To visualize the entire shape of the intraepithelial nerve fibers, whole mount preparations of the dog urethra were produced and immunostained with an antiserum against CGRP, one of the predominant substances contained in the nerves.
The immunoreactive nerves in the lamina propria were smooth (non-beaded) in appearance and weak in immunoreaction. Within the epithelium, they displayed typical beaded profiles and were intense in immunoreaction. The intraepithelial fibers branched and wound into an extensive network with wide meshes (“reticular terminal”). The bead-like swellings included large ones resembling Herring bodies in hypophyseal neurosecretory fibers.
Another type of nerve terminal, consisting of fine and weakly immunopositive fibers, was also found in the epithelium. These branched in dendritic or in dense bouquet-like fashion, occupying smaller areas (“bouquet-like terminals). Vesicular swellings often characterized these terminals, though they were smaller and more uniform in size and far less in their amount of immunoreactive substance than were the swellings in the reticular terminals. Both types of nerve terminals originated from the same nerve trunk.
The connection between the reticular and bouquet-like terminals, which may presumably represent secretory and receptive parts, respectively, morphologically supports the possible occurrence of an axon reflex in the urethral CGRP neurons.
Our whole mount preparations, when doubly stained with CGRP and serotonin antibodies, further revealed the CGRP-positive reticular terminals being closely associated with serotonin- or CGRP-immunoreactive paraneurons dispersed in the epithelium.

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In order to clarify periodic changes in the localization of enzymes engaged in estrogen biosynthesis during the estrous cycle, immunohistochemical and fine structural studies were performed using estrous cycling and pregnant hamster ovaries. Results showed that ovulation takes place at midnight between Day 4 and Day 1 in the regular 4 day-cycle hamster. Immunoreactivity for aromatase is localized in the granulosa cells of the secondary follicle and granulosa lutein cells during the morning (10:00am) of Day 1 to the evening (5:00pm) of Day 4; in the night (9:00pm) of Day 4, only the granulosa cells of the Graafian follicle showed a strong immunoreaction.
As for 17α-lyase, theca interna cells of the secondary follicle are immunopositive throughout Day 2 to the morning (10:00am) of Day 4. Only a few cells in the theca interna of the Graafian follicles are immunopositive in the evening (5:00pm) of Day 4. No positive cells for this enzyme were detected in the night (9:00pm) of Day 4 or morning (10:00am) of Day 1. The rapid decrease of estrogen biosynthesis occurring just before ovulation is considered to be due to the disappearance of 17α-lyase in the theca interna cells of the ovary.
On Day 10 of pregnancy, the granulosa cells of the secondary follicles and both the granulosa and theca lutein cells of the corpora lutea are immunostained with the aromatase antibody, while the theca interna cells of the secondary follicles reacted positively to the 17α-lyase antibody.
Only the granulosa and theca interna cells from the large preovulatory Graafian follicle of Day 4 (proestrus) which are positively stained for aromatase as well as 17α-lyase show ultrastructural features typical of steroid secretory cells.

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The distribution and terminal formation of nerves in the periodontal ligament of dog incisors and canines were investigated by immunohistochemistry for neurofilament protein (NFP) and by electron microscopy. The NFP-immunoreactive nerve fibers were found to be densely distributed in the apical third of the periodontal ligament, while they were sparse in the coronal two thirds. Most of the nerve endings in the periodontal ligament showed a tree-like appearance and resembled those nerve endings demonstrated in the periodontal ligament of human and monkey under the category of free nerve endings. Presumable axon terminals of these were slightly thicker than preterminal portions, running along periodontal collagen fibers and tapering within them. In light microscopic images, at least, they differed from the Ruffini endings which are commonly seen in rodents, displaying a glove-like configuration with extremely expanded tips. Under the electron microscope, however, the tree-like endings of the dog appeared similar to the Ruffini endings of rodents: their terminals were filled with mitochondria, covered with a cytoplasmic process of a Schwann cell, and surrounded by collagen fibers. These ultrastructural findings, combined with the results of previous physiological studies suggest that the nerve endings demonstrated in the present study can be identified as Ruffini endings. It is even stressed that the dog-type of Ruffini ending can be regarded as a representative of the sensory receptors in the mammalian periodontal ligament. In addition to these endings, knobbed endings, corpuscular (lamellated and glomerular) endings, and free nerve endings were rarely encountered in the periodontal ligament of incisors and canines of the dog.

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Demineralized bone matrix gelatin (BMG) was implanted into the skeletal muscle of Sprague-Dawley (S. D.) rats, and histological changes were examined 3, 5, 7, 10 and 15 days later. Before bone formation, a specific calcification process was found in most of the BMG from day 5 and 7 after implantation. The heterotopic calcified sites were not always consistent with the sites of the alkaline phosphatase activity. It was considered that this calcification progresses without any cellular components, and we distinguished this type of calcification as “acellular mineral deposition” from the calcification which occurs in new bone formation. This “acellular mineral deposition” was first observed as small spherical calcified deposits in the BMG on day 7 after implantation; these deposits then gradually grew and fused with each other.
Some multinucleated cells appeared near the site of calcification on day 7 after implantation, but osteoblasts or osteoblast-like cells were scarcely observed around the calcified deposits in BMG until day 7. Vascularization was often observed near the “acellular mineral deposition” and the new bone formation. Fourier transform infrared spectroscopy showed that the calcified deposits in BMG were composed of hydroxyapatite, carbonateapatite and other calcium phosphate components, and that the first two components became prominent with time. It is believed that the “acellular mineral deposition” is due to the deposition of calcium and phosphate into the BMG by a process of heterogenic nucleation that does not involve osteoblasts or matrix vesicles. Bone formation induced by the BMG occurred after the “acellular mineral deposition.” The experimental calcification shown in this paper seems a useful model for the study of biocalcification.

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Cytoskeletal architecture of the matrix cell and neuroblast in the wall of midbrain of 4-6 day-old chick embryos was examined by electron microscopy and immunohistochemistry. The matrix cell, the undifferentiated stem cell later producing neurons and glial cells in the central nervous system, is characterized ultrastructurally by abundant free ribosomes and a poorly developed cytomembrane system. A few microtubules running in random directions are observed in the matrix cell body. In the cell processes, microtubules are oriented longitudinally, and linked with each other by cross-bridges, presumably composed of microtububeassociated proteins (MAPs). The cell processes contain abundant cytoplasmic filaments including a large amount of actin filaments which adhere to the plasma membrane of junctional complexes located immediately below the inner surface of the neural tube.
In the neuroblast which has been differentiated from the matrix cell, the cytomembranous organelles, especially rough endoplasmic reticulum are markedly better developed than in the matrix cell; microtubules are more numerous in the cell body. The cell process contains many microtubules with cross-bridges and a few intermediate filaments, which are relatively characteristic of the cytoskeleton of the neuroblast.
Phalloidin-staining and immunohistochemistry showed that the neuroblast was richer in F-actin, β-tubulin, MAP1, MAP2, tau, calspectin, and synapsin I than the matrix cell. As the matrix cell differentiates into the neuroblast, both the cytoskeletal and cytomembranous systems proved to develop features, characteristic of a neuron.

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The ultrastructure of the Type I cells in paraganglia of rat recurrent laryngeal nerve (RLN) was studied after the administration of 5-hydroxydopamine (5-OHDA) and 6-hydroxydopamine (6-OHDA).
Normal Type I cells of RLN-paraganglia contained abundant organelles and their cytoplasm was characterized by the presence of numerous membranebounded dense-cored vesicles (DCVs). The DCVs were round in profile (diameter 107.67±0.06nm, all values expressed as mean±s. e. m. in the present study) and possessed dense cores of moderate to low electron density. After 5-OHDA treatment (single injection, 100mg/kg b. w., i. v.), the majority of DCVs were filled with a material of high electron density. No significant difference was observed between the profile diameter of the DCVs in 5-OHDA-treated rats (104.96±0.06nm) and that in normal rats. After 6-OHDA treatment (three injections, 100mg/kg b. w. each at 12h intervals i. p.), no significant alteration in the electron density of the core was noted. However, most of the DCVs were enlarged and round, elliptical or irregular in profile (190.57±2.77nm×130.34±2.09nm). The dense core of DCVs was centrally or eccentrically located in DCVs.
The results of the present study indicate that: 1) there is only one type of granulated glomus cell (i. e., Type I cells) in the rat RLN-paraganglia under normal physiological condition; and 2) since the ultrastructural morphology of DCVs in Type I cells of rat RLN-paraganglia is altered after 5-OHDA or 6-OHDA treatment, these cells may possess mechanisms for the uptake of false adrenergic neurotransmitter and/or neurotoxin.

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This paper demonstrates that, in the mediation of light, the suprachiasmatic nucleus (SCN) functionally associates with the anterior periventricular and parvocellular paraventricular neuron systems in rats. Intact rats (group 1) and rats undergoing a hemicomplete cutting of the SCN (group 2) were housed in a dark room (2-3 weeks) and killed after an exposure to light for 10, 30 or 60min. Other intact animals (group 3) kept in a dark room (2 weeks) were exposed to light for 10min, then stored 60min in the dark room, and killed in darkness. The SCN, anterior periventricular nucleus, and parvocellular paraventricular nucleus were examined immunohistochemically using antisera for vasoactive intestinal polypeptide (VIP), arginine vasopressin, somatostatin, rat corticotropin releasing factor (rCRF), and c-fos protein. In comparison with animals kept in darkness, animals exposed for 10 and 30min to light indicated a remarkable reduction of VIP immunoreactivity in the SCN and some increase of CRF immunoreactivity in the parvocellular paraventricular nucleus. The diminution of VIP immunoreactivity did not occur in the isolated SCN of group 2 animals. In group 3, a 10 min-light exposure induced a remarkable enhancement of nuclear c-fos immunoreactivity in neurons in the ventrolateral region of the SCN, in the anterior periventricular nucleus, and in the parvocellular paraventricular nucleus, most strongly in the SCN. Double immunolabeling methods have shown that VIP, somatostatin, and CRF neurons in the respective nuclei were c-fos positive.

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The intracellular localization and translocation of the 1α, 25-dihydroxyvitamin D₃ receptor (1α, 25(OH)₂D₃ receptor) in osteoblasts of mouse parietal bone and MC3T3-E1 cells were examined immunocytochemically using a rat monoclonal antibody to 1α, 25(OH)₂D₃ receptor. In osteoblasts of parietal bones in vivo, immunoreactivity for 1α, 25(OH)₂D₃ receptor was detected not only in the nuclei but also in lysosomal structures, and also sparsely in the cytoplasmic matrix. The transport of 1α, 25(OH)₂D₃ receptor was investigated immunocytochemically after incubation with 1α, 25(OH)₂ D₃. In osteoblasts of parietal bones, after 1min incubation with 10^-8M 1α, 25(OH)2D₃, the perinuclear cytoplasm showed intense immunoreactivity for 1α, 25(OH)₂ D₃ receptor. After 10min incubation, immunoreactivity was intense in the nuclei, especially in the heterochromatin. In MC3T3-E1 cells, after 1min incubation with 1α, 25(OH)₂D₃, immunoreactivity for 1α, 25(OH)₂ D₃ receptor was found in the form of a fibrillar structure extending radially to the periphery of the cells. The immunostaining pattern of anti-1α, 25(OH)₂D₃ receptor was similar to the distribution of microtubules stained with anti-β-tubulin antibody. After 10min incubation, the nuclei showed intense immunoreactivity for 1α, 25(OH)₂D₃ receptor. Incubation with colchicine dissociated the fibrillar structures and inhibited the intranuclear localization of the 1α, 25(OH)₂D₃ receptor. These findings suggest that the 1α, 25(OH)₂D₃ receptor is located in the nuclei, in lysosomal structures and also sparsely in the cytoplasmic matrix of osteoblasts in vivo, and that the receptor is transported to the perinuclear cytoplasm via microtubules to be then translocated into the nucleus, especially into the heterochromatin.

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The structure and distribution of cellular components in the mediastinal milky spots of C57BL/6 mice were studied by electron microscopy and immunohistochemistry. The mediastinal milky spots were generally covered with plump mesothelial cells with hemidesmosome-like structures in small projections of the cytoplasm, and consisted mainly of clusters of lymphocytes, macrophages and fibroblasts. Macrophages and lymphocytes were often seen passing between mesothelial cells and migrating into the pleural cavity. Neither high endothelial venules (HEY) nor lymphatics were present in the mediastinal milky spots. Thy-1 (CD5)-, L3/T4 (CD4)- and Lyt-2 (CD8)-positive cells were located predominantly at the basal area adjacent to fat cells under the lymphoid tissue. IgG- and Ia-positive cells dominated throughout the lymphoid tissue, whereas IgM-positive cells were observed in the lymphoid tissue other than the basal area. Mediastinal milky spots showed a specific distribution of lymphocyte subsets: the T cell area comprised the basal area, and the B cell area was mainly other areas.
The mean percentages of CD4-, CD8-, IgM-, IgG- and IgA-positive cells in the mediastinal milky spots were 16.5%, 20.1%, 28.8%, 37.3% and 2.1%, respectively; the percentage of B cells was higher than that of T cells. IgG- and IgM-positive cells were the main lymphocyte subsets in the mediastinal milky spots. The most characteristic finding was that IgA-positive cells were a very minor cellular component.

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The osteoinductive effects of bone morphogenetic protein (BMP, derived from murine osteosarcoma) were studied with regard to its use combined with β-tricalcium phosphate (β-TCP). BMP and β-TCP were molded into pellets by the “pressure method”, originated by us and transplanted to ddY mice. Control mice received interdorsal muscular implantations of either the BMP or β-TCP pellets. The animals were sacrificed 1, 2 and 3 weeks after grafting, for radiological, histochemical, and ultrastructural observations. The BMP-β-TCP compound pellets induced faster cartilage and bone formation, whereas these activities were slower when pellets made solely of BMP were used. The β-TCP pellets demonstrated no osteoinductive properties. Observations revealed two types of β-TCP resorbing multinuclear giant cells. One was osteoclastic, expressing calcitonin receptors, having numerous mitochondria and ruffled border-like structures; the other was not osteoclastic in nature. In animals grafted with the compound pellets, a great number of osteoclastic cells gathered on the pellets, much earlier than those grafted with the pellets made of BMP alone. Then, osteoblastic bone formation over the cement lines followed an osteoclastic resorption of both β-TCP and newly formed bone. In contrast, BMP induced few osteoclastic cells, resulting in slower bone coupling. Furthermore, the faster bone formation induced by the compound pellets seemed to be associated with the presence of β-TCP. Porous by nature, β-TCP would entrap BMP within its micropores, and thus, the intrinsically diffusible BMP is retained and its action consequently prolonged. In addition, the compound pellet offered increased surface contact between BMP and mesenchymal cells. Therefore, BMP-β-TCP compound pellets induce cartilage and bone formation more rapidly than does BMP alone.

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