Rabbit ovarian microvasculature, with particular regard to developing and atretic follicles, was studied by scanning electron microscopy (SEM) of vascular corrosion casts.
The microvascular network of the follicles was arranged in typical round plexuses of varying shape and size. Four different morphological types of vascular plexuses supplied the follicles: Type 1 (100-250μm in diameter) consisted of a simple net of thin capillaries that delimited a small empty central cavity. Type 2 (diameter>250μm) consisted of a multilayered capillary wall delimiting a large empty central cavity. This wall presented an inner layer made of uniformly distributed dilated and tortuous capillaries with numerous angiogenetic sprouts. Type 3 (diameter of 100-300μm) lacked the central cavity and comprised randomly arranged thin capillaries. Type 4 (diameter>250μm) consisted of a multilayered capillary wall delimiting a central cavity. Its inner layer was made of capillaries not homogeneous in size and course (thin in some areas, sinusoidal in others, sometimes highly dilated). In addition, the wall showed large interruptions (avascular areas) and focal invasion of the central cavity by newly formed vessels.
Types 1 and 2 showed the gradual transformation of thin capillaries into sinusoids as has been demonstrated in evolutive follicles. Types 3 and 4, described here for the first time, probably represent the vascular supply to atretic follicles. In particular, Type 3 supplied follicles undergoing obliterating atretic degeneration, whereas Type 4 supplied atretic follicles with hypertrophy of the theca layer. In fact, follicular atresia is a pleiomorphic phenomenon which ends in a regression of the follicles (obliterant atresia), but which may induce a temporary follicular hyperactivation (luteinizing atresia). These changes in the microvasculature prelude the formation of the so called “interstitial gland of the ovary”. Furthermore, these data prove that: 1) both thecal capillary vasodilatation and angiogenetic processes basically support the gradual increase of ovarian blood flow during follicle growth; 2) microvascular changes of atretic follicles are possibly related to a type of inflammatory reaction since they seem to be a consequential rather than primary cause of atresia.

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Light microscopy of tissue sections stained either with cationic iron colloid (pH 1.0-2.0) and nuclear fast red or with this colloid and thionin showed that the adult rat brain contains a considerable number of neurons which are strongly negative-charged by being coated with sulfated proteoglycans such as chondroitin sulfates. These neurons are distributed mainly in the cerebral cortex, hippocampus, zona incerta, medial and lateral cerebellar nuclei, ventral pontine nuclei and certain other areas. In the hippocampal formation, the strongly negative-charged cells seem identical with the GABAergic inhibitory interneurons reactive to the lectin Vicia villosa agglutinin. Neurons, including the GABAergic Purkinje's cells, of the cerebellar cortex showed no reaction to our cationic iron colloid at pH values of 1.0-2.0. Many non-GABAergic pyramidal cells in the lamina ganglionalis of cerebral cortex and many non-GABAergic large neurons of the ventral pontine nuclei were highly reactive to our colloid at pH values of 1.0-2.0. This suggests that our cationic iron colloid at pH values of 1.0-2.0 mainly stains certain subtypes of GABAergic neurons as well as some non-GABAergic neurons projecting long associational or commissural fibers.

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The occurrence of neurons with strongly negative-charged surface-coats was confirmed in the human brain.
Cerebral cortical tissue pieces (Area 19 of Brodmann), which had been removed in surgery from a 45-year-old Japanese man with meningioma were fixed with formalin. These specimens were cut into sections, stained with fine cationic iron colloid at pH value 1.0-2.0, treated for Prussian blue reaction, counter stained with carbolthionin, and observed with a light microscope. The observations indicated that some large-sized pyramidal cells in the ganglionic lamina were strongly nagativecharged or coated with sulfated proteoglycans, though where these cells projected to could not be determined.

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The rate of migration of immature granule cells of the rat olfactory bulb and polarity of cell-organelles in the migrating granule cells were investigated by ³H-thymidine autoradiographic and electron microscopic methods. The time lag in migration between two points was determined by cross-correlation analysis of labeling indices of the two areas. Granule cells were estimated to take 6 days to migrate rostralwardly from the subependymal layer at the anterior wall of the lateral ventricle to the center of the bulb, and an additional 1 to 6 days to migrate radially from the subependymal layer to the granular layer of the bulb. These results showed that the rate of rostralward migration of granule cells was faster than that of their radial migration. Golgielectron microscopic as well as routine electron microscopic studies on migrating granule cells revealed that centrioles and Golgi apparatus were located at the base of the leading process that possesses a growth cone at its tip.

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A morphological study of the lymphatic system in the diaphragm of adult Japanese monkeys was carried out using intraperitoneal injection with India ink, light microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques. For SEM examination of the subperitoneal connective tissue, the peritoneal mesothelium was stripped by either the NaOH/sonication microdissection techniques or the NaOH digestion method.
Light microscopy of 1μm Epon sections and TEM of thin sections demonstrated that lymphatic capillaries were distributed in the subperitoneal connective tissue of the muscular and tendinous portions. Gaps (stomata) between neighboring cells of the peritoneal mesothelium were occasionally encountered. In SEM observations, stomata with oval shapes and various sizes were abundantly seen in the peritoneal mesothelium, but were absent from the pleural mesothelium. It deserves particular note that a large number of foramina were detected in the subperitoneal connective tissue. The cluster of the foramina was similar in appearance to the “macula cribriformis” depicted by KIHARA (1956). Carbon particles injected into the monkey peritoneal cavity passed through the peritoneal stomata and then the macula cribriformis, ultimately to enter the lymphatic capillaries. This paper also discusses the functional implication of the lymphatic system in the diaphragm.

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The effects of tenotomy on the surface morphology of muscle fibers including myotendinous junctions in the rat soleus muscle were studied by scanning electron microscopy (SEM). Using potassium hydroxide (KOH) and collagenase, the extracellular materials were successfully removed to expose the surface of muscle fibers. When the soleus muscle was tenotomized at both proximal and distal ends, virtually all muscle fibers showed marked alterations of the fiber surface characterized by the formation of numerous transverse grooves and folds along their length. Narrow longitudinal grooves and folds of the sarcolemma were also observed. At myotendinous junctions, the fiber ends showed an overall rounded shape with several short sarcoplasmic processes, indicating that the processes were significantly retracted. These changes were clearly recognizable at 5 days after tenotomy, and most apparent at one week. Thin-section electron microscopy of the same SEM samples demonstrated that such folding of the sarcolemma was not directly related to the sarcomere pattern of the underlying myofibrils, suggesting that, once formed, the folds and grooves were retained for a certain period of time. At 2 and 3 weeks the surface morphology of the fibers underwent a recovery process of restoring the smooth surface on which the cross-striations of the underlying myofibrils were seen. At the fiber ends, sarcoplasmic processes regrew into slender, wavy and short forms. Such sarcoplasmic processes were greater in number and more elaborate than those in the control muscle. At 5 and 6 weeks the fiber surface resumed an almost normal morphology, except that the sarcoplasmic processes at the fiber end were still shorter and more numerous than those in the control. These observations support our previous results obtained by thin-section electron microscopy that the myotendinous junction undergoes a series of morphological changes of collapse and regrowth of the sarcoplasmic processes, reflecting changes in the underlying myofibrils. In conclusion, the changes in the surface morphology of tenotomized muscle fibers were well correlated chronologically to those of myofibrils such as the central core lesion.

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The three-dimensional structure of the Sertoli cell in the Korean Jindo dog was investigated by scanning and transmission electron microscopy. Additionally, type-A and type-B Sertoli cells were simulated by three-dimensional computer graphic imaging to clarify the anatomical relationship between Sertoli cell and germ cells. Descriptions in the present study are based on examination of the reconstructed type-A cells at stages IV, V and VI of the spermiogenetic cycle and stages VII, VIII and IX of the reconstructed type-B Sertoli cells. Morphologically, three types of Sertoli cell processes were evident: 1) flat club-like, elliptical processes bifurcating and trifurcating randomly; 2) slender cord-like, tubular processes; and 3) sheet-like processes. The sheet-like processes rested upon more than half the surface of each round spermatid located in the proximity of the Sertoli cell. Curiously, just before spermiation, the apical club-like processes shifted from their initial position at the spermatid head and subsequently covered the disengaged residual body, after which the residual body was no longer evident in the tubule. Though the mechanism for this elimination is not known, the process suggests a reciprocity between the Sertoli and germ cells. An anatomical dissimilarity between type-A and type-B Sertoli cells appeared in the apical region, with the apical club-like processes of the type-B Sertoli cell given off by sheet-like processes as well as by the Sertoli column. At the base of all Sertoli cells, sophisticated sheet-like processes paralleled the basal lamina, tenaciously adhering end-to-end to the sheet-like processes of adjacent Sertoli cells. Mid-column involvement between the Sertoli cell and germ cells appeared somewhat less cohesive than that in the basal and apical regions.

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The kinetics of rat macrophage proliferation in the inflamed pancreas was analysed using a duct-ligation pancreatitis model. We performed a double immunostaining of pancreatic cryosections using a panel of monoclonal antibodies to either macrophage-specific (ED1, ED2) or macrophage-related (CR3 and Ia) antigens in combination with a cell marker of DNA synthesis (5-bromo-2′-deoxyuridine, BrdU). One hour labeling with BrdU revealed each recorded macrophage phenotype to have a very high labeling index (12-28%), peaking on day 2 after induction of pancreatitis. The percentage of cach proliferating phenotype also reached 20-40% of the total BrdU⁺ cells on day 2. The proliferating macrophages consisted of heterogeneous subpopuations including monocyte-like cells and resident macrophages. Their growth occurred in a relatively synchronized fashion, and seemed to be triggered by common proliferative signals.

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Our previous study demonstrated that in the small intestine of guinea pigs, apoptotic epithelial cells at the villus tips were phagocytosed by lamina propria macrophages, leaving only apical cytoplasmic plates, which thereafter were domed and extruded into the lumen. This finding contrasts with the generally accepted view that effete epithelial cells are simply exfoliated into the lumen. In order to explain this discrepancy, the present study examined luminal cell elements of the small intestine in the guinea pig, rat and mouse; the latter two have been favored species for studying the kinetics of intestinal cells.
Light and electron microscopic observations indicated that the luminal fluid of the guinea pig contained numerous cytoplasmic fragments covered with long microvilli and not containing a nucleus; these fragments corresponded with the apical cytoplasm of apoptotic epithelial cells. In the rat and mouse, in contrast, luminal cell elements were represented by round cell bodies possessing a nucleus and microvillous border; the nucleus displayed compaction and segregation of chromatin at the periphery, a microscopic figure characteristic of apoptosis.
As far as the rat and mouse are concerned, the present findings support the accepted view that epithelial cells undergoing apoptosis are exfoliated as total, nucleus-containing cells. In the guinea pig, in contrast, only an apical thin plate of effete cells is shed off, as our previous studies have suggested.

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Vascular architecture in the periodontal ligament of hamster incisors was investigated by use of vascular casts under a scanning electron microscope (SEM). In addition to ordinary nutrient blood vessels, anastomosing vessels of large caliber developed, surrounding the incisor. From their characteristic configuration, these vessels were regarded as “sinusoids”. The plexus of sinusoidal vessels was connected with capillaries in the papillary layer of the enamel organ at the labial periodontal ligament, and with veins penetrating into the alveolar bone on the lingual side. Transmission electron microscopic (TEM) observation showed that the sinusoidal wall was composed of only a thin layer of endothelial cells, lacking a smooth muscular element, and surrounded by densely arranged collagen fibers. Although the frequent association of Ruffini-type nerve endings with siunsoidal vessels was noted, neither direct contact nor specialized structures between these was recognizable. A possible function of the periodontal sinusoids is discussed on the basis of their distribution and ultrastructural evidence.

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Glycoconjugates were histochemically studied in the distal colon of developing ICR mice in view of the presence of goblet and vacuolated cells. Alcian blue, high iron diamine and periodic acid-Schiff stainings were performed to characterize glycoconjugates. In addition, two lectins, Ulex europeus agglutinin I and Limax flavus agglutinin, were applied to detect fucosyl and sialyl residues, respectively. The reactivities to these stainings, noted from day 18 of gestation, did not seem to undergo any major change throughout their development. The present results suggest that: 1) goblet cells secrete sulfated glycoconjugates containing fucosyl and sialyl residues as terminal sugars; 2) vacuolated cells have glycoconjugates containing sialyl residues but no or few fucosyl residues nor sulfonic groups, and certain sialyl residues in the glycoconjugates are probably O-acetylated or O-acylated at least in part; and 3) the brush border of absorptive cells contains glycoconjugates sialylated and fucosylated to some extent but rarely sulfated.
Since glycoconjugates elaborated by goblet and vacuolated cells differ from each other, one should be fully aware of the presence of these two types of mucinproducing cells in the distal colon.

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This report documents ultrastructural changes in the postacrosomal region of the hamster and mouse sperm head at the initial stage of gamete interaction in vivo. Prominent structures were sequentially visualized: first, the periodic substructure crossbridging the postacrosomal sheath to the overlying plasma membrane, and then the small, electron-dense, granular structures lining the outer surface of the postacrosomal sheath. The periodic substructure became visible at the restricted region where the sperm plasma membrane was just prior to or in the act of detaching from the periodic substructure. The granular substances lined up along the external face of the postacrosomal sheath immediately after the detachment of the sperm plasma membrane, but before the complete degradation of the periodic substructure. These structural changes were completed before sperm nuclear decondensation started. The region where the granular structures were visualized was close to that of the antigen recognized by the monoclonal antibody MN13, which is supposed to be involved in the association of the periodic substructure with the overlying plasma membrane (TOSHIMORI et al., 1991). Based upon these results, we present the progress of events at the initial stage of gamete interaction.

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