an innervation of the hypendymal region is discussed as well as possible nervous connections with the pineal gland.This work was supported by grants from Statens almindelige Videnskabsfond, Copenhagen.     

A microprobe analysis of Gomori-positive glial cells in the rat arcuate nucleus

Summary Astrocytes and microglial cells in the arcuate nucleus of the rat hypothalamus contain lipofuscin-like granules which react with chrome alum gallocyanin and exhibit endogenous peroxidase activity. These granules were assessed with energy dispersive X-ray microanalysis and compared to neuronal dense bodies and glial cytoplasm. The granules are distinguished by a consistent content of sulphur and a frequent presence of calcium. The localization of other elements such as iron, copper, potassium and chlorine is impaired by methodical difficulties. The sulphur content as well as the endogenous peroxidase activity is interpreted as indicating a special variant of lipofuscin. The presence of calcium is discussed with respect to recent concepts of glia as a regulator of the ionic environment of the CNS.     

Lymphocyte emperipolesis into autologous monocytes in leukocyte cultures exposed to mitogenic lectins

Lymphocyte-monocyte synergistic interaction in cooperative response to mitogens and antigens is well established. This paper describes a less known--antagonistic (effector-target)--lymphocyte-monocyte interaction that came into existence in a leukocyte culture after the commencement of cellular response to concanavalin A, phytohemagglutinin and Wistaria floribunda mitogen. An invasion of lymphocytes into monocytes and monocyte polykaryons has been found 24-48 h after exposure to mitogens. The invasion is not followed by lysosome fusion with lymphocyte-bearing vacuoles, but is associated with a sequential destruction of the vacuole wall, and eventual disintegration of some affected cells. The expression of pan-T-cell surface antigens, staining patterns for nonspecific esterase and acid phosphatase as well as ultrastructural features show that the lymphocytes entering into and those located within monocytes and polykaryons represent activated T-cells. The presence of developed Golgi complexes associated with coated and smooth vesicles, and lysosomal bodies with microvesicles, tubular arrays or dense cores suggest that these T-cells belong to subpopulations which possess cytolytic activities. The lymphocyte invasion is considered cytolytic emperipolesis directed towards some autologous cells of the mononuclear phagocyte series. Its extent depends upon the mitogen concentration, and density of cell population in the culture. It also shows individual variability. The relationship of cytolytic emperipolesis to phagocytosis, and its possible significance as a mechanism of cell-mediated elimination of undesirable cells is discussed.     

Xeromorphic features of three stem assimilants in relation to their ecology

The anatomy of the assimilating stems of three brooms (Fabaceae) all show distinct xeromorphic features in their tissues, but these are developed to different degrees. The epidermis consists of very thick, cutinized outer walls, deeply sunken stomata, and a dense layer of trichomes, at least in younger steins. In older sterns some of the stomata are blocked by cell-plugs. The chlorenchyma is multilayered. The main supporting tissue consists of peripheral gelatinous fibre strands. It is argued that these may function in water storage. Parenchymatous water storage cells are present in the pith of two species. The structure and function of the xeromorphic features are discussed in relation to the ecology of the plants.     

Neurofilamentous network and filamentous matrix preserved and isolated by different techniques from squid giant axon

The contribution of the neurofilamentous network to the structure of the squid giant axon was analyzed electron-microscopically. Axial 10-nm filaments cross-linked by radial 5-nm bridges form a network that is present in preparations prepared by a variety of techniques. The axoplasm is differentiated into dense and less dense regions. In the presence of Co(II) ions, the neurofilamentous network was remarkably well preserved and appeared to be associated with a dense web of fine filament matrix, which also was identified in extracted axoplasm and in fractions enriched with neurofilament protein complex. In the presence of La(III) ions, the neurofilamentous network had a coarse and open appearance. The stereo images of extracted and critical-point dried axoplasm suggested that the neurofilamentous network contains ordered lattice-like regions. Extracted preparations of extruded axoplasm and fractions enriched with neurofilament protein complex suggested that the properties of the network are determined by the neurofilament protein complex. It is proposed that the neurofilamentous network is the essential determinant of the form of the axon, and that the order within the network is determined by the radial components of the network. The structures observed in the different preparations are not artifacts, but rather are related closely to their native state in the axon.     

Ultrastructure of the APUD-like cells of the frog thymus gland

Within the thymus gland of the European common frog, Rana temporaria, cells with endocrine- like appearance have been found. At the ultrastructural level the most characteristic feature of their cytoplasm is the presence of secretory granules. Some cells possess irregular electron lucent granules with an eccentrically located dense core while others possess smaller electron dense granules. The cytoplasm contains also cisterns of rough endoplasmic reticulum, free ribosomes, and small mitochondria. The cells possess irregular nuclei with the pronounced nucleoli. These endocrine-like cells are connected by desmosomes with neighbouring non-granulated epithelial cells. Ultrastructural features of the cells described here resemble those seen in polypeptide hormone-secreting cells belonging to the family of cells of the APUD (Amine Precursor Uptake and Decarboxylation) series.     

CORRELATION BETWEEN FIBER LENGTH, ULTRASTRUCTURE, AND THE LENGTH-TENSION RELATIONSHIP OF MAMMALIAN SMOOTH MUSCLE

The length-tension relationship was determined for strips of guinea pig taenia coli and correlated with the length and ultrastructural organization of the component fibers. The mean fiber length in "stretched" strips (passive ≥ active tension) was 30% greater than that for fibers in "unstretched" strips (active >> passive tension). In stretched fibers the dense bodies and 100 A diameter myofilaments were consolidated into a mass near the center of fibers in cross-sectional profile. The thick myofilaments were segregated into the periphery of the fiber profiles. In unstretched fibers the dense bodies-100 A diameter filaments and the thick myofilaments were uniformly distributed throughout cross-sectional profiles. A tentative model is proposed to account for the change in fiber length and ultrastructural organization that accompanies stretch. The basic features of the model require the dense bodies to be linked together into a network by the 100 A diameter filaments. The functional consequences of stretching the fibers are discussed in relation to the model proposed for this network.     

Neglected features of probable taxonomic value in Podostemaceae: The case of Polypleurum

The paper reports on hitherto largely neglected discriminative characters in Polypleurum stylosum, P. elongatum, P. minus, as well as the problematic P. munnarense (Podostemaceae, subfam. Podostemoideae) from India and Sri Lanka. The characters discussed include dimorphism of floriferous and vegetative shoots, leaf dimorphism, presence of peculiar floating trichomes on vegetative leaves, and presence of silica bodies in root and shoot tissue. In addition, anatomical pecularities (well-developed cuticle covering the ovary and ovules, cell inclusions in the form of droplets, and presence of idioblasts with firm inclusions of unknown compounds) and specific morphological features (presence of an apical septum in the gynoecium) are documented. All these features point to different developmental and physiological properties of the species concerned, thus signaling separate evolutionary pathways. They may help to elucidate phylogenetic relationships at various taxonomic levels and may prove useful for the delimitation of the genus.     

CONTENT AND ULTRASTRUCTURAL LOCALIZATION OF TRANSITIONAL METALS IN ASCIDIAN OVARY

The content of some transitional metals (Ti, V, Cr. Mn. Fe, Cu) in the ovary of Ascidia malaca and Ciona intestinalis and in the gonads of Polycarpa gracilis has been reported. The metals were localized histospectrographically and ultrastructurally. In the ovaries of C. intestinalis . the quantitative analysis shows a variable content of metals related to the ovary maturative cycle. The iron is localized in the electron dense cytoplasmic granules of A. malaca oocytcs and in the cytoplasmic dense formation of P. gracilis test cells. Cl, S, Si and P are present in the test, follicles and inierovarian cells of all examined species.
The results are discussed in relation to the zoological position of these species. The presence of metals and Ascidiacee phylogenesis might be correlated.     

Nuclear bodies in Douglas fir (Pseudotsuga menziesii Mirb.) microspores

The identification of nucleolar proteins and immunocytochemical localization of small nuclear ribonucleoprotein (snRNP) elements revealed the presence of three types of nuclear bodies in Douglas fir microspore nuclei. One type consists of structures resembling Cajal bodies (CBs) and contains nucleolar proteins as well as snRNPs and U2 snRNA. The second type is bizonal bodies, which are nuclear bodies also linked with the splicing system. The bizonal body comprises two parts: the first contains Sm proteins and stains strongly with silver stain, and the second resembles CBs in terms of the degree of silver staining and molecular composition. Douglas fir is the second species after larch where the presence of bizonal bodies has been demonstrated. Pseudotsuga menziesii Mirb and Larix decidua Mill are species with one of the longest microsporogenesis processes known in plants. The presence of bizonal bodies in both species may be linked to the intensification of the splicing processes in microspores with an exceptionally long cell cycle. The third type of structure is dense bodies, whose morphology and degree of silver staining strongly indicate their functional and spatial relationship to the dense part of bizonal bodies.