Fine structural cytology of the adenohypophysis in rat and man

The present review deals with the use of electron microscopy in the identification of pituitary cell types as well as the assessment of their functional state, in rat and man. Application of immunoelectron microscopy, especially immunogold techniques, utilizing multiple labeling in establishing differentiation and hormone content of cell types, is emphasized. Recent evidence of plurihormonality in various pituitary cell types indicates that the once axiomatic one cell-one hormone theory is untenable and that the present perception of pituitary cell types and their function requires modification. Detection of hormonal and nonhormonal substances in pituitary cell types, not associated with their known endocrine function, suggests that hypophysial cells may have yet unknown roles, possibly in the realm of paracrine and autocrine regulation.     

Preparation of carbon films from evaporated fibers and rods at fore-vacuum pressure

Fore-vacuum (pressure ≥ 3 Pa) evaporation of carbon fibers and rods to thin films has been tested and the resulting thicknesses recorded. A modified sputter coater was used as a vacuum evaporator. The quality of the carbon films was evaluated by bright-field and dark-field electron microscopy. Although high-vacuum-evaporated carbon films are superior in quality, low-vacuum-evaporated carbon films were found fully acceptable for routine work in bright field, for both TEM and SEM purposes. Apart from being time-saving, the method presented has the obvious advantage, in SEM preparations, that carbon coating and metal sputtering can be carried out in the same unit without breaking the vacuum.     

Morphology of the minipig kidney

The minipig has a multilobar kidney with a wide cortex and short papilla. The vascular bundles are of a simple type. Although short and long looped nephrons are both present, the short looped kind predominates. The minipig has many morphological similarities to dog and human kidneys. One particularly unique feature of the minipig papillary collecting duct cells, however, is the presence of electron-dense granules in the basal cytoplasm which appear to be secreted into the lateral intercellular spaces, perhaps forming a water-tight seal in a manner analogous to membrane-coating granules found in the epidermis of skin.     

Immunohistochemical and histochemical findings favoring the occurrence of autocrine/paracrine as well as nerve-related cholinergic effects in chronic painful patellar tendon tendinosis

The pathogenesis of the pain in patellar tendon tendinosis ("jumper's knee") is unclear. We have recently presented new information about the sensory nervous system in the human patellar tendon, but there is very little information regarding the possible occurrence of a cholinergic system in this tendon. In the present study, specimens of pain-free normal tendons and chronically painful tendinosis tendons were examined by different immunohistochemical and histochemical methods. Antibodies against the M(2) receptor, choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT) were applied, and staining for demonstration of activity of acetylcholinesterase (AChE) was also utilized. It was found that immunoreactions for the M(2) receptor could be detected intracellularly in both blood vessel cells and tenocytes, especially in tendinosis specimens. Furthermore, in the tendinosis specimens, some tenocytes were seen to exhibit immunoreaction for ChAT and VAChT. AChE reactions were seen in fine nerve fibers associated with small blood vessels in both the normal control tendons and the tendinosis tendons. The observations suggest that there is both a nerve related and a local cholinergic system in the human patellar tendon. As ChAT and VAChT immunoreactions were detected in tenocytes of tendinosis tendons, these cells might be a source of local acetylcholine (Ach) production. As both tenocytes and blood vessel cells were found to exhibit immunoreactions for the M(2) receptor, it is likely that both of these tissue cells may be influenced by ACh. Thus, in conclusion, there appears to be an upregulation of the cholinergic system, and an occurrence of autocrine/paracrine effects in this system, in the tendinosis patellar tendon.     

Scanning electron microscopy of nerve fibers in human fetal cochlea

The cochleas of four human fetuses ranging 22–25 weeks gestation were studied by scanning electron microscopy (SEM) for the purpose of obtaining a better understanding of the nerve fiber arrangement in the human ear. After critical point drying, the specimens were dissected and the floor of the tunnel of Corti and the outer wall of Nuel's space were exposed for observation. Upper cochlear turns, especially the apical turn, seemed to be still immature. Observed nerve fibers were classified into three types:

• 1 Spiral fibers: Fibers traveling basalward and following the shape of the cochlea were found in both the tunnel of Corti and Nuel's space and believed to be the afferent nerves responsible for innervating the outer hair cells
• 2 Radial fibers: radiating outward from the osseous spiral lamina—one such radial fiber transversing high in the tunnel space (supposedly the efferent nerve servicing the outer hair cells), and another sort of radial fiber (found crossing the tunnel floor), the nature of which was uncertain.
• 3 Irregular fibers: Consisting of thin, randomly running fibers within the cochlea. The destination of these fibers was not determined, but possibly they represent transitory nerve branchings of afferent or more probably efferent nerves, which would later regress during maturation.

     

Ultrastructural demonstration of exocytosis in the intact rat adrenal medulla

Evidence is presented for morphological proof of exocytosis in the rat adrenal medulla in situ. Techniques were modified to allow perfusion of the intact adrenal gland with secretagogues (or electrical stimulation) followed by tannic acid. Unstimulated specimens demonstrated exocytotic (omega-shaped) profiles filled with flocculent material. This flocculation was also seen in the intercellular space. Stimulation of the adrenal medulla also resulted in the appearance of exocytotic profiles and an accumulation of the flocculent mass. This was often most evident in the subendothelial space. This is the first demonstration of exocytosis in the rat adrenal medulla by electron microscopy. The techniques used in this study will be useful for studying the pathway of secretory products of the adrenal chromaffin cell before they enter the vascular system.     

Protozoal infections in the acquired immunodeficiency syndrome

Several protozoa have emerged as the major opportunistic infections and cause of death in patients with acquired immunodeficiency syndrome (AIDS). Pneumocystis carinii pneumonia is the leading cause of death in AIDS patients. Electron microscopy (EM) usually shows numerous trophozoites and cysts of Pneumocystis filling up the entire alveolar space, while only cysts are seen under the light microscope. The focal thickening of cyst wall of Pneumocystis, as demonstrated by EM and manifested as a “parentheses” shaped structure with silver stain, serves as a diagnostic marker for Pneumocystis. Freeze-fracture EM has demonstrated the intimate contact between Pneumocystis trophozoites and the type I pneumocytes, which may contribute to the alveolar-capillary block, leading to severe respiratory distress. However, EM is seldom needed for the diagnosis of this infection. Toxoplasma encephalitis, which is an unusual clinical manifestation in cases of toxoplasmosis reported previously, has become a common complication and one of the major causes of death in patients with AIDS. Because subclinical infection by Toxoplasma is common, serologic tests usually offer no definite answers as to whether the infection is acute or chronic, active or past. The small size and its non-specificity in both morphology and tissue affinity make light microscopic diagnosis of toxoplasmosis difficult. Only immunologic staining, such as immunoperoxidase and immunofluorescence, can help to achieve a definite positive identification of the organism. When special antibodies or facility for such staining is not available, EM is the final resort for identifying Toxoplasma by showing the apical complex with the characteristic sausageshaped rhoptries. Cryptosporidiosis, practically unknown before the AIDS outbreak, has become one of the most common intestinal protozoa in both immunocompromised and immunocompetent patients. The protracted and sometimes fatal course of cryptosporidiosis in immunocompromised patients can be explained by the presence of autoinfective oocysts (thin-walled oocysts), as detected by EM, and by recycling of first-generation schizonts observed experimentally. While diagnosis of cryptosporidiosis can be made by detection of oocysts in stools in most cases, EM is still the last resort for a definitive identification of Cryptosporidium species. While the incidence of isosporiasis is still low, it has been found more frequently in patients with AIDS than in the general population. The parasite, Isospora belli, being a coccidian as is the Cryptosporidium species, is similar to the latter in its life cycle and clinical manifestations. However, the morphology of its diagnostic stage, the oocyst, is quite different from Cryptosporidium and it is much larger than the latter. The oocyst of Isospora belli, usually containing one sporoblast, can be detected by light microscopy in stools. Microsporidiosis, having been known only recently, is also relatively common in immunocompromised patients, including four patients with AIDS. Although this protozoan can be detected by light microscopy and its polar granules, identified by the periodic acid-Schiff or methenamine silver stain, are characteristic, a definitive diagnosis of microsporidiosis still depends on EM.     

A computer program for the systematic sampling of sections in microscopic morphometry

A computer program is described that facilitates systematic and unbiased sampling in morphometry. Input data are coordinates of the four corners of a section as displayed in the position indicators of the microscope stage. Outputs are coordinate values in the form of a regular lattice that describes where to place the stage and perform the sampling on the section. In addition, some other data are provided by the program, such as total area of section, length of sides, etc. The program is written in BASIC but can be easily converted to other computer languages.     

Scanning force microscopy of chromatin fibers in air and in liquid

We have adapted specimen preparation techniques of conventional electron microscopy for visualizing chromatin structures in the scanning force microscope (SFM) in air and in liquid. The beaded substructure of the nucleoprotein filament was obtained after hypotonic lysis of chicken erythrocytes and air drying, whereas supranucleosomal structures were preserved after treatment of cell nuclei with detergent. In the latter case, the nucleosomes were still distinct but appeared more condensed. A modified droplet diffusion-spreading technique of chromatin from Namalwa cells (a human B-lymphoid line) yielded a uniform filamentous morphology and similar fiber appearance. A reversible swelling of spread chromatin was observed upon exposure of air-dried samples to solutions differing in salt concentrations.     

A microcomputer program in “BASIC” for the accurate determination of the height of small particles

A formula is derived to enable the calculation of the true height of an object, such as a shadowed latex bead, from electron micrographs. Knowing only the angle of shadowing and the length of the evaporated shadow, and by substituting these values in the derived formula, a microcomputer may be programmed to carry out the necessary computations. An example of such a microcomputer program is given. The correct determination of the height of particles by electron microscopy using the shadowing technique is one of the most accurate methods available for the determination of small particle height.     

Cholinergic synapses in the central nervous system: Studies of the immunocytochemical localization of choline acetyltransferase

Cholinergic synapses can be identified in immunocytochemical preparations by the use of monoclonal antibodies and specific antisera to choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine (ACh) and a specific marker for cholinergic neurons. Electron microscopic studies demonstrate that the fibers and varicosities observed in light microscopic preparations of many brain regions are small-diameter unmyelinated axons and vesicle-containing boutons. The labeled boutons generally contain clear vesicles and one or more mitochondrial profiles. Many of these boutons form synaptic contacts, and the synapses are frequently of the symmetric type, displaying thin postsynaptic densities and relatively short contact zones. However, ChAT-labeled synapses with asymmetric junctions are also observed, and their frequency varies among different brain regions. Unlabeled dendritic shafts are the most common postsynaptic elements in virtually all regions examined although other neuronal elements, including dendritic spines and neuronal somata, also receive some cholinergic innervation. ChAT-labeled boutons form synaptic contacts with several different types of unlabeled neurons within the same brain region. Such findings are consistent with a generally diffuse pattern of cholinergic innervation in many parts of the central nervous system. Despite many similarities in the characteristics of ChAT-labeled synapses, there appears to be some heterogeneity in the cholinergic innervation within as well as among brain regions. Differences are observed in the sizes of ChAT-immunoreactive boutons, the types of synaptic contacts, and the predominant postsynaptic elements. Thus, the cholinergic system presents interesting challenges for future studies of the morphological organization and related function of cholinergic synapses.     

Molecular genetics and structure of the human immunodeficiency virus

A novel human lymphotropic virus capable of crippling the immune system by infecting and destroying T4 antigen-positive cells is now known to be the etiologic agent of the acquired immune deficiency syndrome (AIDS). The AIDS or human immunodeficiency virus (HIV) belongs to a family of RNA viruses called retroviruses. Several strains of HIV have been molecularly cloned, and DNA sequence comparisons have established that the proviral DNA genome is 9.7 kilobase pairs. The genome possesses characteristic retrovirus features including structural genes, flanked by long terminal repeats, in the order gag, pol, and env and, in addition, four unique nonstructural genes, several of which appear to be essential in regulating virus replication. Electron microscopy has played an important role in elucidating structural, genetic, and molecular properties of HIV and has aided in its classification as a member of the Lentivirnae retrovirus subfamily. Heteroduplex mapping methodologies pertinent to these findings are described. Although the relationships show considerable divergence, the similarities between HIV and lentiviruses are profound and encompass an indistinguishable morphology, genome sequence homology and topography, genomic diversity, and overlapping biology, including a preference for infecting cells of the immune system, a cytopathic effect in vitro, and the ability to produce a persistent, slowly progressing, degenerative disease in vivo. The newest HIV class (HIV-2) has recently been molecularly characterized. HIV-2 also bears all the hallmarks of a lentivirus but is more closely related to simian immunodeficiency viruses than the previously described HIV-1, despite a similar biology. The HIV-lentivirus phylogenetic relationship has broad implications for the AIDS disease process and has given new importance to the study of the natural history and pathogenesis of animal lentiviruses in searching for clues to prevent the spread of AIDS.     

Viral infections in the acquired immunodeficiency syndrome

The following communication is a tripartite synopsis of the role of viral infection in the acquired immunodeficiency syndrome (AIDS). The first section describes the impact of viral opportunistic infection in AIDS; for each virus, clinical presentation and diagnosis, laboratory diagnostic approaches (with emphasis on electron microscopy), and therapeutic interventions attempted to date are discussed. The second segment explores current theories on the pathogenesis of AIDS, and describes diagnostic and therapeutic approaches to the syndrome itself. The final section catalogues ultrastructural anomalies in the cells of AIDS patients, many of which have been mistakenly identified as etiologic agents.     

趋磁细菌AMB-1磁小体纯化及扫描和透视电镜形态观察

培养并收集趋磁细菌AMB-1,采用超声波破碎菌体的方法提取磁小体,并对磁小体进行洗涤、分离、纯化。对趋磁细菌AMB-1,进行扫描电镜观察,同时对趋磁细菌AMB-1和纯化的磁小体进行透射电镜观察。电镜下见,趋磁细菌AMB-1为螺菌,无规则排列;磁小体位于菌细胞内,沿菌体长轴排列成链状。纯化的磁小体为立方-八面体,均匀分散于悬浮液中,表面有完整外膜包裹。     

Ultrastructure of the seminiferous epithelium and intertubular tissue of the human testis

The ultrastructural features of the human testis are reviewed with emphasis upon the process of spermatogenesis and the cytology of the Leydig cells. The seminiferous epithelium is structurally partitioned by the Sertoli cells into basal and adluminal compartments via the specialized tight junctions between the Sertoli cells. Spermatogonia reside in the basal compartment, and, via a series of cell divisions, produce the primary spermatocytes, which at the commencement of their development move into the adluminal compartment, and thus the lengthy process of meiotic maturation is initiated. The fine structure of primary spermatocytes is described together with the complex transformation of the spermatids into spermatozoa during the process of spermiogenesis. Earlier studies of the organization of the human seminiferous epithelium showed that germ cells at different developmental stages formed identifiable collections termed cell associations or stages, but since several stages were seen in a single tubule cross-section, this gave the impression of an extremely irregular pattern of spermatogenic development. When the topographic arrangement of germ cells was re-examined with the aid of computer modelling, a highly ordered distribution was revealed, conforming to a helical pattern based on the geometry of spirals. Thus spermatogenesis in the human testis is subjected to a precise regulation in keeping with the ordered arrangement of the germ cells seen in other mammalian species. The intertubular tissue of the human testis is composed of loose connective tissue containing blood vessels, occasional lymph capillaries, macro-phages, mast cells, and the Leydig cells which occur either as single cells or form small clusters. The Leydig cell cytoplasm contains an abundant supply of smooth endoplasmic reticulum and mitochondria with tubular cristae, both features being characteristic of steroidogenic cells. Human Leydig cells contain large Reinke crystalloids of variable size and number, but their function remains obscure. The frequent occurrence of paracrystalline inclusions within the cytoplasm of the human Leydig cell suggests that these elements are precursors of the Reinke crystalloids.     

A computer program for morphometry of circular and elliptical profiles on the microscope

We describe a short computer program, which is written in Pascal language, to measure the diameter of circular and elliptical profiles in sections. Coordinate pairs on the microscope stage, corresponding to two or three points of a profile, are input to obtain its diameter. The program enables one to take measurements directly on the microscope, thereby reducing photographic work and caliper measurement. While the program is largely designed for electron microscopy, it also may be useful for light microscopy morphometry.     

Contamination of thin sections: Some observations on the cause and elimination of “embedding pepper”

A method is described for eliminating a form of contamination called embedding pepper that appears in, or on, thin sections following poststaining in lead citrate. The pepper is present most often in the matrixes of mitochondria, peroxisomes, and red blood cells. Formation of pepper can be prevented by treating the section with 0.5 N HCl or 1.0% ethylenediaminete-traacetic acid (EDTA) for 1–5 min before staining in uranyl acetate or lead citrate.     

TEM studies of the nitrided Ni-Ti surface layer

The structure of surface layer, obtained on the nearly equiatomic Ni‐Ti alloy after nitriding under glow discharge conditions at temperatures 700 or 800 °C, was investigated. The structural characterization of the intruded layer was performed on cross‐sectional thin foils by the use of the transmission and scanning electron microscopes. The obtained results show that the nitrided layers consist mainly of the nanocrystalline TiN phase and small amount of Ti₂N. Between the nitrided layers and β‐NiTi matrix an intermediate Ti₂Ni phase layer was observed.