Distribution and ontogeny of VIP-like immunoreactivity in the gastro-entero-pancreatic system of a cartilaginous fish Scyliorhinus stellaris

Summary The presence, distribution and development of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the gastro-entero-pancreatic system of a cartilaginous fish Scyliorhinus stellaris (L.) was investigated by immunohistochemical methods utilizing mammalian VIP antisera. In the gut VIP-like immunoreactivity was observed in both nerves and endocrine cells. Endocrine cells with VIP-like material were only detected in the intestinal epithelium while nerve fibres containing VIP-like material were noted along the whole gastro-entero-pancreatic system, being more numerous in the pyloric sphincter and in the intestinal portion. Immunoreactive nerve cell bodies were encountered in the stomach and intestinal portions localized in the submucosa and in the myenteric plexus. Intestinal immunoreactive endocrine cells were already present in the first developmental stage considered (embryos aged 4 months). They grow in number and before birth reach a frequency higher than in adults. Nerves and cell bodies showing VIP-like immunoreactivity, appear later, before birth, as a few elements in the smooth muscular layer, but only after birth their distribution and frequency are similar to those found in adults. The faint immunofluorescence shown by the immunoreactive endocrine cells and their developmental pattern, which is always different from that observed in nervous elements, suggest the presence of at least two VIP-like substances in the gastro-entero-pancreatic system of S. stellaris.     

Light and electron microscopic study on the endocrine cells of the pancreas in a marine teleost,Fugu rubripes rubripes

Summary The pancreatic endocrine tissue of Fugu rubripes rubripes consists of numerous round principal islets (Brockmann bodies) of various sizes scattered around the gall-bladder. The endocrine cells are divided into A-, B-, D-, and F_f-cells. Each cell type was identified by comparing thick and thin sections in both light and electron microscopy. Aldehyde-fuchsin positive B-cells contain numerous round secretory granules (average diameter 300 nm) each of which has a round compact core of moderate density; a narrow space exists between this core and the limiting membrane. Grimelius' silver positive A cells contain round secretory granules (average diameter 360 nm) with a hexagonal or tetragonal crystalline core (average diameter 170 nm) of high density; the silver grains preferentially appear in the space between the limiting membrane and the core. The crystalline core of each -granule often contains an appendix-like structure of variable shape. D cells blackened by the silver impregnation method of Hellman and Hellerström (1960) have round secretory granules (average diameter 320 nm) filled with a flocculent material of low density. The fourth cell type (F_f-cell) has a clear cytoplasm after differential staining for light microscopy. By electron microscopy, this cell has elongated fusiform secretory granules (520 nm average length × 230 nm average width) filled with numerous filaments arranged in parallel with the longitudinal axis. Figures suggesting granule formation in the sacs of the Golgi apparatus were obtained in all of islet cell types. Equivalents of emiocytotic release of secretory granules were encountered in the A and F_f cells.     

Use of immunocytochemical staining of somatostatin for correlative light and electron microscopic investigation of D cells in the pancreatic islet of Xiphophorus helleri H. (Teleostei)

Summary Somatostatin-containing cells have been demonstrated by immunocytochemistry in semithin sections of the pancreatic islet of the teleost fish, Xiphophorus helleri. These cells were shown by correlative light and electron microscopy to be identical with D cells previously defined in this species by the silver impregnation method of Hellman and Hellerström.Supported in part by grants from the British Council and from the Medical Research Council of Great Britain     

Immunocytochemical study of the endocrine pancreas in the grey kangaroo (Macropus fuliginosus)

Summary The endocrine pancreas of the grey kangaroo,Macropus fuliginosus, was investigated by means of immunocytochemistry using the PAP method on the same section at the light- and electron-microscopic levels. Semithin plastic sections were stained individually with primary antibodies for insulin, glucagon, somatostatin and pancreatic polypeptide (PP), and then photographed. Sections were osmicated, re-embedded in BEEM capsules, and ultrathin sections made and examined. The same labelled cells as in the semithin sections were localised in the thin sections, photographs taken and the morphology of secretory granules studied. The insulin cells were pleomorphic; their secretory granules displayed an electron-dense core surrounded by an empty halo. The glucagon cells possessed granules with an electron-dense core usually surrounded by a halo of less dense granular material. Somatostatin cells had larger, less dense secretory granules. The PP cells showed small, dense secretory granules. In order for an ultrastructural study to be considered reliable for the definite identification of endocrine cell types, it is essential that it be corroborted by correlated immunocytochemical data at the light-and electron-microscopic levels.     

Differences in black pigmentation in lepidopteran cuticles as revealed by light and electron microscopy

Summary Black cuticles of larvae and pupae from various Lepidoptera were studied by light and electron microscopy. There are striking differences in the representation of black pigmentation, especially at the ultrastructural level. Two types may be described: 1. With the light microscope black melanin-like grana, electron-dense electron microscopically, are found in the distal parts of the exocuticle. This type is demonstrated in larvae of Celerio euphorbiae, Papilio machaon, and Phalera bucephala. 2. With the light microscope a dark homogeneous layer in the distal exocuticle can be recognized, however, electron microscopically no structures correlated with this dark pigment layer. This type of pigmentation was present in pupae of Pieris brassicae and Aglais urticae; in Pieris larvae the dark pigmented layer appeared to be limited to the epicuticle. In Celerio processes of the epidermal cells are involved in transporting precursors to the exocuticle. The conclusion was reached that black pigmentation in cuticles is based on different mechanisms as proposed by structural features. The two likely mechanisms are melanization and sclerotization.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 87, project A1, granted to Prof. Bückmann)     

Structural analysis of EcoRI-DNA complexes as revealed by electron microscopy

Electron microscopy of negatively stained isolated restriction enzyme EcoRI revealed particle projections with triangular or square outlines, indicating that the enzyme, in its tetrameric state, is tetrahedron-like. The two dimers making up the tetramer appear to be arranged in two planes orthogonal to each other. Complexes formed by EcoRI with the plasmids pBR322 or pGW10 were investigated by electron microscopic spreading techniques. In the presence of Mg²⁺, EcoRI was bound to the DNA molecules to form pearl necklace-like aggregates. The number of bound EcoRI particles was much higher as the sum of EcoRI-and 5..AATT..3 sites (with exceptions, the 5..AATT..3 sites may function as one type of EcoRI^* sites) along the DNAs, indicating unspecific binding. In the absence of Mg²⁺, EcoRI was bound to the DNA only at the recognition site for EcoRI and the sites where the tetranucleotide sequence 5..AATT..3 was present. A direct correlation of the local concentrations of the bases A and T within the flanking sequences of the binding sites with the frequency of EcoRI to the DNA was observed. Dimers and tetramers of the enzyme was found to bind to the DNA. Tetramers occasionally exhibited two binding sites for DNA as indicated by the observation of DNA loops originating at the sites of bound tetrameric EcoRI particles.Abbreviations BAC Benzyldimethylalkylammoniumchloride - bp base pairs - Kb kilobases - SDS sodium dodecylsulfate Enzymes (EC 3.1.23.13) Restrictionendonuclease EcoRI - (EC 3.1.23.21) Restrictionendonuclease HindIII - (EC 3.1.23.37) Restrictionendonuclease SalGI Dedicated Professor H. G. Schlegel on occasion of this 60th birthday     

Mitosis in the dermatophyteMicrosporum canis as revealed by freeze substitution electron microscopy

Summary Mitosis in the dermatophyteMicrosporum canis was studied by freeze substitution and electron microscopy, and analyzed by three dimensional reconstruction from serial sections of the mitotic nuclei. The interphase nucleus has associated nucleus-associated organelle (NAO) on a portion of the outer surface of the nuclear envelope, subjacent to which there was dense intranuclear material. The NAO divided and separated on the envelope, and a spindle was formed. The spindle was composed mostly of microtubules extended between opposite NAOs. Pairing of kinetochores was observed in the spindle from an early stage of development, when chromosomes were not so condensed, and remained unchanged while chromosome condensation proceeded until metaphase. Before the completion of nuclear division, daughter nuclei were connected by a narrow spindle channel, and then the nucleolus, whose structure underwent minimal change during mitosis, was eliminated into the cytoplasm.     

New types of islet cells in a cyclostome,Petromyzon marinus L.

Four types of acidophilic granular cells, in addition to B-cells, are identified in the islet organ of anadromous specimens of two subspecies of Petromyzon marinus by light and electron microscopy. Three of these acidophils (PI, PII and PIV-cells) occur in both the cranial and hepatic islets while a fourth type (PIII-cell) has only been found in the hepatic islet of some animals. The granules of the PI-cells stain with ponceau de xylidine, give a distinct tryptophan reaction and in ultrastructural examination show large, dense granules. The PII-cells contain unusual crystals and appear to be a non-secretory stage of the PI. The PIII-cells stain deep-red and acid fuchsin. They contain very large, dense granules and some lysosomes. PIV-cells stain selectively with phosphotungstic acid-hematoxylin and ultrastructurally, contain small, more or less dense granules. It appears that PI- and PIV-cells develop directly from B-cells, while the PIII-cells derive from PI-cells. despite their direct or indirect origin from B-cells, the PI-, PIII- and PIV-cells show characteristic features of functionally independent endocrine cells. Petromyzon marinus may be an ideal model for the understanding of phylogenetic and pathological interrelationships between islet and gastrointestinal hormones. It is clear that the interpretation of the islet organ of the cyclostomes, which has been generally considered a source of insulin only, requires a revaluation.     

The structure of the gills of the elasmobranch,Scyliorhinus canicula (L.)

Summary The anatomy of the blood supply to the gills of the dogfish, Scyliorhinus canicula, is described. The anatomical basis for a counter-current exchange system at the respiratory surfaces is reported. Within the interbranchial septum there is a capillary network joining all the afferent branchial arterioles of the gill. The structure of the walls of the corpus cavernosum is found to be of smooth muscle cells supported by a basal lamina and connective tissue and lined by endothelial cells containing phagocytic vesicles. Both the capillary network and corpus cavernosum are suggested to function in smoothing the pressure pulses of the blood flow. Pre- and post-lamellar vessels and pre- and post-lamellar sphincters are described. The sphincters are thought to control the number of secondary lamellae physiologically in the respiratory circuit, and by retaining blood within nonperfused lamellae to act in conjunction with pillar cells (contracting in antagonism to the hydrostatic skeleton of the blood) to maintain the rigidity of secondary lamellae in the water current.Whorls of cells of unknown function are found within the interbranchial septum. In the epithelium lining the water channel large cells having a complexly branching plasma membrane and a very large central vacuole occurs. The cytoplasm lining the lumen contains numerous vacuoles each surrounded by a double membrane.This work formed part of a thesis submitted for the degree of Master of Science at the University of Bristol. I should like to thank Professor G.M. Hughes for the use of facilities in the Department of Zoology, University of Bristol.     

Light and electron microscopic investigations of six types of glandular cells of the bovine adenohypophysis

Summary Twelve bovine adenohypophyses were prepared for light and electron microscopy of the cell types of pars distalis. Correlation between the light and electron microscopy was effected by use of alternate thin and thick sections. Cytological changes in the experimental animals were used as criteria for the identification of six different types of secretory cells.Two types of acidophils, alpha and epsilon cells, are recognized in peripheral area of the pars distalis by light and electron microscopy. The alpha cells contain orangeophilic secretory granules of a maximum diameter of 400–450 m and correspond to ordinary acidophils (STH cells). The second type, epsilon cells, contains larger, fuchsinophilic granules of 600 to 900 m in diameter, increase in number and granulation after pregnancy and thyroidectomy, and are thought to be prolactin cells (LTH cells).Two types of amphophils, zeta and delta 1 cells, were found in the central area of the pars distalis. The zeta cells contain smaller numbers of amphophilic, cored granules (200 m maximum diameter) and based on the comparison with literature on other species of animals, are designated as ACTH cells. The delta 1 cells are round or oval and contain very dense, spherical granules (250–300 m) which are stained red or reddish purple with PAS, aldehyde thionin and PAS-methyl blue methods. They show extreme enlargement and bizarre cytoplasmic appearance after castration and are designated tentatively as LH gonadotrophs or LH cells.Two types of basophils, beta and delta 2 cells, were also identified by correlative light and electron microscopy. The beta cells are polygonal in outline, distributed exclusively in the zona tuberalis and contain large, less dense secretory granules (300–400 m) which are stained selectively with Gomori's aldehyde fuchsin. After thyroidectomy, they lose their secretory granules and are transformed into large, vacuolated thyroidectomy cells. They are therefore, identified as thyrotrophs or TSH cells. The delta 2 cells are round, oval or polygonal in shape and contain basophilic granules ranging from 220 to 300 m in diameter. They show extreme enlargement and vacuolization due to the dilation of endoplasmic reticulum, after castration, and are designated tentatively as FSH gonadotrophs or FSH cells.The investigation reported herein was supported by a Scientific Research Grant (No. 291049) from the Ministry of Education of Japan.     

Immunocytochemical localization of vasotocin-like immunoreactivity in the brain of the cartilaginous fish,Scyliorhinus caniculus

Summary The distribution of vasotocin-like peptides in the central nervous system of the cartilaginous fish Scyliorhinus canicula was determined by indirect immunofluorescence and peroxidase anti-peroxidase techniques, using a specific antiserum raised in rabbits against synthetic vasotocin. Immunoreactive perikarya were mainly detected in the anterior hypothalamus, within the midcaudal part of the preoptic nucleus. The most rostral positive cell bodies were located in the dorso-lateral parts of the preoptic area, whereas at a more caudal level, they took a ventro-medial position within the deepest layers of the nucleus. Throughout the preoptic region these cells varied in shape according to their location. Occasionally, scattered vasotocin-like immunopositive cells were also identified in the nucleus periventricularis hypothalami. Vasotocin immunoreactivity was detected in numerous varicose nerve fibers of the preopticohypophysial tract. These fibers were seen to course through the medio-basal hypothalamus and caudally, after having passed the hypophysial stem, they reached the neurointermediate lobe of the pituitary. Numerous immunoreactive fibers were also observed within the rostro-medial region of the median eminence. At this level the fibers were in close proximity to the capillary loops. In the preoptic region, some stained cells exibited short processes that appeared to contact non-reactive perikarya. By comparing the distribution of vasotocin- and corticotropin-releasing factor immunoreactivity on adjacent then serial sections, it was revealed that these peptides, in S. canicula, do not coexist in the same perikarya. The present results, are compared with those obtained in other vertebrate groups, and their possible functional implications are discussed.     

Contrasting pattern of somatic and zygotic embryo development in alfalfa (Medicago sativa L.) as revealed by scanning electron microscopy

Scanning electron microscopy has been used to investigate the morphological changes occurring during the development of alfalfa somatic embryos. Embryos were initiated from callus, transferred to suspension culture and matured on solid agar medium. This developmental pattern was compared to that of zygotic embryos developing in ovulo. Somatic embryos begin as distinct pro-embryos within the callus tissue pieces placed in suspension culture. They become globular and heart-shaped while on solid agar medium and then undergo cotyledon elongation and maturation. Somatic embryos develop comparatively slower at early stages of development and faster at the later stages than zygotic embryos. They lack a well-defined suspensor and have a very rough, poorly-differentiated epidermis, the first layer of which is lost after pro-embryo formation. The cotyledons of somatic embryos are multiple and poorlydeveloped; there appears to be a correlation between the amount of surface roughness of the developing embryo and the extent to which polycotyledony occurs.     

The ultrastructure of the cell types in the endocrine pancreas of the horse

Summary The islets of Langerhans of the equine pancreas were examined with the electron microscope after immersion or perfusion fixation. Five cell types could be distinguished after fixation by either technique: 1. A-cells, situated at the center of the islets, 2. B-cells, containing mostly pale granules and constituting the principal cell type of the periphery of the islets, 3. D-cells, also located mainly at the periphery of the islets, 4. G-cells, found at the edge of the islets and in the exocrine pancreas, and 5. S-cells, (small granule cells), which are relatively few in number and occur only in the islets. The function and age-dependent modifications of these cells are discussed. The formation of light and dark cells and of mixed cells are regarded as artifact, since cells of this type occur only under the condition of immersion fixation.Dedicated to Professor Dr. Drs. h.c. W. Bargmann on the occasion of his 70th birthday.     

The cell cycle of Bacillus subtilis as studied by electron microscopy

Bacillus subtilis strain Marburg was grown exponentially with a doubling time of 65 min. To follow the time course of various cell cycle events, cells were collected by agar filtration and were then classified according to length. The DNA replication cycle was determined by a quantitative analysis of radioautograms of tritiated thymidine pulse labeled cells. The DNA replication period was found to be 45 min. This period is preceded and followed by periods without DNA synthesis of about 10 min.The morphology and segregation of nucleoplasmic bodies was studied in thin sections. B. subtilis contains two sets of genomes. DNA replication and DNA segregation seem to go hand in hand and DNA segregation is completed shortly after termination of DNA replication.Cell division and cell separation were investigated in whole mount preparations (agar filtration) and in thin sections. Cell division starts about 20 min after cell birth; cell separation starts at about 45 min and before completion of the septum.     

Morphology,morphometry and electron microscopy of HeLa cells infected with bovine Mycoplasma

Summary The host-parasite relationship of HeLa M cells artificially infected with a bovine species of Mycoplasma was studied by light microscopy, transmission electron microscopy and scanning electron microscopy. The use of morphometry to quantitate some of the findings was explored. The parasites were seen in locations extracellular to the cell surface. The detection of small numbers of organisms by light microscopy was well demonstrated by use of the fluorescent antibody technique. Scanning electron microscopy proved to be an excellent method for revealing the surface details of cell-parasite morphology. Ultra-thin sections showed that the parasites are aligned mostly parallel to the plasma membrane of the host cell but separated by a gap of 10 nm. Morphometry indicated an average of 69 organisms per cell surface occupying 1.7% of the surface area. An increase of 26% in diameter of the HeLa cells, possibly as a result of infection, was observed.The authors wish to thank Christiana Ulness and Andrea Erickson for expert technical assistance and Arnold Schmidt for the operation of the scanning electron microscope. This work was supported by grants from the U.S.P.H.S.: AI 09586, AI 10743, and AI 06720     

Occurrence of bombesin-like immunoreactivity in the brain of the cartilaginous fish,Scyliorhinus canicula

Summary The presence and distribution of bombesin-like material were investigated in the brain of the cartilaginous fishScyliorhinus canicula using conventional immunocytochemical techniques. Perikarya containing bombesin-like immunoreactivity were identified in the hypothalamus, within the magnocellular component of the preoptic nucleus. Some immunopositive elements appeared to be of cerebrospinal fluid-contacting type. Beaded immunoreactive fibers were seen crossing the ventral telencephalon and the whole hypothalamus. An important tract of fibers was found in the infundibular floor and in the median eminence, in close contact with the vascular system of the pituitary portal plexus. A moderate number of positive fibers innervated the habenular complex and the dorsal wall of the posterior tuberculum. These findings indicate that a neuropeptide strictly related to amphibian bombesin is located in specific hypothalamic neurons ofS. canicula. The distribution of the immunoreactive fibers and terminals suggests that, in fish, this peptide, may be involved in neuroendocrine and neuromodulator functions.     

Localization of corticotropin-releasing hormone (CRF)-like immunoreactivity in the central nervous system of the elasmobranch fish,Scyliorhinus canicula

Summary The occurrence and localization of immunoreactive corticotropin-releasing factor (CRF) in the brain and pituitary of the elasmobranch fish Scyliorhinus canicula, were studied by means of specific radioimmunoassay and immunohistochemistry using the indirect immunofluorescence method. Brain and pituitary extracts showed a good cross-reactivity with the ovine CRF antiserum, but serial dilutions of tissue samples did not completely parallel the standard curve. Relatively high concentrations of CRF-like material were found within the pituitary, diencephalon, and telencephalon. CRF-like immunoreactive perikarya were observed in the preoptic nucleus and in the nucleus lateralis tuberis. Numerous immunoreactive cells appeared to be of the CSF-contacting type. CRF-like immunopositive fibers were seen to run through the hypothalamus within the ventro-medial floor of the infundibular region. A dense plexus of immunoreactive nerve endings terminated in the median eminence and the neurointermediate lobe of the pituitary. These results indicate that a neurosecretory system containing CRF-like immunoreactivity exists in the brain of elasmobranchs, a group of vertebrates which has diverged early from the evolutionary line leading to mammals. In addition, our data support the notion that a CRF-like molecule is involved in the regulation of corticotropic and melanotropic cell activity in this primitive species of fish.     

Principal cell types in the pancreatic islet of a teleost fish,Xiphophorus helleri H.

Summary By means of correlative light and electron microscopy, five pancreatic islet cell categories are described in the teleost fish, Xiphophorus helleri, each of which has specific light microscopic appearance and fine structure. Different histochemical techniques have been used, including immunofluorescence with antiporcine insulin and glucagon sera. In addition to B- and A₁-cells, two categories of A₂-cells have been observed, both reacting with antiporcine glucagon serum: A₂-cells with round granules gave a positive reaction for tryptophan; A₂-cells with crystalline granules gave a negative reaction with the same staining technique on the same section. The clear cells, the last category, were not specifically stained by any of the staining methods carried out in this investigation. The influence of fixation on staining affinities and on ultrastructure was shown to be considerable.Supported in part by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (grant La 229/4) and by the Deutscher Akademischer Austauschdienst, Bonn-Bad Godesberg.     

The crater defect in boar spermatozoa: A correlative study with transmission electron microscopy,scanning electron microscopy,and light microscopy

Nuclear vacuoles resembling the “crater defect” described in bull spermatozoa were observed in 14 boars. Both the incidence of the defect and semen quality were monitored with phase contrast microscopy over a three-month period. The percentages of cratered spermatozoa varied widely both among boars and in ejaculates from the same boar taken on different days. The presence of cratered spermatozoa at a level of 5% or more appeared to be associated with low semen quality. The defect was studied with scanning and transmission electron microscopy and was found to consist of nuclear invaginations, about 0.5 μm in diameter, containing some scanty amorphous electron-dense material. In boars showing a high incidence of spermatozoa with crater defects, abnormalities of the acrosome and perforatorium were common.     

Tachykinin-like immunoreactivity in the sinus venosus of the dogfish (Scyliorhinus canicula)

The sinus venosus of the elasmobranch heart is characterized by the presence of large bundles of unmyelinated nerve fibres that bulge into the cardiac lumen, below the endocardium. In the dogfish (Scyliorhinus canicula), these fibres contain numerous dense-core membrane-bounded granules of about 200 nm in diameter. Most intramural ganglion cells of the sinus venosus also show densely packed granules similar to those found in the subendocardial fibres. We have observed strong substance-P-like immunoreactivity in the large fibre bundles and in the perikarya of the ganglion cells. Preabsorption of the antisera with fragment 7–11 of substance P has shown that the antisera recognize the tachykinin canonic sequence. Our findings suggest that an undetermined tachykinin is secreted in the elasmobranch heart, and that it is probably released into the blood stream in the context of a little-known neuroendocrine system.