In order to obtain a more precise microtopographical surface map of the epithelium of the rabbit vaginal mucosa, investigations by light microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) have been systematically correlated. The epithelium was examined from the “portio vaginalis cervicis uteri” down to the “vestibulum”. This study shows that the upper 2/3 of the vagina —which, in this species, is very long, measuring 13-14cm—is lined by a single epithelial layer of tall ciliated and microvillous cells closely resembling the endocervical epithelium with which it is continuous. Moreover, these ciliated and microvillous cells also cover mucosal infoldings in the upper part of the vagina, especially those on the ectocervix and in the fornices, and scattered vaginal crypts. In particular, the ciliated elements decrease in number below the fornices, so that in large areas of the middle part of the vagina only microvillous cells are recognizable. Prior to the squamo-columnar junction, however, the ciliated cells increase again. This study also reveals that in the rabbit the squamo-columnar junction is located at the level of the pubic symphysis and that a squamous pluristratified epithelium covers only the lower 1/3 of the inner surface of the vaginal wall.
In the estrous, i. e., precoital rabbit, the microvillous cells show little sign of secretion, whereas after mating they exhibit remarkable secretory features. These seem to increase progressively with postcoital stages (5h, 24h and 10 days) in the form of extensive mucification. These secretions often come in contact with spermatozoa retained in the mucosal infoldings and crypts, and are similar to those occurring in the endometrium, where they clearly depend upon progesterone activity. These epithelial features, different from those of other mammals, including humans, suggest that the greater part of the rabbit vagina accomplishes functions other than serving for copulation and as a fetal passageway. The present findings support the view that the rabbit vagina also plays a role as a reservoir of spermatozoa and in maintaining their viability, like the endometrium and ecdocervix.

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Intrapineal neurons and intracapsular and intrapineal myelinated fibers were studied by light microscopy in male cotton rats (Sigmodon hispidus) at various postnatal ages. Until 300 days of age, neurons are encountered mainly in the distal third of the organ; neuronal nuclei are largest in this region. The remaining regions contain only a few neurons with smaller nuclei. In all regions, nuclear sizes of the neurons increase, and neuronal numbers reduce by half until 100-200 days. Neurons thus consist of different populations of cells, and the aging changes in neuronal nuclei seem to be related to the differentiation of the organ. The disappearance of neurons and the enlargement of the nuclei of remaining neurons proceed simultaneously with age. Intracapsular and intrapineal myelinated fibers appear at 20 days and their density increases with age. As the myelinated fibers are distributed mainly in the distal region and its capsule, these fibers are related topographically to intrapineal neurons. The intracapsular myelinated fibers continue into bilateral bundles of myelinated fibers found under the transverse sinuses, corresponding to the nervi conarii. Since the intracapsular fibers and the fibers traced between the capsule and the parenchyma bifurcate in a proximal direction, the intracapsular fibers are considered to run proximally, some entering the organ.

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Electron microscopic observation was performed to examine whether spermiogenesis in the hamster might be affected by a short term vasectomy. When viewed by light microscopy, spermiogenesis was temporarily inhibited at 2 weeks, though to a limited extent among individuals, and had apparently recovered to the control level at 4 and 8 weeks after vasectomy in all hamsters. It was revealed at the electron microscopic level that mature spermatozoa with an abnormal flagellum were intermingled among numerous normal spermatozoa. The flagellum of the mature spermatozoa was composed of four different components: a mitochondrial sheath, outer dense fiber, fibrous sheath and axoneme. Abnormalities of the mitochondrial sheath were of three types: its discontinuity, displasia and deformation. The appearance of these abnormalities increased with the time after vasectomy and finally reached 52% as the highest value at 8 weeks. Additional noteworthy findings were the bisectioning of the outer dense fibers 2, 4 and 7, the fusion of two or three fibers, and the partial deletion of the fibrous sheath. These defects were characteristic in the vasectomized hamsters. Possible correlating microenvironmental factors are discussed as to how these abnormalities of the four flagellar components occur in the vasectomized hamsters.

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The effects of dietary zinc deficiency on testicular protein secretion, mainly that by Sertoli cells, were examined by electron microscopy and two-dimensional polyacrylamide gel electrophoresis of [³⁵S] methionine-labeled secretory proteins from mouse testes.
Zinc deficiency caused a significant decrease in the gonadosomatic index and a distinct increase in deoxyribonucleic acid concentration. Sertoli cells maintained normal fine-structural features; junctional complexes among Sertoli cells continued to divide seminiferous tubules into basal and adluminal compartments in the zinc-deficient mouse testes. Severe atrophic changes were observed in spermatogenic cells after meiotic division in the adluminal compartment, but not in spermatogonia located in the basal compartment. Zinc replacement treatment caused spermatogenesis to recover normally.
Although total protein secretion was not affected by zinc deficiency, one polypeptide spot appeared due mainly to the loss of its target spermatogenic cells. The present study indicates that zinc is indispensable for spermatogenic cells after meiosis and that testicular protein secretory functions can be preserved in the absence of zinc.

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The present study was designed to demonstrate the short term mineralization of dentin and enamel, and to investigate the effects of sodium β-glycerophosphate (Na-β-GPO₄) on calcification in a serum-free, chemically-defined medium. The first mandibular molars at the bell stage dissected from 18-day-old mouse embryos were used as explants, which were cultured by an improved flotation method. Calcification of enamel in the 18-day-old embryonic molars occurred within the 6th day of culture. In another experiment, the molar germs were cultured in a serum-free, chemically-defined medium supplemented with 1, 5 and 10mM Na-β-GPO₄. Promotion of tooth mineralization was recognizable at very low concentrations, such as 1mM Na-β-GPO₄, in 18-day-old embryonic tooth germs.
The culturing system reported here shortens the time required for dentin and enamel calcification to one half or one third of that reported previously and therefore should prove useful for examining regulations for cytodifferentiation and morphogenesis in tooth germs and the mineralization of dentin and enamel.

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Primordial germ cells (PGCs) were studied by electron microscopy in human embryos at 27 and 30 days of gestation.
PGCs were mainly found in the hindgut epithelium, some at the stage of separation from the endoderm, and others in migration through the mesenchyme of the dorsal mesentery between the primitive intestinal wall and the coelomic epithelium, including the coelomic angle. A few PGCs were still located in the endodermal epithelium. Before the process of separation and migration, PGCs appeared clearly different from neighboring somatic cells (endodermal, mesenchymal and epithelial cells of the hindgut and the coelomic layer). PGCs located in the endodermal epithelium were large and showed an irregular cell body often provided with filopodia-like processes that contacted somatic cells to form small areas of focal junctions. As a rule, PGCs possessed a large, round nucleus containing one or two conspicuous nucleoli. Lipid droplets, abundant glycogen particles, ribosomes and mitochondria were often observed in their cytoplasm.
A detailed ultrastructural analysis revealed a delicate fibrillar coat frequently present on the free surface of PGCs during their migratory phase through the mesenchyme of the dorsal mesentery. This surface coat, likely corresponding to a glycocalyx, appeared as a particular, conspicuous filamentous layer of 30nm thickness, mainly in the leading pseudopodial projections of PGCs migrating toward their target.
The surface coat of PGCs can be associated with the binding sites of specific macromolecular components of the extracellular matrix, including fibronectin, and hence plays a role in PGCs recognition and migration during this special embryonic phase.
Therefore, it is suggested that PGCs, with their fibrillar surface coat, might be able to provide their own substrate-adhesion or cell recognition molecules which are a necessary prerequisite for cell movement through the migratory pathway in the mesentery toward the genital anlage.

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A KOH-collagenase or simple KOH digestion method was employed for scanning electron microscope (SEM) studies of elastin components in the rat thoracic aorta, mouse urinary bladder, and human ductus deferens. Immersion of the fixed tissues in 30% KOH solution for 8-10min at 60°C, with or without subsequent collagenase treatment, successfully removed collagen fibrils and basal laminae while leaving cellular and elastin elements unchanged at their original shapes and locations.
The internal elastic lamina of the rat aorta appeared as a solid sheet formed by elastin fibrils 0.1-0.2μm thick, while the medial elastic laminae were more fibrous because of the presence of numerous fine elastin fibers on their surface. Adventitial elastin fibers were of a cord-like shape complicatedly entangled among the adventitial fibroblasts. These fibers were seen as bundles of fibrils 0.1-0.2μm thick.
In the mouse urinary bladder, elastin formed a thin lace-like sheet just beneath the serosal covering of the peritoneum. This sheet was composed of small bundles of fine (0.1-0.2μm thick) fibrils.
The external connective tissue of the human ductus deferens was made up of a three-dimensional loose network of elastin fibers 0.1-1.5μm thick. These fibers also appeared as bundles of the fine fibrils.
These findings indicate that the present method is useful for SEM studies of elastin as well as cellular components in various tissues and organs. This study also maintains that elastin fibers and laminae are basically composed of unit fibrils of 0.1-0.2μm thickness. As elastin components are arranged specific to individual organs and tissues, it is reasonable that these components are concerned in the characteristic mechanical properties of these tissues and organs.

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By means of peroxidase-anti-peroxidase (PAP) immunohistochemistry with both the neurofilament (NF) triplet (small: 68K, medium: 150K, high: 200K) antisera and the antiserum against spot 35-calbindin, developmental changes in expression of the immunoreactivity for the NF triplet proteins in different domains of the Purkinje cells was examined in the cerebella of postnatal rats. From birth till the postnatal day 6 (P6) the somata and dendrites exhibited moderately positive immunoreaction for small and medium NF subunits. In contrast, the incubation for the high NF subunit resulted in a negative immunoreaction for the somata and dendrites of the Purkinje cells at those stages. On P8 and P10 they were weakly immunoreactive for all NF triplet. Thereafter the intensity of their immunoreaction decreased progressively and the Purkinje cell somata and dendrites were immunonegative for all NF triplet proteins on P21, when the Purkinje cell attained an adult appearance in morphology.
On the other hand, the Purkinje cell axons, which can be identified selectively by the positive immunoreaction for the spot 35-calbindin throughout the course of the postnatal development, exhibited positive immunoreactivity for all the NF triplet on the 9th, 21th and 50th postnatal days in the cerebellar white matter. The development of the slow axoplasmic transport and the posttranslational modification of the NF triplet proteins are discussed as possible mechanisms underlying the differential expression of the immunoreactivity for the NF triplet proteins in different domains of the Purkinje cells during postnatal development.

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A number of novel honeycomb structures were encountered among collagen bundles in the capsule lumen of the Golgi tendon organs in the adult Chinese hamster. They varied in size and shape and consisted of a hexagonal aggregation of columnar components (or units), usually perpendicularly disposed with respect to the long axis of the tendon organ. They measured 90-100nm along their inner diameter and less than 1.0μm in length, separated by partitions of electron dense materials about 30nm wide. Each columnar unit consisted of a central, globular, electron dense material (20-30nm in diameter) with a constant periodicity of 30-40nm and many (16-20) radially-arranged filaments (about 6nm in diameter), showing a flower-like pattern. The honeycomb structures were attached to or fused with collagen fibrils or fibers at their margins, and were also attached to the microfibrils and basal laminae of the sensory endings, Schwann cells, and the innermost cell layer of the outer capsule. These findings suggest that the honeycomb structures might contribute to a complicated three-dimensional connection of collagen bundles as a whole, and that this connection might efficiently cause the mechanical deformation of the sensory endings by their squeezing in muscular contractions or passive stretching.

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The intercalated portion of the rat liver was studied by scanning electron microscopy (SEM) after removal of interlobular connective tissue by acid or alkaline hydrolysis. Biliary intercalated portions have generally been regarded as short straight links lying between the bile capillary network and the interlobular duct. The biliary system as observed by SEM lacked such specialized segments for linking. Instead, it contained long intercalated ductules taking winding and branching courses. The ductular branches frequently anastomosed with each other to form an extensive plexus along the limiting plate. The ductules repeatedly connected with the plate on their courses as well as at their terminals. This disposition of the ductules probably potentiates their tolerance to luminal obstruction. At the junction between the ductule and the limiting plate, ductular cells and hepatocytes shared the biliary lumen. The lumen sometimes approached the base of the ductule, providing a possible route for bile leakage.
The intercalated ductule was composed of low fusi-form epithelial cells throughout its length, meeting its classical criteria by light microscopy. Its basal surface was furrowed with narrow grooves along cell boundaries. The ductular cells extended numerous microplicae in the basal grooves and on their lateral surfaces, suggesting their secretory function.

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Dehydration of specimens with ethanol or acetone makes it impossible to detect manganese superoxide dismutase (Mn-SOD) by immunohistochemistry. To circumvent obstacles and demonstrate localization by post-embedding immuno-electron microscopy, a rapid freezing and freeze-substitution technique was employed using Lowicryl K4M embedding medium. This was effective enough to allow the specific observation of immunogold particles for Mn-SOD on the mitochondria of cardiac muscle cells and WI-38 cells (human normal fetal lung diploid cells). This method preserved the antigen-antibody binding activity of Mn-SOD even after dehydration. Therefore, rapid freezing and freeze-substitution is useful for post-embedding immuno-electron microscopy of Mn-SOD and can further be employed for other antigens previously difficult to detect by conventional methods.

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