The bovine subcommissural organ: cytochemical and immunochemical characterization of the secretory process.

Specific glycoproteins of the bovine subcommissural organ (SCO) were studied by means of various techniques: light and electron microscopy, immunoaffinity chromatography, electrophoresis and Western blotting. Use of lectins (Con A, WGA, PHA-E and -L, LCA) allowed to specify the synthesis and release of complex-type glycoproteins that bear high-mannose-carbohydrate chains in their precursor forms and probably triantennary carbohydrate chains in their mature forms. Antibodies raised against SCO extracts were characterized by means of various tests and used to purify specific compounds. Immunopurified fractions using A99 polyclonal antibody contained numerous polypeptides reactive with Con A, their apparent molecular weight (MW) ranging from 240 to 50 kDa. Only two glycopeptides were strongly labeled with WGA (98 and 52/54 kDa MW). Immunopurified fractions using C1B8A8 monoclonal antibody, specific of the complex-type glycoproteins at different steps of glycosylation, showed three specific Con A-reactive polypeptides at 88, 54 and 34 kDa MW. Only the 34 kDa glycopeptide was strongly labeled with WGA. The latter could correspond to the monomeric form of the secreted compound. Electrophoretical analyses of Reissner's fiber material allowed the detection of a WGA-positive smear in the upper part of the blots, suggesting that the complex-type glycoproteins, when released into the CSF, constitute a stable polymer.

Ontogenesis of the secretory epithelium of the bovine subcommissural organ. A histofluorescence study using lectins and monoclonal antibodies.

A spatio-temporal analysis of the differentiation of a group of specialized (secretory) ependymal cells in the subcommissural organ (SCO) of the brain was undertaken in the bovine using a monoclonal antibody (C1B8A8) which is specific of the secretory process in this organ. In addition, lectins (concanavalin agglutinin (Con A), Lens culinaris agglutinin (LCA), wheat germ agglutinin (WGA), and Phaseolus vulgaris agglutinin (PHA] were used to analyse the maturation of the carbohydrate moieties of the secretory product (subcommissuralin). Monoclonal antibody NC-1 specific to a complex carbohydrate epitope including a terminal 3-sulfoglucuronyl residue similar to HNK-1 was also tested to compare the reactivity of the SCO with that of other brain structures. These cells express a specific antigen related to the known secretory activity of the SCO during early embryogenesis (2 months). This antigen is recognized by C1B8A8 antibody and by Con A suggesting that high mannose-type glycoproteins are synthesized at this stage. Later on (approximately 3.5 months), appearance of C1B8A8, WGA, LCA, L- and E-PHA-positive material in the apical lining of the ependymal cells, close to the ventricular cavity, suggests that maturation of the complex-type glycoproteins (Asn-linked) occurs at this stage. Presence of secretory material in the CSF and Reissner's fibre could be detected using the same probes at a stage of 4 months. As early as 2 months NC-1-positive material was detected in the ependyma of the mesencephalic roof, while no reaction occurred in the SCO epithelium. This suggests that the carbohydrate moieties of subcommissuralin is different from that of ependymins beta and gamma. Using specific monoclonal antibodies, molecular characterization of subcommissuralin and experimental analyses on its accurate role in brain development will further our tentative comparison with ependymins. The secretory ependymal cells in the SCO express a particular phenotype and could represent an increasing model to study cell differentiation in the brain.

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

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.