Metallothionein (MT) in the human male excretory ducts of the testis, such as the tubuli seminiferi recti, rete testis, ductuli efferentes, ductus epididymidis, and ductus deferens was examined immunohistochemically in 15 patients (one, testicular injury; one, testicular torsion; six, testicular tumor; seven, prostate cancer). The immunoreaction for MT was identified in Sertoli cells of the tubuli recti, and in the epithelial cells of rete testis, ductuli efferentes, ductus epididymidis at the tail portion, and ductus deferens near the epididymal tail. The immunoreaction in the tail was found mainly in the principal cells, and the secretory products near the surface of epithelial cells, and in the lumen. The MT immunoreaction in the epididymal head and body was observed to occur in the basal cells only. This result suggested that the epithelial cells have a different physiological function among the various portions of the excretory ducts of human testis.

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The laser scanning microscope (LSM) was applied to observe microautoradiographs. High quality laser scanned differential interference contrast (DIC) images of autoradiographs from the mouse kidney after injection of ³H-fucose, and of mouse skeletal muscle after injection of ¹²⁵I-insulin, were obtained. In the confocal LSM mode, reflectance from developed silver grains was observed. In a merged image of the reflectance and the DIC, it was possible to distinguish between silver grains and tissues in the specimen clearly, and to discern their spatial relationship. It is anticipated that the technique can be applied to microautoradiography at light microscope level.

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In order to further clarify the biological significance of glutathione-peroxidase (GSH-PO) in the prostate, we have studied the immunocytochemical localization of GSH-PO in glandular epithelial cells of the ventral prostate in castrated rats treated with testosterone or testosterone plus 17β-estradiol (E₂). In the control rat ventral prostate, GSH-PO was predominantly observed in the glandular epithelial cells. The intensity of GSH-PO staining in the glandular epithelial cells were remarkably depleted two days after castration, and it was clearly recovered by daily injection of testosterone, 1mg/head, or testosterone, 1mg/head, plus E₂, 0.01mg/head. Furthermore, glandular hyperplasia of the prostate was definitely detected in the castrated rats with testosterone plus E₂ for six weeks. In this case, the intensity of GSH-PO staining was markedly increased and the number of GSH-PO-positive granules increased remarkably, as compared with those of other experimental groups. These findings strongly suggest that castration causes marked suppression of GSH-PO protein synthesis, and lipid peroxides (or peroxidation), in the glandular epithelial cells, may be increased by testosterone-or testosterone plus E₂-administration.

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We studied cerebral perforating arteries in 18 autopsied cases of cerebral infarction, using monoclonal antibodies, antiactin antibody HHF 35, and antimacrophage antibody HAM 56. Degeneration and focal necrosis of smooth muscle cells were in the form of initial arterial lesions in patients with cerebral infarctions. These lesions were mostly detected in the outer layers of the media, and eventually developed into widespread necrosis. Following the progression of the medial changes, monocytes, possibly migrating from Virchow-Robin's space, accumulated in the adventitia. In advanced lesions, monocytes in the blood stream penetrated through the endothelial cells and accumulated in the subendothelial space and the media. Monocytes migrating from Virchow-Robin's space, as well as from the blood stream, thickened the arterial wall and narrowed the lumen, while accelerating fibrosis and plasma deposition. Applying immunocytochemical techniques we note that monocytes, HAM 56 positive cells, may have played a leading role in the development of occlusive arterial lesions, resulting in cerebral infarction.

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The present study was undertaken to explore whether tumor cells of villous adenomas of the human colon showed an organized expression of differentiation markers. Eleven cases of villous adenomas were examined by employing histochemical techniques, which are useful for characterizing the colonic mucosa, including high iron diamine-alcian blue pH 2.5, lectin stains with DBA, GSA-I and UEA-I, immunostainings for blood group A determinant, lysozyme, and proliferating cell nuclear antigen (PCNA). The results demonstrated that the adenoma tissues showed the organized expression of these sugar moieties or antigens similar to those found in the normal colon crypts. Namely, tumor cells in the upper portions of tumor tissues revealed characteristic features of normal epithelia, lining the upper compartment of the crypts, and contained sialomucins, which possessed DBA reactivity. Tumor cells in the lower portions, on the other hand, resembled epithelia lining the lower compartments of the crypts, contained sulfomucins, showed GSA-I and UEA-I reactivities, and stained for blood group A determinant and lysozyme. PCNA-positive cells were more abundant in the lower portions of the tumor tissues than in the upper portions. It seems likely that cellular differentiation, similar to normal crypt cells, occurs in the tumor tissues.

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Bacterial endotoxin induces alteration of intracellular calcium homeostasis and function in the liver. Male Wistar rats were injected intravenously with E. coli endotoxin (5mg/Kg), and these livers were removed 1, 4, 8, and 24hr later. Measurement of liver and serum malondialdehyde (MDA), an index peroxide, was carried out after endotoxin administration. Using the method of Ando et al., histochemical localization of Ca⁺⁺ ATPase activity was observed, and the specificity of the reaction was tested, using incubation media lacking Ca⁺⁺ or ATP, or by substituting ADP for ATP and including quercetin. Lipid peroxide in liver was increased 4-fold at 24hr, and was slightly increased in serum at 8hr after endotoxin administration. Histochemically, Ca⁺⁺ ATPase activity was demonstrated on the plasma membrane, bile canaliculi, mitochondria of liver cell, and Kupffer cell in control liver.
After endotoxin administration, Ca⁺⁺ ATPase activity of the plasma membrane, bile canaliculi, and mitochondria in hepatocytes and Kupffer cells reduced. These results suggest that decreased Ca⁺⁺ ATPase activity and increased peroxide may contribute to the hepatic injury associated with the concentration of cytosolic calcium.

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Electron microscopic cytochemical studies have been made on sulfated glycosaminoglycans in mast cell granules from the rat peritoneal cavity by means of combined alcian blue (AB) pH 1.0-phosphotungstic acid (PTA) staining and enzyme (heparinase or chondroitinase ABC) digestion or chemical modification (nitrous acid) procedures. The results obtained revealed that the mast cell populations from the rat peritoneal cavity are grouped into at least three cell types on the basis of the molecular species of sulfated glycosaminoglycans contained in the cytoplasmic granules, the first cell type occupying the majority of the cell population with cytoplasmic granules containing primarily heparin, the second cell type being of an exceedingly small proportion with cytoplasmic granules containing primarily isomeric chondroitin sulfate (D and/or E), and the third cell type (intermediates between the first and second types) constituting a relatively small proportion with cytoplasmic granules containing two kinds of cytoplasmic granules containing heparin and isomeric chondroitin sulfate (D and/or E) respectively. In addition, unique substructures of cytoplasmic granules containing different sulfated glycosaminoglycans were disclosed such as tubular or thin thread-like elements, interstitial vesicles and surface spine-like figures.
The present results are taken to represent the first electron microscopic cytochemical evidence that mast cell populations are heterogeneous in terms of molecular species of sulfated glycosaminoglycans contained in the cytoplasmic granules in mammals.

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Ultrastructural changes and enzyme-cytochemical localization of Chang hepatoma in rat, human hepatoma, and aflatoxin B₁(AFB₁)-treated rat hepatocytes are demonstrated by electron microscopy. Both dark and light cell types appear in immersion-fixed samples, as well as in perfusion-fixed ones, in normal and experimental samples. The light cell contains pale cytoplasm rich in glycogen, less G6Pase activity in ER, and much sensitivity to AFB₁-treatments. Dark hepatocytes make up the major cell population in ascites-form and solid-form of Chang hepatoma, induced by chronic feeding with 3′-methyl-4-dimethyl-aminoazobenzene. Dense cytoplasmic ground substances, ER, Golgi apparatus, and normal mitochondria are encountered in dense cells. Enzyme-cytochemical labelings indicate that each of the activities of acid phosphatase, glucose-6-phosphatase, and thiamine pyrophosphatase are localized in both cell types. This work confirmed the existence of two populations of dark and light hepatocytes in normal and abnormal livers.

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Ethanolic phosphotungstic acid (EPTA) method and G-6-Pase method were used to localize cytochemically the atrial specific granules (ASG) and sarcoplasmic reticulum (SR) of rat atrial cardiocytes under the electron microscope, and at the same time, the calcium (Ca) in ASG and SR was analyzed quantitatively by analytical electron microscopy (AEM). In these methods, the procedure of osmium tetroxide fixation was shortened or even abolished to keep more combined Ca. The Ca concentration in ASG and in the terminal cisterns of SR (TSR) was 64±16 and 99±21mM/kg dry wt respectively. These values are approximate to those reported in earlier works that were done on cryosections. It is suggested that these cytochemical methods could reserve a large part of combined Ca in the intracellular Ca store, and they are much simpler than the low temperature techniques of biological specimen preparation. Therefore, these methods are useful for AEM microanalysis of elements in organelles.

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