at the fishing port of Málaga. Antibodies raised against secretory glycoproteins extracted from the dogfish SCO exclusively detect class-specific epitopes, thus ...
Cell Tissue Res (1996) 283:75–84
© Springer-Verlag 1996
192102.200 071225.664 190503.200 190503.600 011420.499 091313.625 190325*200
Secretory glycoproteins of the subcommissural organ of the dogfish (Scyliorhinus canicula): evidence for the existence of precursor and processed forms M.D. López-Avalos1, J. Pérez2, J.M. Pérez-Fígares1, B. Peruzzo3, J.M. Grondona2, E.M. Rodríguez1,3 1
Departamento de Biología Celular y Genética, Facultad de Ciencias, Universidad de Málaga, E-29071 Málaga, Spain Departamento de Biología Animal, Facultad de Ciencias, Universidad de Málaga, E-29071 Málaga, Spain 3 Instituto de Histología y Patología, Universidad Austral de Chile, Valdivia, Chile 2
&misc:Received: 16 July 1995 / Accepted: 15 September 1995
&p.1:Abstract. The subcommissural organ of the dogfish, Scyliorhinus canicula (L), has been investigated by use of antibodies and lectins applied to blots and tissue sections processed for light and electron microscopy. Antibodies have been raised against each of the bands that have previously been identified in immunoblots by the use of antisera raised against secretory glycoproteins extracted from the dogfish subcommissural organ, viz., the 600-kDa band and two gel regions including the 475 to 400-kDa and the 145-kDa bands obtained from preparative gels; they are referred to as Ab-600, Ab-475/400, and Ab-145. These antisera and the lectins concanavalin A and wheat germ agglutinin have been used for the staining of: (1) blots of extracts of the dogfish subcommissural organ and optic tectum; (2) tissue sections of the dogfish brain. The findings indicate that the bands of 600, 475 and 400 kDa contain compounds that should be regarded as secretory glycoproteins of the dogfish subcommissural organ. The 600-kDa and 400-kDa bands are labeled by concanavalin A; wheat germ agglutinin labels the 475-kDa band strongly and the other two weakly. Ab-600 reacts with the bands at 600, 475 and 400 kDa and stains materials stored in the rough endoplasmic reticulum and secretory granules of 200–600 nm in diameter. The 600-kDa compound is probably a precursor form. Ab-475/400 stains the same three bands revealed by Ab-600; immunocytochemically, it reacts with two types of secretory granules (200–600 and 800–1200 nm in diameter) but it does not label the rough endoplasmic reticulum. Ab-145 reveals the bands at 600, 475 and 400 kDa and a diffuse zone in the region of 145 kDa; in light-microscopic immunocytochemistry, it behaves as Ab-475/400. The 475-kDa and 400-kDa glycoproteins, and a compound of approximately 145 kDa thus probably correspond to processed forms. Ab-475/400 stains This work was supported by grant PB 93-0979 from DGICYT (Spain); grant from the Instituto de Cooperación Iberoamericana (Spain) to J.M.P.-F. and E.M.R.; grant 95/1591 from FIS (Spain) to J.M.P.-F.; and grant 194-0892 from FONDECYT (Chile) to E.M.R. Correspondence to: J.M. Pérez-Fígares&/fn-block:
granules present in cell processes ending on local blood vessels and at the leptomeninges. Since this antiserum selectively labels secretory granules, this finding may be taken as evidence for a basal route of secretion. &kwd:Key words: Subcommissural organ – Secretory glycoproteins – Antibodies – Immunochemistry – Immunocytochemistry – Dogfish, Scyliorhinus canicula (Elasmobranchii)
Introduction The subcommissural organ (SCO) is a circumventricular organ present throughout the vertebrate phylum (Oksche 1961; Leonhardt 1980; Rodríguez et al. 1992). It secretes N-linked complex-type glycoproteins (Herrera and Rodríguez 1990). The bulk of this secretion is released into the ventricular cerebrospinal fluid (CSF) where it condenses into a fibrous structure known as Reissner’s fiber (RF) (Sterba and Ermisch 1969). RF grows caudally by addition of newly released molecules to its proximal end (Ermisch et al. 1971) and extends along the aqueduct, fourth ventricle, and central canal of the spinal cord. Biochemical studies using a set of specific antibodies and lectins have been performed on the materials secreted by the SCO of the sheep (Meiniel et al. 1986), bovine (Rodríguez et al. 1987; Nualart et al. 1991; Hein et al. 1993; Meiniel et al. 1993), and chick embryos (Karoumi et al. 1990; Meiniel et al. 1993). Such studies on the SCO of lower vertebrate species are however missing. This has led us to seek a species of fish that offers advantages for performing such an investigation. The dogfish Scyliorhinus canicula has been selected because it has a well-developed SCO and is avalaible in large quantities at the fishing port of Málaga. Antibodies raised against secretory glycoproteins extracted from the dogfish SCO exclusively detect class-specific epitopes, thus indicating, for the first time, that important species differences in the nature of the SCO secretion might occur (Pérez et al.
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1993; Grondona et al. 1994a). Immunoblot studies have revealed the presence of two high molecular weight glycoproteins in the dogfish SCO (Grondona et al. 1994b). The existence of two additional secretory compounds of lower molecular mass has also been suggested. The present investigation has been designed to obtain further information about the number, molecular mass, and processing (precursor and processed forms) of the compounds secreted by the dogfish SCO. For this purpose, specific antibodies have been raised against each of the compounds identified in a previous report (Grondona et al. 1994b). These antibodies have been used for blot and immunocytochemical studies of the SCO of the dogfish.
methylsulfonyl fluoride (Merck, Darmstadt, Germany), 1 µM leupeptin (Sigma), and 1 µM pepstatin (Sigma). Each extract contained 100–200 SCOs in 1 ml extraction medium. Optic tectum extracts were prepared following the same procedure. The protein content of extracts was determined according to Bradford (1976). The protein concentration of the various SCO extracts averaged 800 µg/100 SCOs.
Antisera
For the biochemical study, the brains of 5000 dogfish, Scyliorhinus canicula, were used. Fish were collected at the fishing port of Málaga, 5–8 h post-mortem, and kept on ice until their arrival in the laboratory. Dissection, homogenization, sonication, and centrifugation were performed according to a procedure described previously (Pérez et al. 1993; Grondona et al. 1994a, b). The extraction medium was 50 mM ammonium bicarbonate and 0.1% sodium dodecyl sulphate (SDS; Serva, Heidelberg, Germany). It contained the following protease inhibitors: 1 mM ethylenediamine tetraacetate (Sigma, St. Louis, Mo., USA), 0.5 mM phenyl-
In a previous study, we reported the production of antisera against crude extracts of the dogfish SCO. After purification of these antisera by immunoabsorption with several brain tissues, they reacted selectively with the secretion of the dogfish SCO (Pérez et al. 1993; Grondona et al. 1994a). These antisera were labeled as ADSO (A=antiserum, D=dogfish, SO=subcommissural organ). Blots of dogfish SCO extracts immunostained with ADSO revealed two bands with an apparent molecular mass of 750 and 380 kDa. When similar blots were immunostained with an antiserum against the constituent glycoproteins of the bovine Reissner’s fiber (AFRU, A=antiserum, FR=fiber of Reissner, U=urea; Rodríguez et al. 1984a), two bands (145 kDa, 35 kDa) were revealed, in addition to the 750-kDa and 380-kDa bands (Grondona et al. 1994b). The loading of 40 µg protein per well (see below) on the stacking gel, instead of 100 µg protein as performed previously (Grondona et al. 1994b), allowed better resolution of the immunoreactive proteins of high molecular weight (see Results). The corrected estimated molecular mass for the largest immunoreactive compound was 600 kDa. Two subsequent immunoreactive bands of 475 and 400 kDa were clearly distinguished (Fig. 1) in the region
Fig. 1. SDS-PAGE of the dogfish subcommissural organ (SCO) and optic tectum (OT). Regions of the gel used for immunization are indicated in the lane stained with Coomassie blue (CB). Blots were processed for immunostained with an antiserum against an extract of the dogfish SCO (ADSO), an anti-bovine Reissner’s fiber serum (AFRU), Ab-600, Ab-475/400, Ab-145, and Ab-35. Par-
allel lanes were processed for concanavalin A (Con A) and wheat germ agglutinin (WGA) binding. Numbers refer to molecular weights in kDa. MW Molecular weight standards; arrows secretory compounds; dotted line non-secretory compound revealed by Ab-475/400 and Con A; arrowheads 35-kDa band; bracket diffuse labeling of a gel region by Ab-145&ig.c:/f
Materials and methods Preparation of SCO extracts
77 at which Grondona et al. (1994b) had described a thick and poorly defined band of 380 kDa. In the present investigation, antisera were developed against each of the following regions of the preparative gels: region 1 contained the 600-kDa band; region 2 included the 475-kDa and 400-kDa bands (because of their proximity, it was impossible to cut them out separately); region 3 included a gel zone of about 4 mm height, together with the 145 kDa band in the center; region 4 included a gel region of 4 mm containing the 35-kDa band. Two preparative SDS-polyacrylamide gels were run (Laemmli 1970), using slab gels 150×150×1.5 mm, containing a polyacrylamide linear gradient of 5–15%. Proteins (1.2 mg) corresponding to 140 SCOs were used in each electrophoresis. Both gels were stained with Coomassie blue. The four zones of the gels were cut out according to Cozzani and Hartmann (1980). The corresponding regions from each gel were pooled. Each of the four pools was washed in phosphate-buffered saline (PBS), pH 7.3, for 12 h, and then homogenized in a glass homogenizer. The homogenate was kept at −20° C until used to immunize four rats (one pool/rat). The protocol of immunization was similar to that used to produce ADSO (Grondona et al. 1994a). The antisera obtained were referred to as Ab-600, Ab-475/400, Ab-145, and Ab-35. The whole procedure to obtain the antisera against these four bands was carried out three times. This resulted in three antisera for each of the bands. The characteristics of the three antisera raised against the same band were essentially the same.
Immunoblotting of dogfish SCO extracts SDS polyacrylamide gel electrophoresis was performed according to Laemmli (1970) using slab gels of 90×60×1.5 mm, containing a 5–15% polyacrylamide linear gradient. Samples of SCO (40 µg, equivalent to 5 SCOs) and optic tectum (40 µg) extracts were dissolved in 60 mM TRIS-HCl, pH 6.8 (Sigma), 2% SDS, 10% glycerol (Sigma), and 5% β-mercaptoethanol (Sigma), and loaded onto the stacking gel. Gels were transferred onto PVDF membranes (Millipore, Bedford, Mass., USA) according to the procedure by Towbin et al. (1979); for further details regarding electrophoresis and electrotransference, see Grondona et al. (1994b). The molecular weight standards used were: glycoproteins of the bovine subcommissural organ (540, 450, and 320 kDa; Nualart et al. 1991); myosin (205 kDa); β-galactosidase (116 kDa); phosphorylase b (97 kDa); ovalbumin (45 kDa); carbonic anhydrase (29 kDa) (Sigma). Blots of SCO and optic tectum were immunostained with the following antisera: (1) ADSO purified by immunoabsorption (Grondona et al. 1994a), 1:2000 dilution; (2) Ab-600, 1:5000 dilution; (3) Ab-475/400, 1:5000 dilution; (4) Ab-145, 1:1000 dilution; (5) Ab-35, 1:1000 dilution; (6) AFRU, 1:1000 dilution. Control blots were incubated with rat pre-immune serum. For immunostaining with antibodies 1–5 (raised in rats), the blots were sequentially incubated with: (1) 5% non-fat milk in PBS buffer, pH 7.3; (2) primary antibody for 18 h; (3) anti-rat IgG developed in rabbit, 1:500 dilution (raised in our laboratory) for 2 h; (4) antirabbit IgG developed in sheep, 1:50 dilution (raised in our laboratory) for 2 h; (5) rabbit PAP (Sigma), 1:200 dilution for 1 h; (6) 0.06% 4-chloro-1-naphthol (Sigma), 0.075% perhydrol (Merck), 20% methanol, in PBS, pH 7.3. AFRU was raised in rabbits; therefore, when this antiserum was used for immunoblotting, step (3) was omitted.
incubated with Con A in the presence of 1 M D-mannose; (2) wheat germ agglutinin (WGA; affinity=glucosamine, sialic acid; Sigma) 1 µg/ml: control blots were incubated with WGA in the presence of 1 M N-acetyl-D-glucosamine. Peroxidase was detected by use of perhydrol and 4-chloro-1-naphthol.
Light-microscopic immunocytochemistry The brains of 15 dogfish Scyliorhinus canicula were fixed by immersion in Bouin’s fluid for 48 h. Some were dehydrated and embedded in paraffin. In others, the SCO-posterior commissure region was dissected out and embedded in butyl-methyl methacrylate (Rodríguez et al. 1984b). Paraffin (8-µm-thick) and methacrylate (1-µm-thick) sections were processed for the immunoperoxidase staining method (Sternberger et al. 1970) using the following primary antisera: (1) ADSO purified by immunoabsorption, 1:2000 dilution; (2) Ab-600, 1:5000 dilution; (3) Ab-475/400, 1:5000 dilution; (4) Ab-145, 1:1000 dilution; (5) Ab-35, 1:1000 dilution. The linking antibodies used were anti-rat IgG developed in rabbit, 1:500 dilution for 1 h, and anti-rabbit IgG developed in sheep, 1:50 dilution for 45 min. Rabbit PAP (Sigma) was used at a 1:200 dilution for 45 min. Diaminobenzidine (DAB; Sigma) was used as electron donor. All antibodies were dissolved in TRISHCl buffer, pH 7.8, containing 0.35% lambda carrageenan (Sigma) and 0.25% Triton X-100 (Sigma) (Sofroniew et al. 1979). Controls included incubation in pre-immune rat serum and omission of the incubation in the primary antiserum.
Electron-microscopic immunocytochemistry The SCO region of four dogfish was dissected out and processed for electron-microscopic immunocytochemistry. These specimens were processed at short (15 min to 30 min) post-mortem times. Fixation was performed in 2% paraformaldehyde, 0.5% glutaraldehyde, and 15% saturated picric acid solution, buffered to pH 7.4 with elasmobranch buffer, by immersion for 24 h at 4° C. Tissue was embedded in Lowicryl K4M (Polyscience, DSA) using benzoyl peroxide (Fluka, Buchs, Switzerland) as accelerator (Peruzzo et al. 1990). Ultrathin sections were immunostained according to the protein-A/gold method. Ab-600 and Ab-475/400 were used as primary antibodies. Both were used at dilutions of 1:8000 and 1:16000. Incubation with non-immune serum, instead of the primary antibody, was used as a control test.
Lectin histochemistry Methacrylate sections (1 µm thick) adjacent to those used for immunocytochemistry were processed for lectin binding. Sections were incubated with peroxidase-labeled lectin (Sigma; Con A 15 µg/ml; WGA 10 µg/ml) dissolved in TRIS-HCl buffer, pH 7.8, for 45 min. DAB was used as electron donor to visualize the peroxidase reaction.
Results SDS-PAGE and blotting
Lectin binding Blots parallel to those used for immunostaining were used for lectin binding. The blots were sequentially incubated with oxidized bovine serum albumin (Sigma) and with peroxidase-labeled lectins for 1 h. The lectins used were (1) concanavalin A (Con A; affinity=mannose, glucose; Sigma) 3 µg/ml: control transfers were
The 600-kDa band reacted with AFRU, ADSO, Ab-600, Ab-475/400, and Ab-145. It did not react with Ab-35. It bound Con A and, although weakly, WGA (Table 1, Fig. 1). The 475-kDa and the 400-kDa bands reacted with all antisera used, but not with Ab-35 (Table 1, Fig. 1). The
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79 Table 1. Reactivity of antisera and lectin binding in blots of the secretory material of the dogfish SCO. AFRU, Anti-bovine RF serum; ADSO, anti-dogfish SCO serum; Ab-600, antiserum against the 600 kDa band; Ab475/400, antiserum against the 475-kDa and 400-kDa bands; Ab-145, antiserum against the 145-kDa band; Con A, concanavalin A (affinity=mannose, glucose); WGA, wheat germ agglutinin (affinity=glucosamine, sialic acid); +, ++, degree of labeling; −, negative reaction&/tbl.c:&
600-kDa band 475-kDa band 400-kDa band 145-kDa region
AFRU ADSO
Ab-600 Ab-475/400 Ab-145 Ab-35 Con A WGA
+ + + +a
++ ++ ++ −
+ + + −
++ ++ ++ −
+ + + +b
− − − −
+ − + +c
+ ++ + +c
a
Seen when loading 100 µg protein in the well (Grondona et al. 1994b) Visualized as a wide zone in the region of the 145-kDa band c Some positive bands in the region of the 145-kDa band b
&/tbl.:
400-kDa band bound Con A and WGA, whereas the 475-kDa band only bound WGA. The reaction of the bands of 600 and 400 kDa to Con A was weak compared to other Con-A-positive bands of the same gel (Fig. 1). In a few blots, a band with a molecular mass larger than 600 kDa reacted weakly with Ab-600, Ab-475/400, and Ab-145 (Fig. 1). Ab-475/400 also reacted with a band of 550 kDa (Fig. 1). A thick diffuse zone in the region of the 145 kDa band reacted with Ab-145 (Table 1, Fig. 1) and occasionally with AFRU. None of the other antisera labeled this zone. WGA and Con A revealed bands in the region of the gel used to raise Ab-145. AFRU, Ab-600 and Ab-475/400 stained the 35-kDa band. This band was also revealed by the rat pre-immune serum. The rat pre-immune serum did not detect any other band in the dogfish SCO extracts. Ab-600, Ab475/400, Ab-145, and Ab-35 revealed no bands in the optic tectum extract (Fig. 1). Immunocytochemistry and lectin histochemistry of the dogfish SCO Light microscopy. &p.1:Ab-600 produced strong selective immunostaining of the dogfish SCO (Fig. 3) similar to that obtained with ADSO purified by immunoabsorption (Fig. 2). Ab-600 stained all regions of the ependymal secretory cells, including the basal processes projecting to the leptomeninges and to blood vessels (Figs. 3, 6).
Figs. 2–5. Transverse sections of the dogfish SCO immunostained with ADSO (1:2000) (2), Ab-600 (1:5000) (3), Ab-475/400 (1:5000) (4), and Ab-145 (1:1000) (5). ADSO and Ab-600 have similar immunostaining patterns; both reveal basal processes of SCO cells to the leptomeninges (thick arrows). Ab-475/400 and Ab-145 stain apical and subapical zones of the SCO cells. Insert: Detailed magnification of the area in the rectangle; Ab-475/400 reveals granules in leptomeningeal processes and endings (fine arrows). Arrowhead indicates an ependymal vascular ending immunoreactive with Ab-600 (3) and with Ab-475/400 (4). ×250&ig.c:/f Figs. 6–7. Sagittal sections of the dogfish SCO immunostained with Ab-600 (1:5000) (6) and Ab-475/400 (1:5000) (7). Immunoreactive basal processes projecting to the leptomeninges are shown (arrows). Insert: Ependymal endings contain granules immunostained by Ab-475/400 (fine arrows). ×340&ig.c:/f
Ab-475/400 revealed immunoreactive material located in the supranuclear region of the ependymal cells. The most apical region of these cells presented the strongest immunoreaction (Figs. 4, 7). Processes projecting to the leptomeninges displayed fine granules immunostained with Ab-475/400 (Figs. 4, 7). Ab-145 immunotained the SCO in a manner similar to Ab-475/400 (Fig. 5). Ab-35 did not react with the SCO. The RF in the central canal of the spinal cord was stained by Ab600, Ab-475/400, and Ab-145. Immunostaining of adjacent 1-µm-thick methacrylate sections with Ab-600 and Ab-475/400 allowed us to establish precisely the intracellular distribution of the immunoreactions. Ab-600 homogeneously stained the perinuclear and supranuclear regions; these regions were mostly occupied by rough endoplasmic reticulum (RER) (cf. Grondona et al. 1994b) (Fig. 8). The apical third of the ependymal cells was strongly immunoreactive (Fig. 8). Additionally, material forming a continuous layer located in the apical-most region and a discontinuous layer located on the free cell surface were also strongly reactive with Ab-600 (Fig. 8). Ab-475/400 did not react with the perinuclear area. It strongly reacted with distinct granules located in the supranuclear region of the ependymal cells (these were not revealed by Ab-600) and with material (1) located in the apical third of the cells, (2) forming a continuous apical layer, and (3) arranged into a loose flocculent layer located extracellularly (Fig. 9). Con A-binding sites (Fig. 10) co-distributed with the Ab-600 immunoreactive material located in the RER region. The apical region of the ependymal cells did not bind Con A (compare Figs. 8 and 10). The material binding WGA (Fig. 11) and that immunoreacting with Ab-475/400 showed the same intracellular distribution (compare Figs. 9 and 11). Electron microscopy. &p.1:Two distinct types of granules can be distinguished in the ependymal cells of the dogfish SCO (cf. Grondona et al. 1994b). Type I granules have a diameter of 200–600 nm and are distributed throughout the supranuclear cytoplasm; they are abundant in the ventricular cell pole. Type II granules range between 800 and 1200 nm in diameter; they are restricted to the medial third of the supranuclear cytoplasm. Ab-600 labeled type I granules (Fig. 12) and the cisternae of the RER (Fig. 14). It did not react with type II granules (Fig. 13).
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Ab-475/400 labeled type I granules (Fig. 15). Although it reacted with type II granules, the label was restricted to the periphery of these granules (Fig. 16). It did not label the RER (Fig. 17). Ab-600 and Ab-475/400 strongly labeled the surface of the microvilli of the SCO cells (Fig. 15; cf. Fig. 9) but they did not label those of the adjacent ependyma (see Fig. 9). When pre-immune serum was used for immunolabeling, no gold particles were seen; the background labeling seen in our preparations (Figs. 12–17), despite the high dilution of the primary antisera, was ascribed to the quality of the preservation of the tissue fixed by immersion. Unfortunately, it was not possible to obtain a living specimen. Discussion The present methods have succeeded in obtaining antisera against the secretory glycoproteins present in bands separated by SDS-PAGE of extracts of the SCO of the dogfish Scyliorhinus canicula. This has allowed us to obtain information about the nature and intracellular distribution of these compounds, extending the information reported by Grondona et al. (1994a, b). Furthermore, better resolution of the electrophoresis run in the present study has led to some corrections in the estimation of the molecular mass of the largest immunoreactive polypeptides (600 kDa instead of 750 kDa, as reported by Grondona et al. 1994b); the thick band previously described as a 380-kDa band (Grondona et al. 1994b) has, in the present study, been resolved into two distinct bands of 475 and 400 kDa. Under the present electrophoretic conditions (40 µg protein/load), the 145-kDa band is not visualized in immunoblots. However, when it is extracted from preparative gels to be used as immunogen, it leads to the production of an antiserum immunostaining the secretory granules of the SCO and the bands at 600, 475 and 400 kDa. The only sound interpretation is that, in the analytic gel, this compound is at a concentration not detectable by immunoblotting. The compounds of 600, 475, and 400 kDa can be regarded as secretory glycoproteins of the SCO on the following grounds: (1) although reacting very weakly, they can be labeled by an anti-bovine RF serum (AFRU) and they bind Con A and WGA; (2) when used as an immu-
Figs. 8–11. Adjacent 1-µm-thick methacrylate sections of the dogfish SCO processed for immunostaining with Ab-600 (1:1000) (8) and Ab-475/400 (1:5000) (9), and for Con A (10) and WGA (11) binding. Ab-600 and Con A label the perinuclear and supranuclear regions (RER) of the SCO cells (stars). The apical marginal zone does not bind Con A but does react with Ab-600 (arrowheads). Microvilli (Mv) are labeled by Ab-600. Both probes also reveal basal processes (asterisks). Ab-475/400 and WGA label small (Sg) and large (Lg) granules and the microvilli of the SCO cells, but not those of the adjacent ependyma (open arrow). They do not label the region occupied by the RER (stars). Small arrowheads Fine leptomeningeal processes labeled by Ab-475/400 and WGA. ×475&ig.c:/f
nogen, each one of the compounds generates antisera that selectively immunostain the secretory material present in cells of the SCO, and the material released into the ventricle (layer of material on the SCO surface and RF); (3) when these antisera are used in immunoblotting, they reveal the same bands shown by AFRU; (4) all these antisera immunostain the SCO of other elasmobranch species (data not shown). The labeling of a 145-kDa compound by AFRU (Grondona et al. 1994b) and the observation that an antiserum raised against the gel region including the band of 145 kDa labels (1) the bands at 600, 475, and 400 kDa, and (2) the secretory granules of the SCO, indicate that a secretory compound might migrate in the region of 145 kDa. Unfortunately, Ab-145, when used in immunoblotting, does not reveal a distinct band, but a diffuse area, in the region of 145 kDa. This has prevented us from correlating the immunolabeling of this gel region with the lectin binding of distinct bands located in this region of the gel. On the other hand, Ab-35 neither reacts with the SCO by immunocytochemistry nor immunostains the bands at 600, 475, 400, or 145 kDa. Consequently, the 35-kDa compound should not be regarded as a secretory product of the dogfish SCO. Similarly, the 550-kDa compound only revealed by Ab-475/400 should not be regarded as a secretory compound, since it is not stained by any other of the antisera; furthermore, an antiserum raised against the 550-kDa band does not immunostain the SCO (result not shown). Grondona et al. (1994b) have postulated that the 750kDa (now 600-kDa) compound is a precursor form. Evidence obtained in the present investigation supports this possibility. It has been shown that the secretory compounds of the SCO of higher vertebrates are N-linked complex-type glycoproteins with sialic acid as the terminal residue (Meiniel and Meiniel 1985; Rodríguez et al. 1986; Meiniel et al. 1988, 1990; Herrera and Rodríguez 1990; Karoumi et al. 1990; Nualart et al. 1991). The precursor forms of these glycoproteins are core-glycosilated glycoproteins with terminal mannose residues. These precursors are located in the RER (Rodríguez et al. 1992). In the mammalian SCO, the bulk of the secretion is stored as precursors in the RER (Rodríguez et al. 1992). Con A has been shown to be a useful tool to detect these precursor forms in tissue sections of the SCO, and consequently to label the RER (Herrera and Rodríguez 1990). The secretory material in the dogfish SCO also appears to be constituted by N-linked complex-type glycoproteins with sialic acid as the terminal residue, as shown by Grondona et al. (1994b). These authors have also demonstrated that Con A: (1) labels the content of the RER of the dogfish SCO, (2) reacts with the 750kDa (now 600-kDa) band. The labeling of the 600-kDa compound by WGA, albeit weakly, has not previously been detected (Grondona et al. 1994b). We have no explanation for this descrepancy, other than to mention the different electrophoretic conditions that have been used in the present study and that have led to a better resolution in the migration of the high molecular mass com-
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pounds. This weak labeling of the 600-kDa compound by WGA argues against its precursor nature, unless the internal glucosamine residues of the glycoprotein secreted by the dogfish SCO display some affinity for WGA under blotting conditions. The present investigation has shown an intracellular co-distribution of the materials reacting with Con A and Ab-600. It thus seems likely that Ab-600 immunoreacts with the precursor forms stored in the RER. Indeed, Ab600 strongly labels the content of the dilated RER cisternae. Additionally, Ab-600 also reacts with type I secretory granules and, in blots, with the compounds of 475 and 400 kDa. The latter compounds might correspond to processed forms of the 600-kDa precursor and might be stored in type I secretory granules. The processed nature of the 475-kDa and 400-kDa compounds is supported by the observation that Ab475/400 and Ab-145 immunoreact with secretory granules but not with the material stored in the RER. Under blotting conditions, this antiserum does react, however, with the 600-kDa compound. Only the 400-kDa compound has the lectin-binding properties of a processed form of an N-linked secretory glycoprotein, i.e., it binds WGA but not Con A. In the mammalian SCO, the bulk of the secretion is stored in the RER as precursor forms, whereas the secretory granules are scarce and probably contain only processed forms (Rodríguez et al. 1992). The large number of type I secretory granules found in the dogfish SCO and their affinity for Ab-475/400, Ab-145, and WGA suggest that, in this species, the bulk of secretory material is stored as processed forms in the secretory granules. Thus, the dogfish SCO probably has a larger pool of readily releasable material, compared with the mammalian SCO. The affinity of type II granules for Ab-475/400 and their lack of affinity for Ab-600, their strong labeling by Con A and WGA (Grondona et al. 1994b), and their location away from the ventricular cell pole support the hypothesis advanced by Grondona et al. (1994b) that they are lysosomes containing secretory material undergoing degradation. This, in turn, would be in accordance with a gland with a large pool of mature secretory material.
Figs. 12–14. Ultrastructural immunocytochemistry of the dogfish SCO immunostained with Ab-600. Label is found on microvilli (Mv), small granules (solid arrows), and RER. Large granules (open arrow) in Fig. 13 are not labeled. V Ventricle; C ciliary roots; J junction complex; N cell nucleus. Fig. 12: ×13500; Fig. 13: ×30000; Fig. 14: ×12200&ig.c:/f Figs. 15–17. Dogfish SCO immunostained with Ab-475/400. (15) Microvilli (Mv) and type I granules (arrows) present in the apical cell pole are strongly labeled. C Ciliary roots; V ventricle. ×11300. Insert: Detailed magnification of two small granules displaying the label preferentially at their periphery (arrows). (16) Type II granules are immunostained peripherally (large arrows). Small arrows Small granules. ×17300. (17) A large dilated RER cisterna (RER) in the perinuclear cytoplasm displays labeling little different from background labeling. Arrow Large granule; N nucleus. ×11700&ig.c:/f
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