A histochemical analysis of gamma-glutamyl transpeptidase (GGT) activity was performed on 42 cases of various human thyroid diseases. In contrast to the negative reaction for GGT in the normal thyroid tissues, a rather widespread distribution of activity was demonstrated in diseased thyroids. Among thyroid neoplasms, papillary carcinoma showed the strongest and most stable activity, while a more variable activity occurred in follicular carcinoma. Anaplastic carcinoma and medullary carcinoma were negligible in the activity. In non-neoplastic thyroid diseases, the activity was localized in distribution and usually less pronounced, as compared with the neoplastic lesions.
This study suggests that the appearance of GGT activity in thyroid tissues reflects not only proliferative or regenerative changes of the follicular epithelium but also functional aspects of the lesions.

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The effects of sea bloom on gill primary lamellae of the teleost, young yellowtail (Seriola quinqueradiata) were investigated from the morphological and histochemical point of view. Mucous goblet cells located on the afferent ridge were significantly impaired by the sea bloom (Gymnodinium), while they were intact on the efferent ridge. Most of the mucous cells epxosed to the sea bloom disappeared from the afferent ridge, and a few rudimental cells remained. Histochemically, these mucous cells contained neutral glycoproteins. Electronmicroscopically, the surface of the afferent ridge exposed to sea bloom was covered entirely with the pavement cells. These observations may have relevance to the manner in which the toxic effects of sea bloom on the teleost are mediated.

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A study of the time of appearance and localization of γ-crystalline was made along with an analysis of component elements in the lens of the developing rat and a correlation between the two was found.
The time of appearance and the histological localization of γ-crystallins in developing rat lenses were studied by the immunofluorescent antibody technique. The first positive immunofluorescent reaction with anti-γ-crystal-lin antibodies was observed at 13 1/2 days in the embryonic lens when cells in the posterior wall of the lens vesicle elongated and differentiated into primary lens fibers as found by Schubert et al. This reaction intensified with the addition of the secondary lens fibers in the lens equator.
An elemental analysis of the same samples (used to detect γ-crystallins) using the energy dispersion-type electron probe microanalyzer, revealed that sulphur increased in amount while phosphorus relatively declined in amount with the progress of the formation of the primary and secondary lens fibers. These quantitative changes in sulphur in the X-ray microanalysis are consistent with the appearance and localisation of γ-crystallins.

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Catecholamine-containing subependymal cells were studied by the silver impregnation technique. We divided them into three types, based on location of their cell bodies in the hypothalamus. The majority of these cell bodies were situated in the subependymal layer, while a few were found in the ependymal layer as well as in the preoptic nucleus. The basal processes of these cells were found to extend in three different directions in the brain; 1) They extended laterally and contacted with neuronal processes in the preoptic area, 2) traveled caudally and lateroventrally forming an element of the preoptic recess organ tract, and 3) ran rostrally and dorsolaterally and distributed further widely to the telencephalon.

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The histochemical organization observed in cross sections of rat masseter muscle was investigated by staining for succinic dehydrogenase. Most primary fasciculi were composed of three muscle fiber types: type A, B and C classified by Stein and Padykula (29). Significant differences in the percentage of type B plus type C fibers (Pbc) and the percentage of type C fibers (Pc) were found in different regions (origin, belly, and insertion), and in different parts (the deepest masseter, anterior deep and posterior deep masseter, and superficial masseter). Moreover, marked differences were found in the percentage distributions of Pbc and Pc of different primary fasciculi in each part of the masseter. In general, the average Pbc and Pc values were highest in the deepest masseter, intermediate in the superficial masseter, and lowest in the deep masseter in the origin and belly portions. In aging rats the three kinds of muscle fibers were easier to distinguish and differences in the average Pbc and Pc values in different muscle parts were greater. Moreover, primary fasciculi with high Pbc and Pc values, or those with low Pbc and Pc values were more numerous than in young adult rats.

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Histochemical localization of cholinesterase (ChE) activity in the baroreceptors of the carotid sinuses of the mouse, rat and cat was studied by light and electron microscopy. ChE-positive sites were identified by light microscopy as brownly-stained small plaques both in the mouse and in the rat, or as intensely-stained large plaques or bands in the cat, extending from the transitional zones between the adventitia and media as far as the outer half of the media. By electron microscopy it was evident that the reaction products were deposited in spaces between the axon terminals and the surrounding Schwann cells as well as on the acellular materials around the Schwann cells, including the Schwann cell basement membrane. Enzyme activity was also noted in the cisternae of the rough endoplasmic reticulum as well as in the nuclear envelope of some Schwann cells associated with the axon terminals. It is thought that ChE is synthesized and released by Schwann cells that are associated with the axon terminal. Inhibitor experiments showed that the enzyme noted in the baroreceptors was non-specific ChE as in the case of other mechanoreceptors such as Meissner and Pacinian corpuscles. The fact that the baroreceptor of the carotid sinus has such an intense ChE activity indicates that this enzyme has some important and specific roles in the functions and the maintenance of the receptor.

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The interaction of estrogens with different duration of action or antiestrogen with the estrogen receptor (ER) in adult rat uterus is described. Estradiol-17β(E₂) or nafoxidine (UA; antiestrogen) were injected into the castrated rats.
E₂ injection: Cytoplasmic ER (ERc) decreased, whereas nuclear ER (ERn) increased at 4hr after the injection. Both the ERc and the ERn returned to the control level at 36hr.
UA injection: ERc decreased, whereas ERn increased at 4hr after the injection. Neither the ERc nor the ERn returned to the control level even at 72hr. UA induced a long term ER retention in the nucleus, accompanied by a sustained depletion of the ERc.
Estradiol dipropionate (ER; long acting estrogen) injection: ERc decreased 1 day after the injection and remained low for 7 days while plasma E₂ was high; then returned to the control level by 14 days. ERn increased 1 day after the injection and continued to be high during the subsequent 14 days. In the present 14-day-experiment, ED inhibited the recycling of the ER from the nucleus to the cytoplasm, but not a synthesis of new receptor.

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Histochemical identification and visualization of steroid hormone receptors remains a goal, despite earlier success with dry- and thaw-mount autoradiography. Applications of various histochemical and immunohistochemical procedures for the identification of steroid hormone target cells and subcellular binding sites are reviewed. Results obtained with dry- and thawmount autoradiography after in vivo administration of [³H] estradiol are contrasted with those obtained by in vitro liquid emulsion autoradiography, incubation with conjugated estradiol, antibodies to estradiol, or antibodies to estradiol “receptor”. Dry- and thaw-mount autoradiography in vivo after single injection of [³H] estradiol, and in vitro after tissue slice incubation with [³H] estradiol consistently show preferential nuclear concentration of radioactivity, little in cytoplasm, and none in nucleoli. In contrast, liquid emulsion autoradiography after in vitro uterine section incubation with [³H] estradiol shows no nuclear uptake, but only labeling of cytoplasm of eosinophils. Eosinophils are also labeled with conjugated estradiol. Histochemical studies with conjugated estradiol or estradiol antibodies show variable results with preferential cytoplasmic and distinct nucleolar labeling of uterine epithelial and stromal cells; however, there is only occasional nuclear labeling, and some lack of identification of target cells, when compared to results obtained with our autoradiographic techniques. The specificity and utility of certain histochemical techniques need to be established, since some of the results are probably related to technique and do not reflect biological events. Development and use of refined histochemical techniques are needed to further contribute to the clarification of the mechanism of steroid hormone action and to provide a tool for the clinical diagnosis of steroid hormone dependent tumors.

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A large number of developed silver grains accumulated on the nuclei and some were scattered in the cell organelles of mouse endometrial, interstitial, and myometrial cells, 30min after ³H-17-β-6, 7-estradiol administration. 2hr later, they showed distinct active reactions in their cytoplasmic fine structures, although developed silver grains were still located in their nuclei. A single fixation method with 2 to 4/osmium tetroxide seemed very effective to preserve the carrier bound estradiol.
Female rats were perfused with PLP (periodate-lysine-paraformaldehyde) or PG (paraformaldehyde and glutaraldehyde) before incubation in radioactive estradiol or its ligands. The perfused uteri were cut in small pieces and incubated in ³H-estradiol or ³H-moxestrol with a 10μCi/ml in phosphate buffer for 2hr at 37°C. They were post-fixed with a mixture of glutaraldehyde and osmium tetroxide for 2hr, then embedded in Epon after dehydration. In both cases, the developed silver grains were homogenously on the cell surfaces, in the cytoplasm, and on the nuclear membranes of endometrial, interstitial, and myometrial cells.
No nuclear accumulation was observed in those experiments. However, a very strong accumulation of labeled compounds was observed in the granules of eosinophilic leucocytes and in the lysosomal granules of tissue macrophages.
These data suggest that the receptors for the estradiol or ligands in the endometrial cells, especially in the nuclei, cannot accept directly without carrier proteins, except in the eosinophilic leucocyte or macrophages. The results may support facts obtained by many biochemical studies. Furthermore, it is possible that the primary receptors for estradiol or its ligands are distributed in the cytoplasm and on the membrane surface of cells, with only a small number in the nuclei. In this case, secondary receptors should exist in the nuclei, which only accept the carrier-bound estradiol or its ligands.

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Acetylcholine receptors (AChRs) at the adult mouse and embryonic chick neuromuscular junctions were visualized by using erabutoxin b (Eb), one of the short-chain neurotoxins from Laticauda semifasciata, and antibody against AChRs of the electric organ from Narke japonica. Visualization of AChRs was performed by the following three methods: (1) staining with rhodaminelabeled Eb (TMR-Eb), (2) staining with horseradish peroxidase-labeled Eb (HRP-Eb), and (3) indirect immunofluorescence microscopy using the antibody against purified AChRs. At the light microscopic level, TMR-Eb offered the most simple and rapid procedure for producing clear images. The HRP-Eb method was suitable for viewing the distribution of AChRs at the ultrastructural level. These procedures revealed that adult neuromuscular junctions had a nearly uniform distribution of AChRs at their postsynaptic membrane, whereas embryonic ones possessed aggregations of small discrete regions of high AChR concentration. Similar regions of AChRs were observed on the nonjunctional myotube surface.

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For the demonstration of acetylcholine receptor (Ach R) at neuromuscular junction using α-bungarotoxin-horseradish peroxidase (α-BT-HRP) conjugate, a modified conjugation method was described to keep the better binding activity to Ach R. For the tissue processing, three methods were compared regarding the binding specificity and preservation of ultrastructure. The best result was obtained when EPON sections were made from glutaraldehyde-fixed tissue after incubation with α-BT-HRP conjugate. The preliminary examination of cryostat sections and subsequent embedding for electron microscopic examination was useful but unsatisfactory for the preservation of membrane structure. Electron microscopic examination revealed that α-BT was localized both on the presyraptic and on the postsynaptic membrane.

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α-Bungarotoxin (α-BGT) binding capacity on slide-moullted brain sections which had been fixed with 4% paraformaldehyde (PFA) or periodate-lysine-paraformaldehyde (PLP) was determined by quantitative light microscopic autoradiography using radioiodinated toxin. The results are consistent with our previous data obtained by a biochemical method on tissue homogenate. This is a new methodology for estimation of α-BGT binding capacity in brain regions of a limited area.

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We performed light and electron microscopic autoradiography of GABA and opiate receptors using ³H-muscimol and ³H-DAMA.
Light microscopically, distribution of muscimol and DAMA binding sites was demonstrated by means of Texture Analysing System (Leitz).
For electron microscopic autoradiography, selection of development method is important. The authors obtained silver grains in a pattern precisely localized to the cytoplasmic membrane, i.e, the receptor site by means of EAA development.
We observed ³H-muscimol binding site in the neuropiles of the cerebellar molecular layer and the substantia nigra ultrastructurally. Receptor sites labeled with silver grains were located not only on the synaptic regions but also on the plasma membranes of the dendrites and axons not corresponding to synaptic sites. ³H-DAMA binding sites in the caudate nucleus and the amygdaloid nucleus were noted on the plasma membranes of the axons, dendrites and nerve cell somas, some corresponding to synapses, others not. Autoradiographical observation of neurotransmitter receptors has still problems to be solved. Nevertheless, this technique is very important to observe the morphological relationship between transmitters and their receptor sites.

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Autoradiography combined with scanning electron microscopy (SEM-ARG) was applied to visualize the Con A receptor sites of human red blood corpuscles. Prepared erythrocytes on a glass slide were coated with emulsion by a dipping technique after evaporation by carbon under clean vacuum condition with a cooling trap of liquid nitrogen. Silver halide crystals in the emulsion were distritubed homogeneously on the surface of the erythrocytes. In these experiments, emulsion degelatinized by centrifugation seemed superior to normal emulsion. The remaining gelatin was removed by distilled water at 45°C after development.
Electroconductive staining with tannic acid and osmium tetroxide was effective in obtaining good electron contrast and also in preventing artifacts due to the autoradiographic procedure. The number of developed silver grains on the surface of the red blood cells was proportional to the length of exposure time. After exposure for 8 weeks, the average number of grains was calculated to be 312.46±76.07 (³H-Con A-labeled specimen) or 372.58±106.05 (¹²⁵I-Con A-labeled specimen) per single red blood corpuscle face. The SEM image of the grains was spherical or filamentous, so it was easy to distinguish the grains from the red blood cell surface. On the specimens labeled with ³H-or ¹²⁵I-Con A, a large number of developed silver grains were distributed homogeneously on the surface of the red blood cells, but on the cells labeled with the same radioactive Con A containing α-metllyl-d-mannoside, there were almost no grains present on the surface. Therefore, the distribution of the developed silver grains probably represents Con A receptor sites.
SEM-ARG seems to be a valuable method for the visualization of receptors on the cell surface, even though several problems still remain.

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