Ultrastructural immunocytochemical study of the massa caudalis of the subcommissural organ-Reissner's fiber complex in lamprey larvae (Geotria australis): Evidence for a terminal vascular route of secretory material

Summary The massa caudalis of the subcommissural organ-Reissner's fiber complex of lamprey larvae (Geotria australis) was studied immunocytochemically at the ultrastructural level by use of the immunoperoxidase-silver methenamine procedure. An antiserum raised against bovine Reissner's fiber was utilized as primary antibody.The caudalmost portion of the central canal and its ampulla caudalis communicate, via wide intercellular spaces in their dorsal wall, with large cavities or lacunae. In addition, distinct openings in the dorsal wall of the ampulla establish an open communication between the latter and the lacunae. The lacunae are lined by slender processes of cells of unknown nature. No junctional complexes can be observed between these cells, which lack a basal lamina. The lacunae communicate with structures resembling blood capillaries, however, they are devoid of a basal lamina. These peculiar vessels, in turn, are in direct communication with characteristic blood capillaries.Reissner's fiber (RF) and its massa caudalis are strongly immunoreactive with the antiserum used. The wide intercellular spaces in the dorsal wall of the central canal and the ampulla, as well as the lumina of the (i) lacunae, (ii) modified vessels and (iii) blood capillaries are filled with a flocculent, strongly immunoreactive material. No immunoreactive material was found outside these structures. Thus, the blood capillaries appear to represent the only final target of RF-material arriving at the ampulla caudalis.Supported by Grant I 38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grant S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile, and Grant 6027 from Fondo Nacional de Desarrollo Científico y Tecnológico, Chile. The authors express their gratitude to Mrs. Elizabeth Santibáñez and Mr. Julio Lamilla for providing the lamprey larvae and to Mr. Humberto Molina for preparing the three-dimensional drawing     

Colloid droplets in the magnocellular secretory neurons of the reptilian hypothalamus: An immunocytochemical and lectin-histochemical study

Summary The immunocytochemical and lectin-binding properties of the magnocellular neurosecretory neurons in the hypothalamus of 2 reptilian species, the snake Natrix maura and the lizard Liolaemus cyanogaster, were investigated. Particular attention was paid to the secretory droplets present in these neurons. Antisera against bovine neurophysins I+II, arginine-vasotocin, and mesotocin were used. The following lectins were applied: concanavalin A (Con A), wheat-germ agglutinin (WGA), and Limax flavus agglutinin (LFA). Adjacent 1-m-thick methacrylate sections were used to investigate the same secretory neuron and the same colloid droplets with all three antisera and all three lectins. Several sections were treated with trypsin and urea before immunostaining or lectin binding. Con A bound to both vasotocin- and mesotocin-immunoreactive neurons, WGA exclusively to vasotocin neurons; neither of these neurons reacted with LFA. The colloid droplets were present in vasotocin neurons but absent in the mesotocin neurons. These secretory droplets showed an affinity for Con A but not for WGA, and reacted with antisera against neurophysins and vasotocin. In Natrix maura, the colloid droplets became reactive with Con A and the antisera used only after pretreatment of the sections with trypsin and urea. Within the hypothalamo-neurohypophyseal system, antiserum against vasotocin and WGA revealed the same fiber bundles. It is concluded (i) that in reptiles the vasotocin-neurophysin precursor is glycosylated, (ii) that vasotocin neurons have the exclusive capacity to form colloid droplets, and (iii) that these droplets are an intracisternal (RER) storage form of the vasotocin-neurophysin precursor.This work was partially supported by Grants BOJA 27/9/88 from the Dirección General de Universidades e Investigación de Junta de Andalucía and DGICYT PB87-0710 from the Comisión Interministerial de Ciencia y Tecnología, Madrid, to P.F.-LL.; and Grant 89-01 from the Dirección de Investigaciones, Universidad Austral de Chile, to E.M.R.     

Complex-type glycoproteins synthesized in the subcommissural organ of mammals

Summary The secretory activity in the subcommissural organ (SCO) of the sheep and cow was examined by means of lectin histochemistry and cytochemistry. Among the various lectins tested, Concanavalin A (Con A) revealed glycoproteins rich in mannosyl residues in the rough endoplasmic reticulum of ependymal and hypendymal cells. One of these Con A-positive glycoproteins may represent the precursor of the specific secretory component elaborated in the SCO, giving rise to Reissner's fiber. Lens culinaris agglutinin (LCA) and Phaseolus vulgaris hemagglutinins (E-PHA and L-PHA), known to bind to oligosaccharides, as well as wheat-germ agglutinin (WGA) revealing neuraminic acid, labeled secretory granules located in the apical part of ependymal and hypendymal cells of ruminants, and also Reissner's fiber. Electron-microscopic visualization of WGA-positive material in the Golgi complex shows that complex-type glycoproteins are synthesized in the subcommissural organ of mammals. The electron-dense material is mainly secreted into the ventricular cavity and gives rise to Reissner's fiber. On the basis of lectin affinity for oligosaccharides, a structure of the complex-type oligosaccharide is proposed.     

Secretory glycoproteins of the rat subcommissural organ are N-linked complex-type glycoproteins. Demonstration by combined use of lectins and specific glycosidases,and by the administration of tunicamycin

Summary Two experimental protocols were used to investigate the secretory glycoproteins of the subcommissural organ (SCO). Protocol I: Lectins, specific exoglycosidases and immunocytochemistry were sequentially applied to the same section or to adjacent semithin sections of the rat SCO fixed in Bouin's fluid and embedded in methacrylate. Lectins used: concanavalin A (con A), wheat germ agglutinin, Limulus polyphemus agglutinin, Ricinus communis agglutinin and Arachis hypogeae agglutinin. Glycosidases used: neuroaminidase, -galactosidase, -mannosidase, -glucosidase and -N-acetyl-glucosaminidase. For immunocytochemistry an antiserum against bovine Reissner's fiber (AFRU) was used. Lectins and glycosidases were used in sequences that allowed the cleaved sugar residue to be identified as well as that appearing exposed as a terminal residue. This approach led to the following conclusions: (1) the terminal sugar chain of the secreted glycoproteins has the sequence sialic acid-galactose-glucosamine-; (2) the con A-binding material present in the rough endoplasmic reticulum corresponds to mannose; (3) the apical secretory granules and Reissner's fibers displayed a strong con A affinity after removing sialic acid, thus indicating the presence of internal mannosyl residues in the secreted material; (4) after removing most of the sugar moieties the secretory material continued to be strongly immunoreactive with AFRU. Protocol II: Rats were injected into the lateral ventricle with Tunicamycin and killed 12, 24, 50 and 60 h after the injection. The SCO of rats from the last two groups showed a complete absence of con A binding sites. The results from the two experiments confirm that the secretory glycoproteins of the rat SCO are N-linked complex-type glycoproteins with the conformation previously suggested (Rodríguez et al. 1986).Supported by Grant I/63-476 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grant S-89-01 from the Dirección de Ivestigaciones, Universidad Austral de Chile, and Grant 0890/88 from FONDECYT, Chile     

Comparative immunocytochemical study of the subcommissural organ

Summary The subcommissural organs (SCO) of 76 specimens belonging to 25 vertebrate species (amphibians, reptiles, birds, mammals) were studied by use of the immunoperoxidase procedure. The primary antiserum was obtained by immunizing rabbits with bovine Reissner's fiber (RF) extracted in a medium containing EDTA, DTT and urea. Antiserum against an aqueous extract of RF was also produced. The presence of immunoreactive material in cell processes and endings was regarded as an indication of a possible route of passage. Special attention was paid to the relative development of the ventricular, leptomeningeal and vascular pathways established by immunoreactive structures.The SCO of submammalian species is characterized by (i) a conspicuous leptomeningeal connection established by ependymal cells, (ii) scarce or missing hypendymal cells, and (iii) a population of ependymal cells establishing close spatial contacts with blood vessels.The SCO of most mammalian species displays the following features: (i) ependymal cells lacking immunoreactive long basal processes, (ii) hypendymal secretory cells occurring either in a scattered arrangement or forming clusters, (iii) an occasional leptomeningeal connection provided by hypendymal cells, and (iv) in certain species numerous contacts of secretory cells with blood vessels. In the hedgehog immunoreactive material was missing in the ependymal formation of the SCO, but present in hypendymal cells and in the choroid plexuses. The SCO of several species of New-and Old-World monkeys displayed immunoreactive material, whereas that of anthropoid apes (chimpanzee, orangutan) and man was completely negative with the antisera used.Supported by Grant I/38 259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RR-82-18 from the Dirección de Investigaciones, Universidad Austral de Chile.The authors wish to thank Mrs. Elizabeth Santibánez and Mr. Genaro Alvial for valuable technical cooperation, and Dr. P. Fernandez-Llebrez, University of Malaga, for providing the specimens of Natrix maura.     

Ontogenetical development of the chick and duck subcommissural organ

Summary The ontogenetical development of the subcommissural organ (SCO) was investigated in chick embryos collected daily from the 1st to the 21st day of incubation. Some duck embryos, and adult chickens and ducks were also studied. Immunocytochemistry using an anti-Reissner's fiber (RF) serum as the primary antibody was the principal method used.In the chick embryos the events occurring at different days of incubation were: day 3 morphologically undifferentiated cells in the dorsal diencephalon displayed immunoreactive material (IRM); days 4 to 6 immunoreactive cells proliferated, formed a multilayered structure and developed processes which traversed the growing posterior commissure and ended at the brain surface; day 7 i) blood vessels penetrated the SCO, ii) scarce hypendymal cells appeared, iii) the first signs of ventricular release of IRM were noticed, iv) appearance of IRM bound to cells of the floor of the Sylvius aqueduct; day 7 to 10 the number of apical granules and amount of extracellular IRM increased progressively; day 11 RF was observed along the Sylvian aqueduct; day 12 RF was present in the lumbar spinal cord; day 13 IRM on the aqueductal floor disappeared; days 10 to 21 i) hypendymal cells proliferated, developed processes and migrated dorsally, ii) ependymal processes elongated and their endings covered the external limiting membrane. In adult specimens the ependymal cells lacked basal processes and the external membrane was contacted by hypendymal cells. The duck SCO appears to follow a similar pattern of development.Supported by Grant I/60 935 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidad Austral de Chile. M.H. was recipient of a personal grant from JNO (29-5-54), which is gratefully acknowledged     

The complex-type glycoprotein secreted by the bovine subcommissural organ: an immunological study using C1B8A8 monoclonal antibody

Summary The secretory pathway of the complex-type glycoprotein specific to the subcommissural organ (SCO) was examined using the monoclonal antibody (Mab) C₁B₈A₈. Immunoreactive material was revealed in various compartments of the secretory ependymocyte, i.e., the endoplasmic reticulum, the Golgi area and the secretory vacuoles. In addition, immunoreactive material was also observed in the ventricular cavity. Evidence of a release both at the apical lining and at the basal process of the SCO ependymocytes suggests that the same protein could be secreted into the cerebrospinal fluid and the perivascular spaces. After immunoaffinity chromatography of soluble extracts of the SCO on Mab C₁B₈A₈ immunoadsorbent columns, three glycopeptides were identified on Western blots; they were concanavalin A (Con A)-positive (88, 54 and 34 kDa) and wheat-germ agglutinin (WGA)-positive (54 and 34 kDa). The Con A-positive glycopeptide (88 kDa) is probably related to the high-mannose-type glycoprotein, the precursor form of the secreted compound, whereas the 54 kDa-glycopeptide that is both Con A- and WGA-positive could represent an intermediate form. The 34 kDa-glycopeptide that is strongly WGA-positive could be related to the monomeric form of the secreted compound. These three glycopeptides were not revealed in eluted fractions of soluble extracts of the ependyma that served as control.     

Spatial and structural interrelationships between secretory cells of the subcommissural organ and blood vessels

Summary In 76 specimens (amphibians, reptilians, mammals) belonging to 25 different vertebrate species, the region of the subcommissural organ (SCO) was investigated with the use of a primary antiserum raised against an extract of bovine Reissner's fiber+the immunoperoxidase procedure according to Sternberger et al. (1970).In the SCO of a toad (Bufo arenarum) and several species of reptiles (lacertilians, ophidians, crocodilians), the ependymal cells were the only type of secretory cell displaying vascular contacts, whereas in mammals ependymal and hypendymal cells established intimate spatial contacts with blood vessels. In Bufo arenarum, but especially in the reptilian species examined, the ependymo-vascular relationship was exerted by a population of ependymal cells having a rather constant location within the SCO and projecting to capillaries that showed a remarkably constant pattern of anatomical distribution. In the SCO of mammals the modality and degree of the structural relationships between secretory cells and blood vessels varied greatly from species to species. In the SCO of the armadillo and dog the secretory tissue was organized as a thick, highly vascularized layer with most of the cells oriented toward the capillaries. A rather opposite situation was found in the SCO of New-and Old-World monkeys, where vascular contacts were restricted to a few ependymal cells.Supported by Grant I/38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidal Austral de Chile     

The caudal spinal cord of coho salmon (Oncorhynchus kisutch): Immunocytochemical evidence of a “caudal serotoninergic system”

Summary The caudal spinal cord of the coho salmon was investigated by means of immunocytochemistry using antisera against serotonin, urotensin I, urotensin II, somatostatin and a urea-extract of bovine Reissner's fiber (AFRU). Populations of serotonin-immunoreactive (IR) neurons were found rostral and dorsal to the urophysis in close spatial association with caudal secretory neurons. Thick, smooth serotonin-IR processes extended toward the external surface of the spinal cord where they displayed conspicuous terminal dilatations. Thin, beaded serotonin-IR fibers appeared to innervate populations of caudal secretory and somatostatin-IR cerebrospinal fluid-contacting neurons. Most caudal neurosecretory cells displayed both urotensin I and urotensin II immunoreactivities; only a minority reacted exclusively with either urotensin I or urotensin II antisera. Urotensin II-IR and somatostatin-IR cerebrospinal fluid (CSF)-contacting neurons were found as an integral component of the central canal wall in the caudal spinal cord and filum terminale; their dendritic processes appeared to contact Reissner's fiber, which displayed a weak AFRU-immunoreactivity while inside the central canal, but became strongly reactive in the interior of the terminal ventricle as it formed the massa caudalis. The distribution of serotoninergic processes points to a regulatory role in the function of caudal secretory and CSF-contacting neurons and to a putative serotonin release into the subarachnoid space and/or meningeal vasculature. It is also suggested that the CSF-contacting neurons of the central canal may participate in a feedback mechanism controlling the secretory activity of the subcommissural organ.Supported by Grant A/1095-1 from the International Foundation for Science, Sweden, to C.Y.; Grant I/63-476 from Volkswagen-Stiftung to E.R.; and Grant S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile     

An ultrastructural search for lectin-binding sites on surfaces of spinach leaf organelles

Organelles isolated from leaves of spinach (Spinacia oleracea L.) were prefixed in glutaraldehyde and then incubated with ferritin conjugates of four lectins — Concanavalin A (Con A), Ricinus communis L. agglutinin, MW 120,000 (RCA), soybean agglutinin (SBA), and wheat germ agglutinin (WGA) — in order to probe their cytoplasmic surfaces for saccharide residues. In each case the major leaf organelles, including microbodies, mitochondria and chloroplast derivatives, failed to exhibit labeling when examined with the electron microscope. Tobacco (Nicotiana tabacum L.) leaf protoplasts, incubated simultaneously with and under identical conditions to the spinach organelles, showed specific labeling of their plasma membranes with all four lectin conjugates, thus establishing the efficacy of the procedure for demonstrating the presence of binding sites when they exist. Further attempts to show binding of one of the lectins, Con A, by labeling with fluorescein-Con A and by organelle agglutination, yielded results consistent with the absence of ultrastructural labeling. It is concluded that no saccharide residues recognized by the four lectins are present on the cytoplasmic surfaces of organelles and that those residues reported to be constituents of intracellular membranes, therefore, are most likely exposed on the luminal (extracytoplasmic) surfaces.Abbreviations Con A Concanavalin A - RCA Ricinus communis agglutinin, MW 120,000 - SBA soybean agglutinin - WGA wheat germ agglutinin     

Light- and electron-microscopic investigation of the rat subcommissural organ grafted under the kidney capsule,with particular reference to immunocytochemistry and lectin histochemistry

Summary There is increasing evidence that, in the rat, a serotonin-mediated neural input may have an inhibitory influence on the secretory activity of the subcommissural organ (SCO). In the present investigation the rat SCO was studied 7, 30 and 90 days after transplantation under the kidney capsule, an area devoid of local serotonin-containing nerves. The grafted tissue was examined by use of immunocytochemistry employing a series of primary antisera, lectin histochemistry and transmission electron microscopy. The grafted SCO survived transplantation and contained, in addition to secretory ependymal and hypendymal SCO-cells, also elements immunoreactive with antisera against glial fibrillary acidic protein or S-100 protein. In transplants, SCO-cells produced a material displaying the characteristic immunocytochemical and lectin-binding properties of SCO-cells observed under in-situ conditions. The ependymal cells lined 1–3 small cavities, which contained secretory material. A fully developed structural equivalent of Reissner's fiber was, however, never found. The immunocytochemical and ultrastructural study of the grafted SCO showed an absence of nerve fibers within the graft and suggested a state of enhanced secretory activity. A network of protruding basal lamina structures connected the secretory cells to the newly formed capillaries revascularizing the SCO. One week after transplantation, long-spacing collagen started to appear in expanded areas of such laminar networks and also in the perivascular space. It is suggested (i) that the formation of long-spacing forms of collagen is triggered by factors provided by the SCO-secretory cells, and (ii) that secretory material of the ependymal and hypendymal cells may reach the reticular extensions of the basal lamina. In contrast to the SCO in situ, the grafted SCO-cells showed a positive immunoreaction for neuron-specific enolase. They became surrounded by a S-100-immunoreactive glial sheath that separated them from other transplanted cell types and the adjacent kidney tissue of the host.Supported by Grant I/63 476 from the Stiftung Volkswagenwerk, Federal Republic of Germany, Grants 187 and 0890/88 from Fondo Nacional de Desarrollo Cientifico y Tecnológico, Chile, and Grant S-85-39 from the Directión de Investigaciones, Universidad Austral de Chile. The authors wish to acknowledge the valuable help of Ms. Elizabeth Santibañez and Mr. Genaro Alvial (Valdivia) and Ms. Inge Lyncker (Giessen)     

Lectin histochemistry of the human fetal subcommissural organ

The subcommissural organ (SCO) of 7 human fetuses, 3 to 6.5 months old, was investigated by means of: (i) immunocytochemistry employing three different antisera against secretory products extracted from the bovine SCO and Reissner's fiber; (ii) lectin binding using concanavalin A (Con A; affinity: mannose, glucose), wheat-germ agglutinin (WGA; affinity: N-acetyl-glucosamine, sialic acid), and Limax flavus agglutinin (LFA; affinity: sialic acid). Sections of bovine SCO were processed simultaneously and examined for comparative purposes. The human fetal SCO displayed lectin-binding properties identical to those in the SCO of other mammals. Thus, Con A-binding sites were restricted to abundant supranuclear structures that most likely corresponded to the rough endoplasmic reticulum, but were missing from granules located in the apical cytoplasm. The latter secretory material was strongly WGA- and LFA-positive and formed a distinct zone in the most apical portion of the ependymal cells. In contrast, this type of reactivity was missing in the adjacent cells of ependyma proper. In the bovine SCO, LFA-positive granules were also aggregated in an apical layer. The secretory material in the bovine SCO, especially its apical granular component, was strongly immunoreactive with the three antisera used; the human fetal SCO, however, lacked this immunoreactivity. It is postulated that the SCO of human fetuses secretes glycoproteins with a carbohydrate chain similar to--and a protein backbone different from--the secretions elaborated by the SCO of other vertebrate species.     

Immunocytochemical and ultrastructural evidence for a neurophysinergic innervation of the subcommissural organ of the snake Natrix maura

Summary The subcommissural organ (SCO) of the snake Natrix maura was studied by use of the immunoperoxidase procedure. Primary antisera against bovine neurophysins (Nps I + II, OXY-Np), oxytocin (OXY), mesotocin (MST), arginine-vasotocin (AVT), somatostatin (SOM), -endorphin (END) and bovine Reissner's fiber were used. A conventional ultrastructural study, with special emphasis on the nerve fibers present in the SCO, was also performed. Nerve fibers containing immunoreactive OXY-Np and MST were seen to reach the SCO. The staining of adjacent sections with the anti-Reissner's fiber serum showed that the OXY-Np- and MST-immunoreactive fibers were distributed among the cell bodies and processes of the ependymal secretory cells. No fibers containing immunoreactive OXY, AVT, SOM or END were found in the SCO. The ultrastructural analysis revealed in the SCO the presence of nerve fibers filled with electron-dense granules, 170–210 nm in diameter. Although a direct apposition between these fibers and the SCO cells was frequently seen, no synaptic differentiations were identified. Structures identical to the Herring bodies (found in the neurohypophysis) were seen in the SCO.This work was partially supported by Grants 1/38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and S-85-39 from the Dirección de Investigaciones, Universidad Austral de Chile, conceded to Esteban M. Rodríguez     

Immunocytochemical detection of Reissner's fiber-like glycoproteins in the subcommissural organ and the floor plate of wildtype and cyclops mutant zebrafish larvae

The subcommissural organ (SCO) and the floor plate (FP) secrete high molecular weight glycoproteins that polymerize in the form of the Reissner's fiber (RF). To study to what extent the absence of the FP affects the expression of these glycoproteins, we have investigated the brain and spinal cord of 48-h and 72-h wildtype and cyclops (cyc) mutant zebrafish larvae by using a polyclonal antiserum against bovine RF. Wildtype larvae showed immunoreactivity in the SCO at the dorsal forebrain-midbrain boundary. In the ventricle, over the SCO surface, thin immunoreactive fibers aggregated into an RF that ran along the third and fourth ventricles and the central canal of the spinal cord until, at its caudal end, the fiber disintegrated and formed a strongly immunoreactive massa caudalis that left the neural tube and invaded the surrounding tissues of the tail fin. The rostral end of the FP, lining the pontine flexure, was also strongly immunoreactive, as was the caudal third of the FP. Cyc mutants showed an immunoreactive SCO and fibrous material in the ventricle, but an RF was missing. There was no label in the ventral midline of the neural tube except in some specimens in which the caudal FP persisted and was immunoreactive. It is concluded that the product of the cyc gene is not required for the expression of SCO glycoproteins but for their polymerization into an RF in the brain ventricles.     

Reissner's fiber and the wall of the central canal in the lumbo-sacral region of the bovine spinal cord

Summary Reissner's fiber (RF) of the subcommissural organ (SCO), the central canal and its bordering structures, and the filum terminale were investigated in the bovine spinal cord by use of transmission electron microscopy, histochemical methods and light-microscopic immunocytochemistry. The primary antisera were raised against the bovine RF, or the SCO proper. Comparative immunocytochemical studies were also performed on the lumbo-sacral region of the rat, rabbit, dog and pig.At all levels of the bovine spinal cord, RF was strongly immunoreactive with both antisera. From cervical to upper sacral levels of the bovine spinal cord there was an increasing number of ependymal cells immunostainable with both antisera. The free surface of the central canal was covered by a layer of immunoreactive material. At sacral levels small subependymal immunoreactive cells were observed. From all these structures sharing the same immunoreactivity, only RF was stained by the paraldehyde-fuchsin and periodicacid-Schiff methods.At the ultrastructural level, ependymal cells with numerous protrusions extending into the central canal were seen in the lower lumbar segments, whereas cells displaying signs of secretory activity were principally found in the ependyma of the upper sacral levels. A few cerebrospinal fluid-contacting neurons were observed at all levels of the spinal cord; they were immunostained with an anti-tubulin serum.The lumbo-sacral segments of the dog, rat and rabbit, either fixed by vascular perfusion or in the same manner as the bovine material, did not show any immunoreactive structure other than RF.The possibilities that the immunoreactive ependymal cells might play a secretory or an absorptive role, or be the result of post-mortem events, are discussed.Supported by Grant I/38259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidad Austral de ChileThe authors wish to thank Dr. Enrique Romeny from the Valdivia abattoir for kindly providing the bovine spinal cords     

Morphology ofEntomophthora muscae protoplasts grownin vitro

Summary Entomophthora muscae (C.) Fres. can be grownin vitro as protoplasts. Light and electron microscopical studies of thein vitro developed protoplasts have demonstrated the absence of an organized wall over the protoplasmic Con A-positive membrane at all stages of growth. The cytological organization is typical of the Entomophthorales with condensed chromatin in the interphase nuclei and small eccentric metaphase spindles. Long strands of endoplasmic reticulum, microubules and vesicles surrounding the plasmalemma may be involved in maintaining the precise shape ofE. muscae protoplast. Starvation of the fungus induces the formation of hyphal bodies after deposition of Con A- and WGA-positive wall material at the plasmalemma surface.Abbreviations Con A concanavalin A - DH Drosophila cell culture medium - FITC fluorescein isothiocyanate - GLEN glucose-lactal-bumin-yeast extract-NaCl culture medium for protoplasts - HBL hyphal body-like protoplasts - MM Mitsuhashi and Maramorosch' insect cell culture medium - PATAg periodic acid-thiocarbohydrazide-silver proteinate technique - PBN phosphate buffer with NaCl - S spherical protoplasts - WGA wheat germ agglutinin     

Pinocytosis and locomotion in amoebae XIV. Demonstration of two different receptor sites on the cell surface ofAmoeba proteus

Summary Two different receptor sites, located on the cell surface ofAmoeba proteus were detected by using fluorescent analog cytochemistry (FAC) and electron microscopy (EM). Bovine serum albumin labeled with fluoresceine-isothiocyanate (FITC-BSA) and unlabeled ferritin bind, in a pH-dependent manner, as cations at the outer filaments of the mucous layer. The anionic receptor sites show a high affinity for Ca-ions which suppress the binding capacity of FITC-BSA and ferritin at low pH-values. The cation receptors obviously play an important role in the initiation of pinocytosis as demonstrated by the internalization, intracellular translocation and sequestration of the FITC-BSA. FITC- or ferritin-labeled concanavalin A (FITC-Con A, ferritin-Con A) bind predominantly in a pH-independent manner at the tips of the outer filaments and the basal zone of the mucous layer. The binding capacity of FITC-Con A is not influenced by external Ca-ions. Other lectins such asDolichos bifloris agglutinin (DBA), peanut agglutinin (PNA),Ricinus communis agglutinin I (RCA I), soybean agglutinin (SBA),Ulex europaeus agglutinin I (UEA I) and wheat germ agglutinin (WGA) are not specifically bound to the cell surface. So far, no experimental evidence has been gathered for the definitive function of a Con-A receptor in the mucos layer ofAmoeba proteus.Abbreviations BSA bovine serum albumin - Con A concanavalin A - CTC chlorotetracycline - DBA Dolichos bifloris agglutinin - DTE dithioeritritol - FITC fluorosceine-isothiocyanate - IEP iso electric point - PIPES 1-4-piperazine-diethane sulfonic acid - PNA peanut agglutinin - RCA I Ricinus communis agglutinin I - SBA soybean agglutinin - Uac uranylacetat - UEA I Ulex europaeus agglutinin I - WGA wheat germ agglutinin     

Light and electron-microscopic immunocytochemistry and lectin histochemistry of the subcommissural organ: Evidence for processing of the secretory material

Summary The subcommissural organ (SCO) of the rat was investigated by use of histochemical and immunocytochemical methods at the light and electron-microscopic levels. Consecutive thin methacrylate sections were stained with the pseudoisocyanin (Psi), immunoperoxidase (IMC; employing an antiserum against Reissner's fiber, AFRU), periodic acid-Schiff (PAS) and periodic acid-silver methenamine (SM) techniques, and reacted with six types of lectins. Psi, SM, concanavalin A (Con A) and IMC were also used for double and triple sequential staining of the same section. Increasing dilutions of AFRU (from 11000 to 1200 000) were used for immunostaining of serial paraffin sections. In addition, ultrastructural localization of (i) Con A-binding sites and (ii) immunoreactive secretory material was performed. Some of these procedures were also applied to the ophidian and canine SCO.Con A-positive, Psi-positive and immunoreactive materials coexisted within the same cisternae of the rough endoplasmic reticulum. The Golgi apparatus lacked Con A-positive and immunoreactive substances. Apical secretory granules and secreted material lying on the surface of the SCO showed (i) the highest affinity for AFRU, but were (ii) Con A-negative, and (iii) wheat-germ agglutinin-, PAS and SM-positive. Reissner's fiber displayed a low affinity for AFRU.It is suggested that the SCO secretes N-linked glycoproteins, the carbohydrate and protein moeities of which undergo (i) a maturation process before being released, and (ii) some kind of modification(s) after their release into the ventricle. The perivascular secretory cells of the dog SCO might secrete a material different from that secreted by the ependymal cells.Supported by Grant I/38 259 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Direction de Investigaciones, Universidad Austral de Chile. The authors wish to thank Mrs. Elizabeth Santibáñez, Mr. Genaro Alvial and Mr. Luis Delannoy (Valdivia), and Mrs. Ragnhild Momberger (Giessen) for valuable technical cooperation     

The terminal organ of the subcommissural complex of chordates: definition and perspectives

The subcommissural organ (SCO) exhibits anatomical characteristics of an endocrine organ: The secretion is released either into the blood (hypendymal capillaries) or the CSF of the 3rd ventricle; excretory ducts are absent; the active secretory activity of the ependymal cells can be regulated by humorally transmitted messages or by neural input. The rate of production of the Reissner's fibre (RF) by the SCO is rather fast, and the secretory material is stored in the ampulla caudalis (AC) and must be continuously discharged accordingly. Structures jointly involved in depletion of the AC and the decomposition and removal of the massa caudalis (MC) are collectively called the terminal organ (TO). The TO of the SCO-complex is formed by an assemblage of different structures in the caudal segment of the spinal cord (neurogenic part) and in the tissues (non-neurogenic part) which encompass this part of the cord. The different parts of the TO are characterized, even at the cellular level, by specializations which support the discharge as well as the dissolution of the material of the MC. The RF may be a detoxicator for the CSF, but also a carrier of hormonally active substances. In this case the TO is a site of release of hormones. The function of the entire complex is still under discussion, particularly its role in endocrine integration.     

Immuno- and lectin-electron-microscopic investigation of the neural lobe of the hypophysis in the snake Natrix maura

Summary By the use of lectin histochemistry, and immunocytochemistry with antisera against bovine neurophysins I and II (NPs), arginine vasotocin (AVT) and mesotocin (MST), the neural lobe of the hypophysis in the snake Natrix maura was investigated at the light- and electron-microscopic levels. While paraldehyde-fuchsin stained virtually all neurosecretory endings, the periodic acid-Schiff reaction revealed only a portion of these elements. Furthermore, concanavalin-A and wheat-germ agglutinin lectins reacted with some but not all terminals. While in electron micrographs lectin-positive neurosecretory endings displayed medium-sized, pale neurosecretory granules, those from lectinnegative endings were larger and denser. The antiserum against the two NPs revealed the entire population of neurosecretory endings. The antiserum to AVT stained more numerous fiber elements than the antiserum to MST. Ultrastructurally, correlations concerning size and electron density can be found, on the one hand, between AVT-immunoreactive and lectin-positive neurosecretory granules and, on the other hand, between MST-immunoreactive and lectinnegative granules. The use of immuno-electron microscopy for the characterization of the different endings in the neural lobe and the presence of carbohydrates in some of them is discussed.This work was supported by the Directión General de Universidades e Investigación de la Junta de Andalucía (Grant BOJA 27/9/88) and the Direction General de Investigación Científica y Técnica (DGICYT Grant PB87 0710) Espaa