The structural organization of the intramural lymphatic networks in the jejunum of the Japanese monkey (Macaca fuscata) was studied by an enzyme-histochemical method using whole-mount preparations of tissue layers. A distinction between the lymphatics and the blood vessels was made by light microscopy using 5′-nucleotidase (5′-Nase)-alkaline phosphatase (ALPase) double staining. In each tissue specimen, the reaction product for 5′-Nase activity was localized in the lymphatic vessels, whereas that for ALPase activity was restricted to blood vessels. The mucosal layer exhibited an irregular network of tubular lymphatics connecting to the central lymphatics in the villi. The submucosal layer revealed a horizontal mesh-like network consisting of tubulo-saccular lymphatics. A dense maze-like network of lymphatics was distributed throughout the myenteric layer. The lymphatic networks were independent of the blood vessels. The present results indicate the usefulness of the enzyme-histochemistry of whole-mount preparations of the laminar layers for demonstration of the structure and distribution of the lymphatic system in the intestinal wall.

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The fine structure of Leydig and Sertoli cells in Japanese black bears, Ursus thibetanus japonicus, was studied at different stages of reproductive activity in different seasons. Structures of Leydig cells in the testes that were spermatogenically active (May and June), inactive (November and January), and resumptive (March and April) were found to be different. The nucleus and smooth endoplasmic reticulum in Leydig cells had regressed when observed in November and January, and began to develop in March and April. Lipid droplets were numerous in Leydig cells in November and January, but few in March and April. These seasonal variations in the subcellular structures of Leydig cells corresponded to those in the steroidogenic activity of the cells. In Sertoli cells, changes in mitochondria and lipid inclusions with the seasons were detected, but the nuclei and other organelles, including the rough endoplasmic reticulum, did not change throughout the year. Lipid inclusions of both electrondense and electron-lucent types in Sertoli cells were numerous in November and few in January. This change was reversely proportional to the number of degenerating cell masses in the luminal space, indicating that most of these lipid inclusions might be the end products of germ cells phagocytosed by the Sertoli cells. The results suggest that the seasonal changes in the morphology of Leydig cells are closely associated with their spermatogenic activity.

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The differentiating choroid plexus in rats at ages ranging from prenatal day 16 to postnatal day 3 was investigated with a special emphasis on the immunolocalization of Na⁺·K⁺ATPase in the choroid epithelium and neocapillarization of the choroid stroma. Immunoreactions for Na⁺·K⁺ATPase were localized on the apical plasma membrane of the simple choroid epithelium throughout the stages examined, but it also appeared on the basolateral plasma membrane till prenatal day 18, prior to the formation of the tight junctions as seen from both a marker experiment using lanthanum chloride and immunoreactions for tight junction-associated protein (ZO-1). This suggests that a polarity of the enzyme localization to the apical plasma membrane may take place after the establishment of the blood-cerebrospinal fluid barrier. Mesenchymal cells in the choroid stroma immunoreacted to the laminin antibody and occasionally contacted each other—forming solid cell cords—or endothelial cells of the preexisting capillaries. The contact areas immunoreacted to this antibody. Since there were few or no mitotic figures of the endothelial cells, the involvement of laminin in a mechanical linkage between the adjacent vasof ormative mesenchymal cells as well as the endothelial lining in a manner suggestive of vasculogenesis was indicated.

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The enzyme NADPH-diaphorase (NADPH-d; a marker of NO producing or nitrergic neurons) and the neuropeptide VIP have been detected in nerve structures of the hen oviduct by histochemical and immunohistochemical techniques performed on cryostatic sections and whole mount preparations.
In the upper four segments of the oviduct, i. e. the infundibulum, magnum, isthmus and shell gland, nitrergic and VIP-positive nerve structures were particularly numerous in the intermuscular and mucosal layers, and were represented by fibres and cell bodies showing mainly a perivascular distribution. Functionally, such perivascular structures were related to the blood flow regulation.
Different types of nitrergic pyrenophora were recognized in the walls of the shell gland on the basis of their peculiar morphology.
In the distal zone of the oviduct, the vagina, nitrergic and VIP-positive nerve fibres were widely diffused in the circular muscle, which was particularly thick in this segment. The source of at least a part of such fibres was probably represented by large nerve cell bodies scattered in the layer and containing NO and VIP colocalized. Functionally these cells retained inhibitory motor neurons causing relaxation of the vaginal smooth muscle.

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The influences of age and lactational period on the disdribution, number and structure of somatomammotrophs (SMTs) and the relationships to changes in somatotroph and lactotroph populations in ovine were studied using immunocytochemical procedures with light and electron microscopy as well as morphometric techniques. Adenohypophyseal glands of 15 individuals of the Segureña bread (female lambs and ewes in anoestrus or at different stages of milk production) were used. SMTs were always seen in the adenohypophysis of sheep, but were scarce in number and more frequently distributed in the anterior area of the gland. Their number decreased with age, increased at the beginning of the lactation, and decreased again in late lactation. Ultrastructurally these cells showed variable morphology and stored GH and PRL in different secretory granules. Data presented in this report suggest that SMTs are a stage between PRL and GH producing cells.

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Sections from the human somatosensory cortex were observed with a light microscope. The neurons were classified into light and dark ones. The light neurons were slightly stained with thionin, luxol fast blue MBS and azocarmine G (80% of all neurons) The dark neurons were more or less shrunken, and stained intensely with these dyes (20% of all neurons). Double staining with luxol fast blue MBS and azocarmine G was especially useful to demonstrate the dark neurons, since it clearly stained even their fine processes. Neither light nor dark neurons were reactive to nick end labeling for detection of DNA fragmentation. Triple staining with Golgi's silver nitrate (or Gallyas's ammoniacal silver carbonate), luxol fast blue MBS and azocarmine G showed that the majority of the dark neurons were argyrophilic (argyrophilic dark neurons, 15% of all neurons), while some of them were not argyrophilic (non-argyrophilic dark neurons, 5% of all neurons). Triple staining also showed that the light neurons were only occasionally argyrophilic (argyrophilic light neurons, 5% of all neurons); usually, the light neurons were not argyrophilic (non-argyrophilic light neurons, 75% of all neurons). The results confirm that dark neurons usually represent certain populations of neurons in the human brain, and that they are basically identical to the argyrophilic neurons. The discussion suggests that the argyrophilic light and dark neurons are excited cells, the non-argyrophilic dark neurons are exhausted cells, and the non-argyrophilic light neurons are resting cells. Triple staining further demonstrated that some glial cells were darkened and stained with Golgi's silver nitrate or Gallyas's silver carbonate. Additional Golgi's silver block staining showed that the argyrophilic neurons stained by the conventional block staining method usually possessed a shrunken cell body, which was characteristic of the dark neurons.

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To analyze the degradation system in epidermal cells during their generation, differentiation, and cell death, immunocytochemical localization of lysosomal cysteine and aspartic proteinases, an endogenous cysteine proteinase inhibitor, cystatin β, and ubiquitin were examined using rat sole skin. By confocal laser microscopy, granular immunodeposits for lysosomal proteinases were well demonstrated in epidermal cells; immunoreactivity for cathepsins B and C was prominent in the lower spinous and basal layers, while that for cathepsins L and D was intense in the upper spinous and granular layers, although immunoreactivity for cathepsin D was also detected in the lower epidermal layers. Immunoreactivity for cathepsin H was weakly detected only in the spinous layer, where there were some intensely immunopositive cells with processes which were also immunopositive for S-100α, indicating that these cells were Langerhans cells. Diffuse immunoreactivity for cystatin β was intense in the spinous and granular layers and weak in the basal layer. In addition, we also examined the localization of ubiquitin, which is a signal peptide for cytosolic proteolysis; clear-cut granular immunodeposits for ubiquitin were detected in spinous and granular cells, and some were co-localized with cathepsin B immunoreactivity. In the basal layer, mitotic cells were strongly immunopositive for ubiquitin. These results suggest that cysteine and aspartic proteinases are involved in the lysosomal system of the epidermis, showing different distributions in the epidermal layers depending on the enzymes examined. Moreover, ubiquitin may be associated with the cell cycle-dependent degradation in basal cells while it also participates in the non-lysosomal proteolysis and probably, lysosomal proteolysis in the spinous and granular cells.

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Cellubrevin is one of the proteins involved in the docking and fusion of secretory granules to the plasma membrane. It has been reported that cellubrevin is widely distributed in both neural and non-neural cells, including insulin-secreting B-cells. This study aims to demonstrate by immunohistochemical techniques that cellubrevin is localized in insulin-secreting cells and further to examine whether it might occur in glucagon- and somatostatin-secreting cells in the pancreatic islet in the rat and mouse. We used the polyclonal antiboby against the N-terminal peptide whose specificity was confirmed by Western blot analysis. Double immunostaining demonstrated that cellubrevin was localized in insulin-containing cells, but both glucagon-containing and somatostatin-containing cells lacked the immunoreactivity. Immuno-electron microscopic analysis revealed the localization of cellubrevin on the margin of secretory granules near the plasma membrane but not in the granules closer to the nucleus. These observations support the view that cellubrevin in the pancreatic islet is expressed on the membrane of the secretory granules in B-cells at the stage of exocytosis.

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We examined the localization of connexin (Cx)43 in the enamel organ during ameloblast development. The specificity of monoclonal anti-Cx43 antibody was elucidated by immunoblot analysis and immunoelectron microscopy. Gold particles for Cx43 were detected by immunoelectron microscopy on gap junctions but not on other structures such as desmosomes. Punctate and intense immunofluorescence for Cx43 was detected in all cell types of the enamel organ. Cx43 expression in ameloblasts showed a transient decrease and then increase during ameloblast development. Double staining of Cx43 and amelogenin, one of the enamel proteins, revealed that immunofluorescence for Cx43 markedly decreased in some late presecretory ameloblasts just prior to enamel formation. Morevover, the localization of Cx43 changed during enamel formation. Cx43 was distributed randomly on the lateral plasma membranes of presecretory ameloblasts, but tended to gather on those corresponding to the supranuclear regions of secretory ameloblasts. Immunofluorescence for Cx43 in maturation ameloblasts appeared linear rather than punctate. These results suggest that Cx43 in the late presecretory ameloblasts is degraded just before enamel formation and then newly synthesized Cx43 is redistributed during the secretory stage. These changes in Cx43 expression may be related to the cellular differentiation of ameloblasts.

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Glucokinase (GK) is believed to play a key role in the control of the hepatic glucose metabolism. To address the mechanism of the regulation of glucose metabolism through GK action, we immunohistochemically studied changes in GK distribution in primary cultures of rat hepatocytes. In hepatocyte monolayers incubated in 5mM glucose, GK staining by the immunoperoxidase method was observed predominantly in the nucleus. When cultured hepatocytes were incubated for 30 min in various concentrations (5-45mM)of glucose, there was an appreciable decrease in nuclear GK immunoreactivity, even at 10mM compared with that at 5mM. After the shift of glucose concentration from 5mM to 25mM, the GK distribution changed timedependently over 1h. A time-dependent change in GK distribution was also observed when the glucose concentration was shifted from 25mM to 5mM. Reversal of GK distribution in response to the change in glucose concentration from 5 to 25mM and vice versa was shown to repeatedly occur. Lower concentrations (0.05-5mM) of fructose, which is known to stimulate glucose phosphorylation by GK, in combination with 5mM glucose, induced the translocation of GK from the nucleus to the cytoplasm. Mannose (20mM), a substrate of GK, and sorbitol (1mM), a stimulator of glucose phosphorylation by GK, induced the translocation of GK from the nucleus to the cytoplasm in the presence of 5mM glucose. L-glucose, galactose, 3-O-methylglucose, and 2-deoxyglucose at 20mM each did not affect the GK distribution observed in the presence of 5mM glucose. The results suggest that GK is present mainly in the nucleus under conditions where GK action is not much needed, whereas the enzyme exists mainly in the cytoplasm under conditions where it must function extensively. Our findings indicate that the shuttling of GK between the nucleus and the cytoplasm is essential for the regulation of the glucose metabolism in the liver.

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