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     

Scanning electron microscope observations of the rat subcommissural organ.

Under the scanning electron microscope, the rat subcommissural organ (SCO) appears as an oval zone, rich in kinocilia and well delimited from the non-specific ependymal epithelium. This zone surrounds the cranial and posterior part of the mesencephalic canal's entrance. The ependymal cells of the SCO show coniform processes with microvilli and kinocilia. In contact with the apical pole of the peripheric SCO-ependymocytes lie scarce supraependymal axons. The Reissner's fiber is composed by the twining of the fibrillary structures emerging from the area of the SCO.     

Secretory glycoproteins of the subcommissural organ of the dogfish (Scyliorhinus canicula): evidence for the existence of precursor and processed forms

The probable presence of oxytocin in the hypothalamo-hypophysial system of two reptilian species, the snake Natrix maura and the turtle Mauremys caspica, was re-investigated. A high-pressure liquid chromatographic analysis of the turtle neural lobe revealed the existence of vasotocin, mesotocin, and a third compound co-eluting with oxytocin. Brains from both species were fixed by vascular perfusion with Bouin's fluid. Adjacent paraffin sections were immunostained using antisera against the following substances: (1) bovine oxytocin-neurophysin; (2) a mixture of bovine oxytocin-neurophysin and vasopressin-neurophysin; (3) dogfish neurophysins; (4) oxytocin; (5) arginine-vasotocin; (6) mesotocin; (7) somatostatin. Immunoreactivity against oxytocin was found in parvocellular neurons of the snake suprachiasmatic nucleus and cerebrospinal-fluid contacting neurons of the medial nucleus of the infundibular recess of both species, the latter immunoreactivity being much more conspicuous in the turtle. Numerous fibers containing immunoreactive oxytocin extended between the medial nucleus of the infundibular recess, and the internal region of the medium eminence and the neural lobe. The oxytocin-immunoreactivity in all locations was completely abolished by preabsorption of the anti-oxytocin serum with three different oxytocin preparations. None of the neurons of the suprachiasmatic and medial nucleus of the infundibular recess, including the oxytocin-immunoreactive elements, reacted with either the antineurophysin sera used, or the anti-vasotocin or anti-mesotocin antibodies. The possible existence of a reptilian oxytocin-neurophysin is discussed. The alternative that, in the reptilian hypothalamus, neurons synthesize a compound closely related to, but different from oxytocin is also considered.     

Activity of specific and non-specific cholinesterases in the subcommissural organ of guinea pig and albino rat

Summary The localizations of specific and non-specific cholinesterases were demonstrated by light and electron microscopical methods in the secretory cells of the subcommissural organ of the guinea pig and albino rat.The activity of non-specific cholinesterase at light microscopical level appeared slightly stronger compared to the activity of the specific cholinesterase. No differences in the localizations of the both enzymes could be noticed.In electron microscopic specimens no differences could be observed between the localization or intensity of the specific and non-specific cholinesterase reactions except some nerve fibres round the secretory hypendymal cells in which only a specific cholinesterase reaction product was noticed. The reaction product was found mainly in the cytoplasmic and nuclear membranes, in the rough and smooth surfaced endoplasmic reticulum and around some secretory granules in the ependymal and hypendymal secretory cells of the subcommissural organ in guinea pig and albino rat.The possible role of cholinesterases in the secretory cells of the subcommissural organ is further discussed. Their participation in the metabolism and/or secretion of the hormonal end products is suggested.     

Comparative study of blood group-recognizing lectins toward ABO blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides

Binding specificities of ABO blood group-recognizing lectins toward blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides were studied by lectin-blotting analysis, enzyme linked immunosorbent assay and lectin-conjugated agarose column chromatography. Human serum albumin conjugated with A- and B-trisaccharides was clearly recognized by Helix pomatia (HPA), Phaseolus lunatus, Dolichos biflorus agglutinins, and Griffonia simplicifolia I agglutinin B(4), respectively. Almost the same results were obtained for human group A and B ovarian cyst and A-active hog gastric mucins, but Glycine max agglutinin only reacted to the group A hog mucin. When human plasma von Willebrand factor (vWF), having Asn-linked blood group antigens, was tested, HPA was highly sensitive to blood group A antigen on the vWF. Ulex europaeus agglutinin I (UEA-I) preferentially bound to the vWF from blood group O plasma. Within the GalNAc-recognizing lectins examined, a biantennary complex-type oligosaccharide having the blood group A structure retarded on an HPA-agarose column, and the affinity was diminished after digestion with alpha-N-acetylgalactosaminidase. This product bound to UEA-I agarose column. These results indicate that HPA and UEA-I are most sensitive for detection of glycoproteins possessing small amounts of blood group A and H antigens and also useful for fractionation of complex-type oligosaccharides with blood group A and H antigens, respectively.     

The metencephalic floor plate of chick embryos expresses two secretory glycoproteins homologous with the two glycoproteins secreted by the subcommissural organ

The nature and the function of the compounds secreted by the floor plate (FP) of the metencephalon are little known. The FP cells of the hindbrain react with antibodies (AFRU) against the glycoproteins secreted by the subcommissural organ (SCO). One of the these proteins, RF-Gly I, is a 540-kDa core glycosylated protein. The aims of the present investigation were to identify by immunoblot the AFRU-immunoreactive compound secreted by the FP of chick embryos, to establish temporal and regional patterns of this secretory activity, and to obtain information about the fate of these compounds. It was established that the SCO and FP of chick embryos secrete two AFRU-immunoreactive compounds of 540 and 230 kDa. The two compounds secreted by the FP have been designated as FP-Gly I and FP-Gly II. The expression of these proteins was circumscribed to the metencephalic FP, and occurred from HH 29 to HH 36. Within the FP cells, FP-Gly I and FP-Gly II were confined to the supranuclear and apical regions, which under the electron microscope displayed numerous cisternae of the rough endoplasmic reticulum and granules. Aggregates of AFRU-immunoreactive material appeared on the free surface of the FP. The possibility that FP-Gly I and FP-Gly II are released into the ventricle to reach distant targets is discussed.     

Analysis of the secretions of the subcommissural organs of several vertebrate species by use of fluorescent lectins

Summary The glycoprotein secretions of the subcommissural organ were analyzed with the use of nine fluorescent lectins, specific to different sugar moieties. After exposure to Concanavalin A a bright fluorescence was observed in the ependymal cells of the subcommissural organs of all vertebrates studied (Lampetra planeri, Ameiurus nebulosus, Bufo bufo, Lacerta vivipara, Gallus gallus, Rattus norvegicus, Ovis aries). The fluorescence is abolished by the competitive sugar, -D-mannopyranosyl. The intensity of the lectin fluorescence decreases from the phylogenetically lower to the higher forms, paralleled by a change in polarity of the secretion from a vascular (lower vertebrates) to a ventricular (higher vertebrates) direction. The strong affinity for Concanavalin A suggests the presence of a glycoprotein rich in mannosyl residues in the ependymal cells and a similarity of composition of this glycoprotein among the vertebrates. Lens culinaris agglutinin and wheat germ agglutinin revealed fluorescent rosettes in the hypendymal cells of the sheep. Binding of both these lectins suggests the presence of a glycoprotein rich in N-acetyl-D-glucosamine.In the underlying ventricular cavity, no fluorescence could be observed, suggesting that the Reissner's fiber does not possess the same carbohydrate constitution as the ependymal secretion of the subcommissural organ.     

Preparation and discharge of secretion in the subcommissural organ of the rat

Summary The secretion of the subcommissural organ (SCO) of the rat was studied by means of immunocytochemistry at the electron-microscopic level with the use of (1) the polar embedding medium Lowicryl K4M at -30° C, (2) the protein A-gold technique, and (3) a rabbit antiserum against bovine Reissner's fiber (see Sterba et al. 1981).Two different substructures of the ependymal and the hypendymal SCO-cells display a positive immunocytochemical reaction: (1) sacs containing flocculent secretion, which originate from the granular endoplasmic reticulum, and (2) vacuoles filled with fine granular secretion, which are pinched off from the Golgi apparatus. The secretory material of the sacs and the vacuoles is discharged both (i) apically into the cerebrospinal fluid and (ii) basally into intercellular spaces of the SCO-hypendyma. The apically released secretion is condensed to a lamina-like formation, which more caudally assumes the form of Reissner's fiber. The route of the basally released secretion remains, however, vague. The periodically striated bodies, which were thought to be morphological mediators of the discharge of the secretion into the capillaries, are never labeled by gold particles.Supported by grants from the Ministry for Science and Technology of the German Democratic RepublicThe expert technical assistance of Mrs. B. Wolff, Mrs. S. Mehnert, Mrs. E. Siebert, Mrs. Ch. Schneider, and Mrs. I. Seifert is gratefully acknowledged     

Concanavalin A-binding glycoproteins in the subcommissural and the pineal organ of the sheep (Ovis aries)

Summary Glycoproteins rich in mannosyl or glucosyl residues were analyzed in the subcommissural organ (SCO) and the pineal organ of the sheep (Ovis aries). By use of concanavalin A labelled with fluorescein isothiocyanate, fluorescent material was found both in ependymal and hypendymal cells of the SCO. In the pineal organ, either isolated or grouped parenchymal cells showed a marked fluorescence. These cells may correspond to ependymal elements also called interstitial cells or supporting cells. In addition, scarce slender, fluorescent processes were observed in the pineal parenchyma. The techniques of electrophoresis and electrotransfer on nitrocellulose paper have been applied to analyze the glycopeptide content of the SCO and the pineal organ in comparison to cerebellar and cerebral fractions solubilized by use of Triton X 100. Approximately 30 different concanavalin A-reactive glycopeptides were revealed in each fraction. In the SCO extract four glycopeptides (30, 54, 72, 100 kd) might correspond to subunits of the glycoprotein(s) characteristically stored in the ependymal cells of the SCO. In addition, two glycopeptides (32/33, 115 kd) are specific to the pineal organ extract. The possible similarity of the concanavalin A-reactive material in both organs is discussed and a putative secretory activity of the pineal ependymal cells is postulated.     

Cell junctions in the subcommissural organ of the rabbit as revealed by use of ruthenium red

Summary Tight junctions were found in the apical junctional complex of the adult rabbit subcommissural organ (SCO) in addition to zonulae adhaerentes and gap junctions of typical ependymal cells. Ventricular perfusion of ruthenium red before fixation was found to give excellent results for distinguishing between gap and tight junctions at the ependymal surface. The implication of tight junctions as a mechanical means of sealing off the SCO area from the cerebrospinal fluid and the use of ruthenium red as a tracer substance are discussed.This work was supported by grants from Statens almindelige Videnskabsfond, Copenhagen.     

Vasopressin- and oxytocin-containing fibres in the pineal gland and subcommissural organ of the rat

Summary Vasopressin and oxytocin were specifically demonstrated in the rat brain using the unlabelled antibody-enzyme method and purification of the first antiserum. Vasopressin and oxytocin fibres extend via the subcommissural organ or habenular commissure into the pineal stalk and terminate in the anterior part of the pineal organ. In addition, immediately adjacent to the subsommissural organ many vasopressin-containing fibres run caudally toward the central grey. These results are discussed in relation to the proposed presence of vasotocin in the pineal gland.This study was supported by the Foundation for Medical Research, FUNGOThe authors wish to thank Dr. D.F. Swaab and Prof. J. Ariëns Kappers for their suggestions and critical remarks     

Development of the ciliary complex and microtubules in the cells of rat subcommissural organ

Summary The fine structure of the rat subcommissural organs from the late stages of gestation through the postnatal to the adult stages was studied with the electron microscope. Emphasis in this report is placed on the development of the cilium with its affiliated structures. With the progress of cytodifferentiation centrioles originally located in the Golgi region migrate to the cell apex, where each then serves as a basal body to form a cilium which has a 9+2 organization of substructures. Thus, each of the mature subcommissural cells is provided with two cilia of motile type. Satellites first appear on one side of the basal body at about 17 fetal days, rapidly increase in number with age, and finally surround the basal body, forming an elaborate latticework. In the perinatal period microtubules progressively increase in number in the distal cytoplasm, which concurrently elongates and forms a prominent projection in the brain ventricle. Closely associated with the microtubules are large clusters of dense granular masses with an undefined border, which bear a close resemblance to the dense masses appearing in the differentiating cells of respiratory epithelium and having been generally assumed to be the precursor substance for centriole replication. However, the mature subcommissural cells contain no centrioles other than the preexisting pair, each of which has organized a cilium. The dense masses in the subcommissural cells are presumed to be involved in the formation of the cytoplasmic microtubules instead of new centrioles.Work supported by the National Science Council, the Republic of China, and by the China Medical Board of New York, Inc. A preliminary report was presented at the 6th International Congress for Electron Microscopy, Kyoto, 1966 (Lin, H.-S., andI-1. Chen: Satellites of the ciliary basal body and microtubules in the cells of the rat subcommissural organ. In: Electron Microscopy (R. Ueda, ed.) Vol. II, 461–462. Tokyo: Maruzen Co., Ltd. 1966).     

The binding of fucose-containing glycoproteins by hepatic lectins. The binding specificity of the rat liver fucose lectin

The parameters that affect the interaction of ligands with a fucose-binding lectin from rat liver have been examined. 125I-Fucosyl-bovine serum albumin (Fuc-BSA) containing 50 residues of fucose/molecule was used as the standard ligand. At low initial concentrations of ligand (10 ng/ml) and lectin (140 ng/ml), the reaction reaches equilibrium at pH 7.8, 23 degrees C, within 40 min. The binding of ligands is Ca2+ dependent with half-maximal binding occurring at 54 microM Ca2+; of several metal ions tested, only Sr2+ partially replaced Ca2+. Binding was maximal between pH 7.6 and 8.6, fell slightly up to pH 10, but fell markedly below pH 7. The lectin-ligand complexes dissociated at low pH, on removal of Ca2+, or in the presence of a large excess of competing ligand. The apparent association constant (Ka) for Fuc-BSA was 1.75 X 10(8) M-1. The fucose content of the Fuc-BSA also influenced binding, with little apparent binding below 24 fucose residues/molecule and maximal binding from 40 to 50 fucose residues/molecule. With knowledge of the parameters influencing binding, sensitive reproducible assays for the lectin were developed. The binding specificity of the lectin was examined by measuring the inhibition of 125I-Fuc-BSA binding by neoglycoproteins, monosaccharides, and glycosides or by direct binding of neoglycoproteins. Galactosides and beta-linked fucosides were the best ligands among the neoglycoproteins, with much weaker binding by mannosyl- or N-acetylglucosaminyl-BSA. On the basis of the pattern of inhibition of Fuc-BSA binding by various monosaccharides and glycosides, it is possible to propose the conformations of saccharides that best fit the lectin-binding site. The C1 conformation of N-acetyl-D-galactosamine fits best, although other not obviously related monosaccharides such as L-fucose, L-arabinose, and D-mannose can also assume conformations that permit them to be effective inhibitors. The pattern of binding of neoglycoproteins to the lectin differs from that of other pure hepatic lectins. Thus, the fucose lectin has a high affinity for Fuc-BSA and galactosyl-BSA but a low affinity for N-acetylglucosaminyl-BSA. The galactose lectin binds only galactosyl-BSA and shows little binding with either N-acetylglucosaminyl-BSA or Fuc-BSA. In contrast, the mannose/N-acetylglucosamine lectin binds N-acetylglucosaminyl-BSA and Fuc-BSA but not galactosyl-BSA.     

Immunohistochemical evidence for neuronal and non-neuronal synthesis of GABA in the rat subcommissural organ

In the past few years, several studies have demonstrated in the rat subcommissural organ the presence of nerve endings and modified ependymocytes showing an uptake of [³H]GABA. The present work was performed to demonstrate in this cerebral zone the possibility of a GABA synthesis by the immunohistochemical localization of glutamate decarboxylase (GAD). GAD-positive reaction was detected with unlabelled antibody-enzyme peroxidase anti-peroxidase. Some nerve terminals containing either clear round vesicles, or sometimes clear round vesicles and some large granular vesicles, exhibited a positive staining. These terminals could belong to GABAergic inputs in the subcommissural organ. The few reactive terminals containing some granular vesicles could be related to the serotoninergic input as suggested previously (Gamrani et al., 1981). Several ependymocytes of this structure contained GAD-like positive reaction; these cells are also capable of taking up [³H]GABA (Gamrani et al., 1981) and present neuronal properties with regard to GABA. However, the presence in their cytoplasm of enolase, a specific glial marker, related them to glial elements. The presence of GABA in these ependymocytes suggests a modulating function of GABA on the secretory activity of the subcommissural organ.     

The binding of fucose-containing glycoproteins by hepatic lectins. Purification of a fucose-binding lectin from rat liver

A lectin with a high affinity for binding ligands through fucose residues has been purified to homogeneity from rat liver. Affinity chromatography of the lectin on fucosyl-bovine serum albumin-agarose is the key step in the purification. Contaminating amounts of a previously described lectin that binds mannose and N-acetylglucosamine are removed from the fucose-binding lectin by either immunoadsorption on anti-mannose/N-acetylglucosamine lectin IgG-agarose or by specific elution of the fucose-binding lectin from fucosyl-bovine serum albumin-agarose. The pure fucose-binding lectin contains two polypeptide subunits with molecular weights of 88,000 and 77,000, respectively, as judged by gel electrophoresis. Peptide maps of the subunits, however, show that they are very similar structurally. In addition, peptide maps show that the fucose lectin is structurally distinct from other rat hepatic lectins. This is supported by the lack of cross-reaction among the different rat liver lectins and their specific antibodies and the inability of specific antibodies to the mannose/N-acetylglucosamine lectin to inhibit the binding of fucosyl-bovine serum albumin by the fucose lectin.     

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.