Light- and electron-microscopic immunocytochemical investigation of the subcommissural organ using a set of monoclonal antibodies against the bovine Reissner's fiber

Abstract

Ten monoclonal antibodies (Mabs) against glycoproteins of the bovine Reissner's fiber (RF) have been used in a structural and ultrastructural immunocytochemical investigation of the bovine subcommissural organ (SCO) and RF. The SCO of other vertebrate species has also been studied. For comparison, polyclonal antibodies against bovine RF (AFRU) were used. The SCO and RF of ox, pig and dogfish and the SCO of dog, rabbit, rat and frog were submitted to light-microscopic immunocytochemistry using AFRU and Mabs. Postembedding ultrastructural immunocytochemistry was applied to sections of bovine SCO using AFRU and Mabs. Bovine SCO consists of ependymal and hypendymal cell layers, the latter being arranged as cell strands across the posterior commissure, or as hypendymal rosette-like structures. All cytoplasmic regions of the ependymal and hypendymal cells were strongly stained with AFRU. Six Mabs showed the same staining pattern as AFRU, one Mab stained RF strongly and SCO weakly, two Mabs stained RF but not SCO, and, finally, one Mab (3B1) exclusively stained the apices of the ependymal and hypendymal cells. All Mabs recognized the SCO and RF of the pig. Two Mabs bound to the SCO of the dog. One Mab stained the SCO of the rabbit and another the SCO of the rat. The SCO of frog and dogfish were totally negative. Bovine SCO stained with AFRU, showed label in the rough endoplasmic reticulum (RER) and the secretory granules (SG) of the ependymal and hypendymal cells. The former, in the form of parallel cisternae, reticulum or concentric rings, was seen throughout all cytoplasmic regions. SG were abundant in the apical pole of the ependymal and hypendymal cells. Only one Mab showed a staining pattern similar to AFRU. Five Mabs showed strong reactions in the SG but weak labeling of the RER. Mab 3B1 showed the label confined to the SG only. Our results suggest that: (i) in the bovine tissue, some epitopes are present in both precursor and processed materials, whereas others are characteristic of mature glycoproteins present in SG and the RF; (ii) the bovine SCO secretes at least two different compounds present in ependymal and hypendymal cells; (iii) both compounds coexist in the same secretory granule; (iv) there are conserved, class-specific, and species-specific epitopes in the glycoproteins secreted by the SCO of vertebrates.

The subcommissural organ

“…Ultrastructural immunocytochemistry using polyclonal and monoclonal antibodies has demonstrated strong labeling of the secretory material located in the dilated RER cisternae (Figs. 15, 16) and the secretory granules located at the apical cell pole (Grondona et al, 1994b;Lösecke et al, 1984;Pérez et al, 1995;Peruzzo et al, 1990;Rodríguez et al, 1986Rodríguez et al, , 1987a and in the vascular and leptomeningeal ependymal endings Rodríguez et al, 1987b). There are monoclonal antibodies that react with the secretory granules but not with the RER content .…”

Analysis and quantification of the secretory products of the subcommissural organ by use of monoclonal antibodies

“…To raise Mabs against the intracellular secretory glycoproteins of the bovine SCO, we have followed the same protocol described for anti-RF Mabs (see details in Pérez et al, 1995Pérez et al, , 1996; SCO secretory glycoproteins purified by immunoaffinity chromatography were used as antigen. Briefly, extracts of bovine SCO were passed through a column of sepharose bound with the anti-RF Mab 2A5 or 3E6 and the bound fraction was used for immunization.…”

“…Ultrastructural immunocytochemistry was performed using as primary antibody a polyclonal antiserum (AFRU) , and the anti-RF Mabs 2A5, 2A8, 1H3, 3E6, 3F1, 3B1, and 1F12 (see Pérez et al, 1995). Mab 2A8 showed a staining pattern similar to that of AFRU.…”

“…Secretory granules appear filled with gold particles for 3B1, but gold particles tended to be located in the periphery of the granules for Mab 3E6 (arrow). ϫ12,000 Reproduced from Pérez et al (1995) with permission of the pubisher. secretion of the SCO of several vertebrate classes (Rodríguez, personal communication).…”

“…At variance, a mono-clonal antibody (Mab) recognize a single epitope of the antigen and this makes them valuable, precise tools for morphological, analytical, and functional studies of the SCO-RF system (Meiniel et al, 1988;Pérez et al, 1995Pérez et al, , 1996. In this work, we describe the production and use of a series of Mabs against both the bovine RF and the secretory glycoproteins extracted from the bovine SCO.…”