Original Paper Acta Anatómica 1992;143:27-40

Department of Anatomy. Faculty of Medicine. University of Hong Kong, Hong Kong

Key Words Lectin Lateral prostate Glycoconjugates Colloidal gold

Localization of Prostatic Glycoconjugates by the Lectin-Gold Method

A bstract The glycoconjugates of the lateral prostate were examined ultrastructurally by lectin-gold histochemistry in combination with a low-temperature embedding technique using Lowicryi K4M. The binding patterns of concanavalin A. wheat germ agglutinin. Griffonia simplicifolia, soybean agglutinin, peanut agglutinin. Ricinus communis agglutinin isolectin I. Griffonia simplicifolia isolectin B,. Ulex europaeus isolectin I and Phaseolus vulgaris agglutinin Phave been documented in the subcellular compartments of the lateral prostate. The results show that the granular endoplasmic reticulum (GER) is rich in glycoproteins with mannosyl residues while the Golgi cisternae. secretory granules and microvilli are less so. The mannose (Man) and N-acetylgucosamine (GIcNAc) residues present in the GER of the epithelial cells may be associated with the initial assembly of the Nlinked oligosaccharides of glycoproteins. The secretory granules exhibited dif­ ferent reactivities to lectins. Most of the lectin-binding sites confined to the limit­ ing membranes may play a role in the transport of plasmalemma glycoconju­ gates to the apical plasma membrane. The epithelial Golgi stack is rich in GIcNAc. galactose (Gal). N-acetylgalactosamine (GalNAc) and sialic acid resi­ dues, and a compartmental organization of the Golgi stack is apparent which might be associated with the sequential addition of sugar residues to the oligo­ saccharides. The plasma membrane contains abundant Man. GIcNAc. Gal. GalNAc and complex carbohydrates, especially in the microvilli, and a differen­ tial lectin labelling was noted between the apical and basolateral plasma mem­ brane. The present study showed that fucosc-containing glycoconjugates were detected in the apical plasma membrane of the lateral prostate. The stromal extracellular matrices as well as the epithelial basement membranes demon­ strated weak lectin reaction. Man, GIcNAc. Gal residues and complex sugars were also noted in the stromal tissues of the lateral prostate including the extra­ cellular matrix, capillaries and smooth muscle.

Received: June 2. 199! Accepted: July 13. 1991

Dr. Y.C. Wong Department of Anatomy. University of Hong Kong 5/F L.i Shu Fan Building. 5 Sassoon Road Hong Kong (Hong Kong)

© 1992 Karger AG. Basel 000 1-5 180/92/143MM127

S 2.75/0

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

L. Chan Y.C. Wong

Materials and Methods

Glycoconjugates are carbohydrate-rich molecules (e.g. glycoproteins and glycolipids) located both intracellularly and extracellularly and also at the cell surface which can be secretory or structural. Cell surface glycoconjugates undergo modification in association with certain cellular and physiological conditions such as cell differentiation, proliferation and under pathological conditions (e.g. neo­ plastic malignancy) [ 1—4]. The carbohydrate residues of glycoconjugates play important roles in activities such as (a) maintaining protein conformation and solubility, (b) stabilizing the polypeptide against uncontrolled proteoly­ sis. (c) mediating biological activity (e.g. immunogenic recognition, non-immunogcnic phagocytosis and receptormediated endoevtosis). (d) intracellular sorting and secre­ tion of glycoproteins and (e) embryonic development and differentiation [5]. The cell surface carbohydrates are involved in cellular differentiation. Teichberg et al. [6] have demonstrated the presence of a P-D-galactoside-binding protein which is involved in the fusion of undifferenti­ ated myoblasts to form differentiated myotubes. It has been suggested that cellular adhesion is mediated by inter­ action of the carbohydrate moiety of surface glycoconju­ gates of one cell with a carbohydrate-binding protein on the surface of another cell. Carbohydrate moieties, both intraand extracellularly and also in the extracellular matrix, are important in cellular interactions and cytodifferentiation. Lectins have been utilized extensively as biochemical tools to isolate glycoproteins, glycolipids and polysaccha­ rides and as probes to investigate the sugar residues of the cell surface. They are especially useful in studies of the quality, distribution, assembly and turnover of glycoconju­ gates in normal and pathological tissues, as well as during embryonic differentiation [7-9], When conjugated to a marker, such as a fluorescent dye. peroxidase or colloidal gold, lectins may be used as histochemical probes to iden­ tify and localize specific carbohydrate residues (10-13]. According to their carbohydrate-binding specificity, lectins are generally classified into five groups: (a) the glucose/ mannose (Man) group, (b) N-acetylglucosaminc (GlcNAc) group, (c) galactosc/N-acctylgalactosamine (Gal/GalNAc) group, (d) L-fucose group, and (c) the sialic acid group. With the use of a number of lectins specific for different sugar terminal groups, the distribution of glycoconjugates of the guinea pig prostate gland has been documented [14], The present study was designed to examine the location and distribution of both the extra- and intracellular glyco­ conjugates of the lateral prostate using a number of bioti­ nylated lectin-gold complexes.

28

Chan/Wong

Preparation o f Tissues for Low-Temperature Embedding Three adult male guinea pigs (Hartley) were killed by an overdose of pentobarbital. The lateral prostate was removed and trimmed into small blocks of about I mm'. The tissue blocks were fixed by immer­ sion in 4% paraformaldehyde and 0.25% glutaraldehyde in 0 .1 M sodium cacodylatc. pH 7.4. at 4 °C for 4 h. After fixation, the tissues were rinsed in 0.1 M sodium caeodylatc buffer with 6.9% sucrose at pi 1 7.4 at 4 C overnight. Some of the tissues w'erc incubated in 0 .1 M NHjCI in cacodylate buffer, pH 7.4, for I h to quench the free alde­ hyde groups 112. 13. 15) prior to dehydration and embedding. For low-temperature embedding in Lowicryl K4M [16-18]. the tis­ sues were dehydrated in a graded series of cthanols by progressively lowering the temperature to -35 °C. The tissues were then infiltrated with Lowicryl K4M at -35 °C overnight. The tissues were embedded in gelatin capsules and allowed to polymerize by ultraviolet irradiation for 2 days under -35 °C in a polymerization chamber. After that the tissues were removed from the freezer, and polymerization was continued with ultraviolet light (curing) for 2-3 more days at room temperature. Staining Procedure fo r Postembedding Labelling o f Glycoconjugates on Thin Sections Thin sections of Lowicryl-K4M-embcddcd tissues were cut and picked up by 200-mesh Formvar-coated or uncoated nickel grids. The gritls were first passed through a series of 3 changes of filtered double glass distilled water for a total of 15 min. They were then treated with 3 changes of 0.05 M Tris buffer solution (TBS). pH 8.2. containing 1% bovine serum albumin (BSA) for 5 min each, before they were in­ cubated to demonstrate the glycoconjugates by either the direct or in­ direct lectin-gold complex methods. Direct Labelling. The method as described by Roth |12. 13| was adapted for present studies. Several lectin-gold complexes were used, includingconcanavalin (Con A), wheat germ agglutinin (WGA). pea­ nut agglutinin (PNA) and Ulexeuropaeus isolectin I (UEA-I). They were diluted by 0 .1 M TBS containing 1% BSA. Thin sections were incubated with lectin-gold complex (1:50 dilution) in 0.1 A7 TBS con­ taining 1% BSA for 1 h at room temperature o ral 4 °Covernight in a moist chamber. The size of gold particles used was 20 nm for Con A and 10 nm for WGA. PNA and UEA-I. After staining with lectingold. the grids were ‘jet'-washed with 0.05 A/ TBS or floated on drops of TBS for 3 changes. 5 min each. The grids were then stained with uranvl acetate (3—4 min) and lead citrate (1-2 min). Indirect LabellingIStreptavidin-Gold Method. The staining proce­ dures were adapted from methods as described by Roth [13] and Bon­ nard et al. |19|. Several biotinylated lectins, including Con A. WGA. Griffoniusimplicifolia (GS-11). soybean agglutinin (SBA). PNA. Ricinus communis agglutinin isolectin I (RCA-I). G. simplicifolia isolectin B, (GS-I-B,), UEA-1 and Pluiseolus vulgaris agglutinin P (P1IA-P). were used. After washing in distilled water and 1% BSA in 0.1 M TBS. pH 8.2. the nickel grids with thin sections were floated on drops of biotinylated lectins (10 pm/ml) in 0.05 M TBS. pH 7.6. containing 0.1 mM CaCI. and MnCI, for 1-2 h at room temperature in a moist chamber. They were washed in 0.05 M TBS. pi I 7.6. for a total of 15 min. The lectin-binding sites were then visualized by incubating with streptavidin-gold (1:50 dilution) in 0.05 A/ TBS, pH 8.2. containing 1% BSA for 1 h at room temperature. The grids were stained with uranyl acetate and lead citrate.

Glycoconjugates of the Lateral Prostate

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

Introduction

Results The fine structure of the lateral prostate has been reported elsewhere [20J and will not be repeated here. In the lateral prostate, gold particles were usually found over the microvilli, secretory granules, lysosomes, the Golgi apparatus, granular (rough) endoplasmic reticulum (GER) of the glandular cells, extracellular material, basal lamina and the sarcoplasm of smooth muscle cells. The locations and densities of gold particles varied somewhat in different structures depending on the lectin used. Most of the lectingold labelling was more or less comparable to the results of light-microscopic lectin staining 114]. Glucosyl- and Mannosyl-Specific Lectin Çoncanavalin A. The microvilli, secretory granules, GER and the lysosomes were heavily labelled by Con-Agold particles (fig. 1. 2). The secretory granules were vari­ able in size and electron densities. The membrane as well as the granular contents of those secretory granules with mod­ erate electron density were more densely labelled (fig. I). The labelling density of gold particles varied among indi­ vidual secretory granules. In some granules, the gold par­ ticles were confined mainly to the limiting membranes (fig. I ). In the Golgi apparatus, a moderate number of gold particles was observed in the cis cisternac. the associated condensing vacuoles and mature secretory granules (fig.3). Con-A-binding sites were present in the GER. especially in the basal cytoplasm (fig.2). The gold particles were seen lying over the cisternal space and the membrane-bound ribosomes. The Con-A-positivc staining of the entire cyto­ plasm of the epithelial cells observed light microscopically corresponded to the positive reaction over the GER. The lateral and basal plasma membranes of secretory epithelial cells were Con A positive, but there were no gold particles over the junctional complexes. The basal cytoplasm was also intensely labelled with Con A. The gold particles were seen over the lysosomes, the small cytoplasmic vesicles and the free ribosomes. Labelling was found over the nuclear envelope, condensed (hetcro-) chromatin and the granular region (pars amorpha) of the nucleolus (fig.4). No labelling was observed at the basal lamina, while weak labelling was observed at the extracellular matrix in the reticular layer.

The plasma membrane and the cytoplasm of the fibroblasts were also positive. In the smooth muscle cells, intense labelling occurred over the free ribosomes and the small vesicles in the sarco­ plasm. The caveoli (small pinocytotic vesicles) and the sarcolemma were also labelled (fig.5). The capillary endothe­ lium was labelled over the cytoplasm, small vesicles and the plasma membrane at both the luminal and stromal sides (fig-6). N-Acetylglucosaminyl-Specific Lectins Wheat Germ Agglutinin. The staining pattern was simi­ lar to that due to Con A. The microvilli, secretory granules, the Golgi apparatus, lysosomcs and the lateral plasma membrane were moderately labelled with WGA. The granular contents of the electron-dense secretory granules showed positive labelling, whereas the labelling of the secretory granules with less electron-dense contents was sparse to negative (fig.7). In the Golgi apparatus moderate numbers of particles were detected on the medial to trans cisternae as well as the Golgi-associated vesicles and con­ densing granules of various sizes (fig. 8). The GER showed no staining. The lateral plasma membrane was WGA posi­ tive. but the staining was uneven. Moderate labelling was usually found in the lower and basal portions (fig.9). Weak staining was observed in the basal lamina and the underly­ ing matrices. The plasma membrane and the cytoplasm of the vascular endothelium was positively labelled (fig. 10). In the smooth muscle cells, WGA-binding sites were also observed in the basal lamina, sarcolemma and the nucleus. Griffonia simplicifolia. Weak reaction with GS-ll was detected over the microvilli and the secretory granules (fig. 11). The Golgi apparatus was weakly stained, and gold particles were equally seen over the trans cisternae as well as the condensing vacuoles. However, the density of gold particles varied from cell to cell. GS-II-binding sites were also detected in the GER. mainly over the membranebound ribosomes (fig. 12). Heterochromatin and the nucleolus were also positively labelled. The labelling was uneven over the basal lamina and the underlying matrix (fig. 13). Positive labelling was found over the myofila­ ments and the small vesicles of the smooth muscle cells (fig. 14) and also over the capillary endothelium and peri­ cytes. N-Acetylgalactosaminyl- or Galactosyl-Specific Lectins Soybean Agglutinin. Intense to moderate labelling was seen over the microvilli and the secretory granules. The labelling over the secretory granules was restricted mainly

29

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

Control sections were prepared either by omitting the lectin-gold complexes (or biotin-labelled lectin) or by incubating the lectin solu­ tions with their specific competing sugars (0.2 M) for I h prior to appli­ cation of the lectins. The stained sections were then examined with a Philips EM 3(H) Electron Microscope at 60 kV or a Jeol JEM 2(HH) EX at 80 kV.

Fig. 3. Sparse labelling of Con-A-binding sites is seen over the Golgi cisternae. mainly on the cis side. A few gold particles are also present over the membrane of condensing vacuoles (CV) on the trans side of the Golgi apparatus. x 31.200. Fig. 4. The heterochromatin, nuclear envelope and the granular region (pars amorpha. PA) of the nucleolus exhibit positive labelling with Con-A-gold. x32.(X)0.

to the limiting membrane; the granular contents showed few gold particles (fig. 15). As with GS-II the Golgi appara­ tus was positively stained, and the SBA-binding sites were mainly located at the trans cisternae. In the basal cyto­ plasm. gold particles were observed over the endocytotic vesicles associated with the basal plasma membrane, lyso­ somes and the lateral plasma membrane. Positive reaction was also found over the basal lamina of both the epithelium and the capillary, extracellular matrices, the surface of fibroblasts, sacroplasm and small endocytotic vesicles of the muscle cells (fig. 16) and the endothelium. Peanut Agglutinin. The microvilli were intensely labelled with gold particles. Hie membranes of the secre­ tory granules showed positive labelling (fig. 17). and the matrix of the granules was also positive. The condensing vacuoles and the Golgi saccules at the maturing faces were

intensely labelled, whereas the Golgi cisternae at the form­ ing faces were mostly unlabelled (fig. 18). However, the labelling intensity seen varied among individual cells. No gold particles were seen over the GER. Weak labelling was shown over some cytoplasmic vesicles. The lateral plasma membranes were moderately labelled. The epithelial basal lamina was unevenly labelled. The fibrillar material as well as the surfaces of collagen fibrils in the lamina propria showed positive PNA-binding sites (fig. 19). Cell surfaces of fibroblasts were also positively labelled. Ricinus communis Isolectin I. RCA-1 intensely stained the microvilli and secretory granules. The staining of the secretory granules was mainly confined to their perimeters (fig.20). Moderate labelling intensity was found over the trans cisternae, condensing vacuoles and associated vesi­ cles of the Golgi apparatus. RCA-I also bound to basal and

30

Chan/Wong

Glvcoconjugatcs of the Lateral Prostate

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

Fig. 1. Con A. This micrograph shows that the microvilli (MV) and the various secretory granules (SG) are densely labelled. Weak labelling is also seen on the lateral plasma membrane, x 32.000. Fig. 2. Micrograph showing the Con-A-binding sites over the GER. Gold particles can be found over both the cisternae and the membrane-bound ribosomes. X 34.400.

Fig. 5. T lie sarcotubulcs and free ribosomes in the sarcoplasm (SP) of the smooth muscle cell are densely labelled with Con A. The small pinocvtotic vesicles (caveoli. C) and the sarcolemma are also positively labelled. X 32.100. Fig. 6. I lie capillary endothelium (NE) shows positive staining with Con A. Dense labelling is seen over the cytoplasmic vesicles and the cytoplasm of a pericyte (P). Gold particles are also found on the plasma membrane of both the endothelium and a pericyte, x 42.80(1.

Fig. 7. The microvilli (MV) show moderate labelling with WGAgold. Gold particles are seen over the electron-dense secretory gran­ ules (SG). x 41,500. Fig. 8. WGA-gold. This micrograph shows that gold particles are mainly seen over the trails cisternac of the Golgi apparatus (G) and small vesicles on the trans side, x 36.700.

lateral plasma membranes, and the cytoplasm adjacent to the basal plasma membrane but not to the basal lamina. The sarcoplasm, the sarcolemma and the extracellular materials between the smooth muscle fibres were also RCA-I positive. Griffonia simplicifolia Isolectin I 6 4. No GS-l-B4-binding sites were seen in any of the intracellular compartments of the epithelial cells. Positive labelling was observed in the capillary endothelium and fibroblasts. Small pinocytotic vesicles and the cytoplasm of the endothelial cells were stained, while the plasma membrane was weakly labelled.

occasional gold particles (fig.21). The staining of the Golgi complex was weak, and only the trans cistcrnae as well as some condensing vacuoles were stained with UEA-1. Weak labelling was also observed on the basal plasma membrane and adjacent cytoplasm. The basal lamina was again nega­ tive. but the collagen fibrils in the reticular layer were posi­ tively labelled with UEA-1. The muscle cells and the endo­ thelium were UEA-I negative.

31

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

Fucosyl-Specific Lectin U. europaeus Isolectin I. The microvilli and luminal plasma membrane reacted positively with UEA-1. The secretory granules were largely negative or showed only

Complex Carbohydrate-Specific Lectin P. vulgaris Agglutinin P. The microvilli were densely labelled with PHA-P. whereas most of the electron-dense secretory granules were negative (fig.22). A few granules with diffuse granular contents were labelled at their peri­ meters. No labelling was seen over the Golgi cisternac; however, a few small vesicles on the trans side were sparse-

Fig. 9- WGA-goId. Micrograph showing the gold particles in the lower part of the epithelial cell, lateral plasma membrane and the cyto­ plasm. Weak and uneven labelling is also noted in the basal lamina (B L ). x 36.7(H).

Fig. 10. The luminal membrane (arrow) of the capillary' endothe­ lium shows positive reaction with WGA-gold. Weak labelling can be seen over the cytosol and the heterochromatin of the nucleus. X41,100.

Fig. 11. T he microvilli (MV) and the secretory granules are weak­ ly labelled by GS-Il. Few gold particles are found in the cytoplasm. X32.100. Fig. 12. GS-Il gold. Micrograph showing the gold particles over the GER. X30,000.

Discussion

32

Chan/Wong

The ultrastructural study of glycoconjugates by lectingold complexes (or biotin-labelled lectin-avidin gold sequence) demonstrated precisely the lectin-binding sites in different subcellular compartments. There has not been any study concerning the subcellular distribution of lectin receptors in the male accessory sex glands. To the best of our knowledge this is the first application of lectin-gold his­ tochemistry in combination with a low-temperature embedding technique using Lowicryl K4M to the study of glycoconjugates in the prostate gland. The results arc in general agreement with the light-microscopic findings [ I4).

Glycoconjugates of the Lateral Prostate

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

ly labelled. The GER in the supranuclear region was nega­ tive. but weak labelling was found over the GER in the basal half of the cells. The lateral and basal membranes as well as the basal cytoplasm were labelled with PH A-P. The basal lamina and the underlying collagen fibrils were also positively labelled (fig.23). The fibroblasts were strongly labelled both at the plasma membrane and cytoplasm. Positive reaction was seen over the sarcolemma and sarco­ plasm at the juxtanuclear region of the smooth muscle. Finally, the endothelium, pericytes and the basal lamina of the capillary were strongly positive (fig.24).

Methodology The present study utilized a postembedding method [12, 15, 21-23] for the ultrastruetural visualization of lectin­ binding sites of the lateral prostate using colloidal gold as marker. The great advantage of postembedding staining is that it allows a wide range of lectins to be tested on a single block of tissue, which is not possible with pre-embedding tech­ niques. Moreover, it is possible to perform double or multi­ ple labelling of different lectin-reactive sites on the same thin sections. The problem of uneven penetration of reagents into the cells which is common in the pre-embed­ ding technique is not encountered in postembedding stain­ ing. However, the hydrophobic properties and extensive

Fig. 15. This micrograph shows the SB A labelling of the microvil­ li. In the secretory granules (SG) gold particles are found mainly on the membrane or periphery of the granules, x 30,300. Fig. 16. SBA-binding sites in the smooth muscle are detected over the heterochromatin of the nucleus (N), the sarcoplasm (SP) in the juxtanuclcar region as well as the small endocytotic vesicle near the sarcolemma. X35.000.

cross-linking of epoxy resins, which block the penetration of reagents and mask the lectin-binding sites, limit their use in postembedding staining. With the recent introduction of hydrophilic resins, Lowicryl K4M and LR White [ 12. 18,24.25]. the problems of penetration and loss of antigenicity arising from the preparation and etching procedure have been largely over­ come. Owing to their high atomic weight, gold particles appear highly electron dense when viewed in the electron microscope which allows precise localization of lectin-reac­ tive sites. Moreover, quantitative evaluation of the staining intensity can be obtained by counting the number of gold particles per square micrometre [12],

33

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

Fig. 13. T his micrograph shows the positive labelling of the basal lamina (BL) and the underlying matrix. GS-II-binding sites are also detected over the cytosol. X41.000. Fig. 14. GS-II binding sites are demonstrated over the myofila­ ments of the smooth muscle (smc). x 36.700.

Nucleus In the present study, Con-A-hinding sites were detected in the nuclei of secretory cells and stromal fibroblasts of the prostate gland. Con-A-binding sites were preferentially associated with the heterochromatin. nuclear envelope and the granular region of the nucleolus. This is in agreement with the biochemical finding that Con A binds to a number of nuclear non-histone glycoproteins in the chromatin [26], These glycoproteins are believed to play some role in the regulation of gene expression. Lens culinares agglutinin (LCA). having a similar sugar specificity to Con A. has been reported to bind to sea urchin embryo chromatin [27]. Furthermore Con-A-binding sites have been observed in

34

Cluin/Wong

Fig. 19. PNA-gold. Micrograph showing the epithelial-stromal interface. Gold particles arc seen over the loosely packed collagen fibrils as well as the fibrillary matrix (*). Uneven labelling is also observed over the epithelial basal lamina (BL) and the cell surface of fibroblast (arrow). x 36,31X1. Fig. 20. Micrograph showing the distribution of RCA-l-binding sites on the microvilli (MV) and the secretory granules. Note that the labelling of the granules is more concentrated at their perimeter (arrow), x 35,7(X).

the nucleus of goblet cells of the chick duodenum [ 12]. However, the distribution of nuclear glycoproteins and their functional implication remains largely unknown. Rani’ll Endoplasmic Reticulum ' Con-A-binding sites were demonstrated over the cister­ nae, as well as the membrane-bound ribosomes, of the GER. Besides Con A receptors, receptors for GS-Il were also observed over the GER of the lateral prostate, indicat­ ing the presence of Man and GIcNAc in this structure. The Con A labelling of the GER was more intense in the basal and nuclear regions. It is known that glycosylation of aspar-

GJycoeonjugates of the Lateral Prostate

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

Fig. 17. In the apical cytoplasm. PNA-blnding sites are demonstrated over the microvilli (MV) and secretory granules (SG). In the secretory granules, the labelling is mostly seen over the limiting mem­ branes. x31.(XX). Fig. 18. In the Golgi apparatus (GA). intense PNA labelling is shown over the condensing vacuoles (arrows), the cytoplasm and the Golgi cisternae at the maturing face. x29.(XK).

Fig. 23. Micrograph showing the PHA-P-reactive sites in the basal part of an epithelial cell. PHA-P-binding sites are located in the basal cytosol, small endocytotic vesicles and basal plasma membrane. Gold particles arc also detected on the basal lamina (BL) and the collagen fibrils (C). x4l.. Fig. 24. Micrograph showing the PHA-P-positive reaction of an endothelial cell (E) and pericyte (P). Note also the presence of gold particles on the basal lamina (BL). x 32.1(X).

agine N-linked oligosaccharide chains of membrane-bound and secretory glycoproteins occurs in the GER. It involves a stepwise assembly of the oligosaccharide chain on dolichvl phosphate followed by en bloc transfer of the oligosac­ charides of N-linked glycoproteins initiated in the GER and completed in the Golgi apparatus [28-30]. However, glycosylation of O-linked oligosaccharides occurs only in the Golgi apparatus [31]. The newly translated and glycosy­ lated proteins are formed within the cisternal lumen of the endoplasmic reticulum with their oligosaccharide chains extending into the lumen. There are two subsets of chains in N-glycosidic linkage, i.e. poly-Man and complex chains, both containing Man at the branching point and linked by GlcNAc to the asparagine. The observation of Con A and

GS-11 binding at the cisternac of the GER confirm the pres­ ence of Man and GlcNAc residues in this structure, which is in agreement with light-microscopic observations [14]. Similar Con A reactivity in the GER has been reported in the goblet cells of the chick duodenum [12]. rat exocrine pancreatic cells [32] and rat epiphyseal chondrocytes [33]. In the present study Con-A-binding sites were observed only in the cisternal space of the GER but also bound to the membrane-associated and free ribosomes suggesting the presence of Man-containing glycoconjugates in the ribo­ somes. The nature of these glycoproteins and their impor­ tance to the function of membrane-attached and free ribo­ somes is not known. It is known that ribosomes are attached to the membrane of the GER by some membrane

35

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

Fig. 21. UEA-I-gold. Micrograph showing the apical part of the epithelial cells. Intense to moderate labelling is present over the microvilli (MV). The labelling of the secretory granules (SG) is sparse or absent, x 48,000. Fig. 22. I he microvilli arc strongly labelled with PHA-P. How­ ever. the labelling of the secretory granules is sparse or absent, x 31,000.

Golgi Apparatus The present study has shown that the epithelial Golgi apparatus of the lateral prostate is rich in Man. GIcNAc. Gal. GalNAc and fucose as indicated by its positive reactiv­ ities to most of the lectins tested including Con A. WGA. GS-II. SBA. PNA. RCA-1 and UEA-I. Variable intensity in the labelling of the Golgi saccules was noted, indicating variation in the degree of glycosylation activity among indi­ vidual epithelial cells. Con-A-binding sites were mostly seen over the cisternac at the forming face (cis to medial), whereas the binding sites for WGA. GS-II, PNA. RCA-I and UEA-1 were mostly observed in the Golgi saccules in the maturing face (medial to trans). In some cases, lectin-binding sites (Con A. WGA and RCA-I) were distributed over all Golgi sac­ cules. This can be explained by the fact that glycoproteins may contain more than one kind of oligosaccharide chain. It is now known that oligosaccharide elongation occurs during sequential movement through the Golgi stack from cis to trans with the stepwise addition of sugars to the grow­ ing oligosaccharides by membrane-bound glycosyltransferases [35, 36]. It has been suggested that the Golgi saccules contain at least three functionally distinct compartments, i.e. cis. medial and trans. The trimming of N-linked oligo­ saccharides from glycoproteins by glucosidases and initial glycosylation of O-linkcd glycoproteins takes place in the cis cisternac. while the formation of the core region of the oligosaccharide chain, and the completion of chain growth by addition of terminal sugar residues, usually Gal. Gal­ NAc and sialic acid, occur in trans cisternae [37. 38]. There is a number of published electron-microscopic studies on the lectin binding of oligosaccharide chains at different stages of their maturation in the Golgi apparatus. Several researchers [ 12,39.40] have reported Con-A-binding sites within the cis cisternae of the Golgi apparatus. On the other hand, Griffiths ct al. |4I] observed a nearly uni­ form distribution of Con-A-binding sites in the Golgi appa­ ratus of hamster kidney cells, while Wasano ct al. [42] reported no Con-A-binding sites in the Golgi apparatus of mucus-secreting cells of the hamster trachea. It is known that Con A binds tightly to Man-rich precursor oligosaccha­ ride chains, to complex Man-type oligosaccharides found in membrane glycoprotein and to lysosomal enzymes. It does not bind to complex chains with three or more GIcNAc branches [37, 43. 44]. The initial transfer of Nlinked precursor oligosaccharides takes place in the GER.

36

Chan/Wong

This oligosaccharide consists of three glucose, nine Man and two N-GlcNAc (Glc-,ManvGlcNAc:). Initial trimming of the oligosaccharides by glucosidases occurs in the GER. After reaching the Golgi complex, the high-Man precursor chains on the cisternal membrane and the secretory glyco­ proteins are partially degraded by mannosidases and re­ modelled by a number of glycosyltransferases [38]. A num­ ber of membrane-bound mannosidases have been identi­ fied in the Golgi apparatus [45. 46). This might explain the Con A binding in the Golgi saccules of the glandular cells of the prostate gland. WGA. which recognizes both GIcNAc and sialic-acidterminating oligosaccharides, bound mostly to the cister­ nae in the maturing face. Tartakoff and Vassalli [39], Grif­ fiths et al. [411and Dunphy et al. [47] have demonstrated by immunocytochcmistry that N-acetylglueosaminosyltransferase was localized in 2-3 medial cisternae. Hcdman et al. [48] have demonstrated the presence of sialoglycoconjugates by Limax flavus agglutinin (LFA) in the trans cisternae of Swiss 3T3 cells. Electron-microscopic autora­ diography has shown that sialic acid is incorporated in the Golgi stack, possibly on its trans side [49]. Roth et al. [50] have reported the ultrastructural immunolocalization of sialvltransferase in one or two trans cisternae in the intesti­ nal epithelial cells and rat hepatocytes. The results of the present study are in general agreement with these previous findings. The present study has demonstrated in the lateral pros­ tate the presence of Gal residues in the Golgi cisternae at the maturing face as well as the condensing vacuoles as shown by their reactivities toward the galactosyl-specific PNA and RCA-I. Compared with RCA-I. PNA exhibited a stronger reactivity to the Golgi cisternae. This is consis­ tent with the current view that galactosyltransférase and sialvltransferase are present in the maturing face of the Golgi stack and that they arc responsible for the addition of Gal and sialic acid residues to the terminal ends of the oli­ gosaccharides [51-53]. Sato and Spicer [54]. using a pre-embedding PNAhorseradish-peroxidasc method, demonstrated the pres­ ence of Gal residues only in the medial cisternae of mouse gastric cells. A number of studies have shown that RCA-I is located in the medial and trans cisternac [40. 41]. Using double-labelling techniques, the galactosyltransferasebinding sites have been shown to coincide with the pres­ ence of thiamine pyrophosphatase in the trans cisternae [15. 55]. Wong and Tam [56, 57] have demonstrated cytochemically thiamine pyrophosphatase activity in the trans cisternae of epithelial cells in the guinea pig seminal vesicle. This indicates a possible co-distribution of galactosyltrans-

Glycoconjugates of the Lateral Prostate

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

glycoproteins (ribophorins) [34]. However, the relation­ ship of Con A binding with these membrane glycoproteins remains to be elucidated.

Secretory Granules The secretory granules showed different reactivities to lectins over the granular matrix and their limiting mem­ branes. Most of the lectin-binding sites of the secretory granules were confined to the limiting membranes. The limiting membranes of the secretory granules showed posi­ tive binding with Con A. SBA. GS-I1. RCA-1. PNA and PH A-P. while the granular matrix was labelled with Con A and WGA. No UEA-l-binding receptors were seen over the granules in the lateral prostate. Biochemical studies have demonstrated the presence of membrane-bound glycoproteins on the membranes of zymogen granules of the rabbit parotid gland [58, 59]. exocrine pancreas [60-62] and chromaffin granules of the adrenal medulla from the rat and ox [63. 64). Lectin histo­ chemistry has also shown the presence of Con A receptors in the membrane of somatotrophic granules from the adenohypophysis [65], RCA receptors on azurophilic gran­ ules from polymorphonuclear leucocytes [66] and WGAbinding sites on the outer membrane of chromaffin gran­ ules from the adrenal medulla [67]. Neiss [68] has demon­ strated histochcmically the presence of a layer rich in vicinol-glycol groups on the inner surface of the limiting mem­ brane in many secretory granules including eosinophilic granules, mast cell granules, lysosomes, renin granules and zymogen granules. The function of these membrane-asso­ ciated glycoconjugates is not known. It has been suggested that they may influence the physiological properties of the membranes such as fluidity, surface charge density anti membrane permeability. They could also play a role in the intracellular regulation of granule transport and their fusion with the apical plasmalemma and in the transport of plasmalemma glycoconjugates to the apical plasma mem­ brane [69. 70). As lectin reactivities of the membranes of the secretory granules were similar to those of the apical plasmalemma and microvilli, it was likely that these mem­ brane-associated glycoconjugates of the secretory granules may be involved in the transport of the plasma membrane glycoconjugates.

Lysosomes The lysosomes found in epithelial cells showed positive reactivities to Con A and WGA in both the limiting mem­ brane and the matrix. Schulte et al. [71) have reported the binding of LCA and Pisum sativum agglutinin (PSA) to lysosomes in the ciliated and secretory cells of the human fallopian tube. These two lectins share a similar specificity to Con A but are more specific for the core region of Nlinked oligosaccharides. It has been well established by bio­ chemical studies that most of the acid hydrolases in lyso­ somes arc glycoproteins [72], Neiss [68] has shown the pres­ ence of a layer rich in vicinol-glycols on the inner surface of lysosomal membranes. Biochemical studies have also shown the existence of glycoproteins in the membranes of lysosomes [73—75], The function of the glycoconjugates on the lysosomal membrane is believed to be a chemical bar­ rier to prevent the attack of membrane by hydrolases [76]. Microvilli The glycoconjugates (glycocalyx) over the microvilli and the apical plasma membrane of the lateral prostate are rich in Man. GlcNAc. GalNAc, Gal. fucose and complex carbohydrates as shown by their positive reactions to Con A, WGA. GS-II. SB A. RCA-I. PNA, UEA-1 and PHA-P. Their reactivities to different classes of lectins indicate the diversity and complexity of the glycocalyx over the apical plasma membrane. Light-microscopic carbohydrate histochemical study has demonstrated that the apical surfaces of the prostatic epithelial cells are rich in vicinol-glycol-containing sugars, including Man. glucose. Gal and fucose. as shown by their strong periodic acid-Schiff reaction [77], A number of functions have been attributed to the gly­ cocalyx in different epithelia. The best example is the gly­ cocalyx in the brush border of the intestinal absorptive cells w'here. besides acting as a protective layer to the cells against the proteolytic enzymes and acid in the chyme, it also contains a number of digestive enzymes [78]. The epi­ thelial cells of the renal tubules exhibit marked variability in their glycocalyx which is related to their specialized func­ tions [79, 80]. Earlier works on ultrastructural histochemistry using cationic substances such as ruthenium red. cationic ferritin and colloidal iron I have revealed that the glycocalyx is rich in complex carbohydrates [81]. Lectins have also been applied to characterize the glycoconjugates in the glycoca­ lyx of various epithelial cells bearing microvilli [12.23. 54. 82-86], Roth and Taatjes [84] have reported differences in LEA binding in the glycocalyx of different segments of renal tubules which may be correlated with their functional activity.

37

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

ferasc and thiamine pyrophosphatase at this site in the guinea pig and may explain the distribution of Gal residues at the Golgi cisternae at the maturing face. Lectin-gold cytochemistry in combination with a lowtemperature embedding technique using Lowicryl K4M, as shown in the present study, is very useful in the study of the distribution of oligosaccharide products within the Golgi stack. It has also provided further evidence in support of the compartimentai organization of the Golgi apparatus [47].

Differences in the sugar content of the glycocalyx have also been detected at different stages of cell differentiation and maturation. During differentiation of embryonic gas­ tric mucoid cells, there is an increase in carbohydrate com­ ponents in the apical glycocalyx which parallels the increase in the number of secretory granules [83|. The lec­ tin reactivities of the microvilli and the apical plasmalemma are similar to those of the limiting membranes of the secre­ tor)' granules suggesting that the limiting membranes of the secretory granules may play a role in transport of the carbo­ hydrate material to the apical plasmalemma [69]. Basal Lamina and Extracellular Matrix The epithelial basal lamina of the lateral prostate showed rather weak reactivities to the lectins tested includ­ ing WGA. GS-II, SBA. PNA and PIIA-P. The underlying fibrillary matrix in the reticular layer also showed weak reactivity. PNA showed the strongest reaction among these lectins. Positive binding was seen over the collagen fibrils, some microfibrillary matrix, basal lamina and the sarcolcmma of smooth muscle ceils. The chemical nature of these PNA-binding sites is not clear: however, oligosaccha­ rides of chondroitin sulphate can react with PNA [87], The positive PNA reaction in the present study may be due to the presence of chondroitin sulphates which arc shown by cationic dyes to be present in the reticular layer and lamina propria [88. 89]. The application of lectins to the ultrastructural cytochemical study of glycosaminoglycans and proteoglycans has been very limited so far. On the other hand, a number of light-microscopic lectin histochemical studies have shown that many lectins bind intensely to stromal connec­ tive tissue as well as to basement membranes [ 14. 90-92]. Takagi ct al. [87] and Velasco et al. [93] have reported that chondroitinase-ABC digestion prior to lectin staining is necessary in order to expose the Gal residues from the link­ age regions. It is. therefore, possible that the lectin-reactive sugar residues or the oligosaccharides of the proteoglycans

arc masked during tissue processing for electron micros­ copy resulting in a low labelling of the extracellular matrix in both glands. Further cytochemical study, such as enzyme digestion, is necessary in order to explore further the use of lectins in glycosaminoglycan or proteoglycan histochemis­ try. Capillaries The capillary endothelia showed positive reactions to Con A. WGA. SBA. RCA-I. GS-I-B, and PHA-P over the luminal-abluminal (stromal) plasmalemma, cytoplasmic vesicles and the pericytes. Differential distribution of lectin receptors was seen on the luminal and abluminal plasma­ lemma of the endothelial cells. Relatively more WGA- and SBA-binding sites were seen over the luminal surface, whereas the abluminal plasmalemma and the endothelial basement membrane contained more Con A and PHA-P receptors. This may indicate a structural polarity of the cap­ illar)-endothelium in terms of their glycoconjugates. How­ ever. the function of the lectin-binding sites and their differ­ ential location in the endothelial cells is at present unknown. Differential labelling of luminal and abluminal plasmalemma of endothelial cells with Con A and Helix pomatia agglutinin (HPA) has been described in mouse brain vessels [94]. Cytochemical observations have shown the differential distribution of enzymes on each side. It has been reported that alkaline phosphatase is present on the luminal plasmalemma [94, 95], whereas on the abluminal surface. 5'-nucleotidase, adenosine triphosphatase and adenylate cyclase arc present [95. 96|. It has also been shown that the plasma-membrane-bound enzyme, 5'nucleotidasc. contains Con-A-binding sites [97. 98]. How­ ever. no enzyme histochemistry or immunocytochemistry has been performed so far on the capillaries or lymphatics in the male accessory sex glands. It remains unclear whether or not the lectin-binding sites located in the capil­ lary endothelia and pericytes are correlated with the pres­ ence of any membrane-associated enzymes.

References

38

3 Howard DR. Ferguson P. BatsakisJG: Carci­ noma-associated cylostruclural antigenic alter­ ations: Detection by lectin binding. Cancer 1981:47:2872-2877. ’ 4 Zieske .ID. Bernstein IA: Modification of cell surface glycoprotein: Addition of fucosvl resi­ dues during epidermal differentiation. J Cell Biol 1982:95:626-631.

Chan/Wong

5 Olden K. Parent JB. White SL: Carbohydrate moieties of glycoproteins. A re-evaluation of their function. Biochim Biophys Acta 1982: 650:209-232. 6 Tcichbcrg VI. Silman I. Bcitsch DD. Rcshelf G: A fi-D-galactoside binding protein from electric organ tissue of F.tcctwphorus clectriaix. Proc Natl Acad Sci USA 1975:72:1383-1387. 7 Brown JC. Hunt RC: Lectins. Int Rev Cvlol 1978:52:277-349.

Glycoconjugatcs of the Lateral Prostate

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

1 Newman RA. Klein PJ. Rudland PI); Rinding of peanut lectin to hreast epithelium, human carcinomas, and a cultured rat mammary stem cell: Use of lectin as a marker of mammary dif­ ferentiation. J Natl Cancer Inst 1979:63: 1339-1346. 2 Atkinson P. I lakimi .1: Alterations in glycopro­ teins of tile cell surface: in Lennar/. W.I (ed): The Biochemistry of Glycoproteins and Pro­ teoglycans. New York. Plenum Press, 1980. pp 191-239.

24 Newman GR. Jasai B. Williams ED: A simple post embedding system for the rapid demon­ stration of tissue antigens under the electron microscope. Histochcm .1 1983:15:54.3-555. 25 Newman GR. Jasani B: Post-embedding immunoenzyme techniques: in Polak JM. Varndell IM (eds): Immunolabelling for Elec­ tron Microscopy. New York. Elsevier. 1984. pp 53-70. 26 Rizzo WB. Buslin M: Lectins as probes of chromatin structure. Binding of concanavalitt A to purified rat liver chromatin. .1 Biol Client 1977:252:7062-7067. 27 Sevaljecvic L. Petrovic SL. Petrovic M: Bind­ ing of lens culinaris lectin to sea urchin embryo chromatin. Experientia 1979:35:193-194. 28 Hanover JA . Lennarz WJ: Transmembrane assembly of membrane and secretory glycopro­ teins. Arch Biochem Biophys 1981:211:1-19. 29 Lennarz WJ: Protein glycosylation in the endo­ plasmic reticulum: Current topological issues. Biochemistry 1987:26:7205-7210. .30 llirschhcrg CB. Snider MD: Topography of glycosylation in the rough endoplasmic reticu­ lum and Golgi apparatus. Annu Rev Biochem 1987:56:63-87. 31 Johnson DC. Spear PG: O-linked oligosaccha­ rides arc acquired hv herpes simplex virus gly­ coproteins in the Ciolgi apparatus. Cell 1983:32:987-997. 32 Pinto da Silva P. Torrisi MR. Kachar B: Freeze-fracture cytochemistry : Localization of wheat germ agglutinin and concanavalitt A binding sites on freeze-fractured pancreatic cells. J Cell Biol 1981:91:361-372. 33 Velasco A. Hidalgo J: Light and electron microscopical localization of concanavalitt A lectin binding sites in rat epiphyseal chondro­ cytes. Histochcm J 1987:19:7-14. 34 Kreihich G. Ojakian G. Rodriguez-Boulan E. Sabatini DD: Recovery of ribophorins and ribosomes in inverted rough vesicles derived front rat liver rough nticrosomes. J Cell Biol 1982:93:111-121. ' 35 Letts PI. Pintcric L. Schachter II: Localization of glycoprotein glvcosyltransferascs in the Golgi apparatus of rat and mouse testis. Biocltim Biophys Acta 1974:372:304-320. 36 StrousGJAM : Initial glycosylation of proteins with acetylgalactosamine! serine linkages. Proc Natl Acad Sci USA 1977;76:2694-2698. 37 Dtinphy VVG. Rothman JE: Compartmentation of asparagine-linked ologosaccltaride pro­ cessing in the Golgi apparatus. J Cell Biol 1983:97:270-275. 38 Kornfeld R. Kornfeld S: Assembly of aspar­ agine-linked oligosaccharides. Annu Rev Bio­ chem 1985:54:631-664. 39 Tartakoff AM. Vassalli P: Lectin-binding sites as markers of Golgi subcompartments: Proximal-to-distal maturation of oligosaccharides. .1 Cell Biol 198.3:97:1243-1248. ' 40 Pavelka M. Ellinger A: Localization of binding sites for concanavalitt A. Riciiuts communis I and Helixpomaliu lectin in the Golgi apparatus of rat small intestinal absorptive cells. J I listochem Cytochem 1985:33:905-914.

4] Griffiths G. Brands R. Burke B. Louvard D: Viral membrane proteins acquire galactose in traits Golgi cisternae during intracellular trans­ port. J Cell Biol 1982:95:781-792. 42 Wasano K. Jankamura K. Yamamoto T: Lec­ tin-gold cytochemistry of mucin oligosaccha­ ride biosynthesis in Golgi apparatus of airway secretory cells of the hamster. Anat Rec 1988; 221:635-644. 43 Baenziger JU . Fiete D: Structural determi­ nants of concanavalin. A specificity for oligo­ saccharides. J Biol Ghent 1979:254:2400-2407. 44 Goldberg DE. Kornfeld S: Evidence for exten­ sive subcellular organization of asparaginelinked oligosaccharide processing and lysosom­ al enzyme phosphorylation. Prix' Natl Acad Sci USA 1986:258:3159-3165. 45 Tulsiani DRP. Oplicim D.l. lousier O: Purifi­ cation and characterization of o-mannosidase from rat liver Golgi membranes. J Biol Chem 1977:252:3227-3233. 46 Tabas I. Kornfeld S: Purification and char­ acterization of a rat liver Golgi cv-mannosidasc capable of processing asparagine-linked oligosaccharides. J Biol Client 1979:254: 11655-11663. 47 Dunphy WG. Brands R. Rothman JE: Attach­ ment of terminal N-aeclylgjucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the Golgi stack. Cel! 1985:40:463-472. 48 Hcdtnan K. Pastan I. Willingham MC: The organelles of the traits domain of the cell. Ultrastructural localization of sialoglycoconjugates using Um ax flacas agglutinin. J llistocltem Cytochem 1986:34:1069-1077. 49 Bennett G . O'Shaugncssy D: The site of incor­ poration of sialic acid residues into glycopro­ tein and the subsequent fates of those mole­ cules in various rat and mouse cell types as shown by radio-autography alter injection of H-acctyl-mannosamine 1. Observations in hépatocytes. J Cell Biol 1981:88:1-5. 50 Roth J. Taatjcs D.I. Lucocq JM. Weinstein J. Paulson J: Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in gly­ cosylation. Cell 1985:43:287-295. 5 1 Kramer MF. Gcuze J.I: Glycoprotein transport in the surface mucous cells of the rat stomach. J Cell Biol 1977;73:533-547. 52 Kramer MF. Gcuze JJ: Comparison of various methods to localize a source of radioactivity in ultrastructure autoradiographs. The site of Hgalactose incorporation in surface mucous cells of the rat stomach. J Histochcm Cytochem 1980:28:381-387. 53 StrousGJAM . Gcuze 11.1. VanHuis GA. Kra­ mer ME: Intracellular site of glycosyl and sul­ fate transferases in the surface mucous cells of the rat stomach: in Gregory JD. Jeanloz RW (cd): Glycoconjugalc Research. New York. Academic Press. 1977. pp 805-808. 54 Sato A. SpicerSS: Ultrastructural visualization of galactosyl residues in various alimentary epi­ thelial cells with the peanut lectin-horseradish peroxidase procedure. I listochemistry 1982:73: 607-624.

.39

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

8 Nicolson (il. The interactions of lectin with animal cell surfaces. Int Rev Cytol 1974:39: 89-190. 9 Lis 11. Sharon N: l.ectins as molecules and as tools. Annu Rev Biochem 1986:55:35-67. 1(1 Leathern AIC. Atkins N.I: Lectin binding to paraffin sections; in Bullock GR. Petrus/ P (cds): Techniques in Immunocytochcmistrv. London. Academic Press. 1983. vol 2. pp 39-70. 11 Roth J: The lectins molecular probes in cell biology and membrane research. Exp Pathol Suppl 1978:3:1-186. 12 Roth .1: Application of lectin-gold complexes for electron microscopic localization of glycoconjugates on thin section. .1 Histochcm C'ylocliem 1983:31:987-999. 13 Roth J: The colloidal gold marker system for light and electron microscopic cytochemistry: in Bullock GR. Pctrusz. P (cds): Techniques in Imrmmocylochemistry New York. Academic Press. 1983. vol 2. pp 217-284. 14 t han I.. Wong YC: Glycoconjugatcs of the lat­ eral prostate of the guinea pig: A lectin histochcmical study. Prostate 1991:19:155-172. 15 Roth .1: Cytochcmical localization of terminal N-acetyl-/f-galactosamine residues in cellular compartments of intestinal goblet cells: Impli­ cations for the topology of O-glycosvIaiion. .1 C ell Biol 1984:98:399-406. 16 Kellenbcrgcr E. Carlemalm E. Villiger W. Roth .1. Garavito M: Low Denaturing Embed­ ding for Electron Microscopy of Thin Sections. Waldkraiburg. Chemischc Wcrkc Lowi GmbH. 1980. 17 Roth .1. Bcndayan M. Carlemalm E. Villiger W. Garavito M: Enhancement of structural preservation and immunooytochemical stain­ ing in low temperature-embedded pancreatic tissue. .1 Histochcm Cylochem 1981:29: 663-671. 18 Carlemalm E. Garavito M. Villiger W: Resin development for electron microscopy and anal­ ysis of embedding at low temperature. J Microsc (Lond) 1982:126:123-143. 19 BonnardC. Papermaslcr DS. Kruchcnbuhl JP: The slreptavidin-biotin bridge technique: Application in light and electron microscope imnumocytochcmistry: in Polak JM. Varndell 1M (eds): Immunolabelling for Electron Microscopy. New York. Elsevier. 1984. pp 95-11L 20 Wong YC. Tse MKW: Line structural and functional study of the prostatic complex of the guinea pig. Acta Anat 1981:109:289-312. 21 Suzuki S. Tsuyama S. Vlurata ! : Post-embed­ ding staining of rat gastric mucous cells with lectins. Histochemistry 1982:73:563-575. 22 Brown D. Roth .1. Orci L: Lectin-gold cyto­ chemistry reveals intercalated cell heteroge­ neity along kidney collecting ducts. Am .1 Phys­ iol I985;248:C348-C356. 23 Joncs CJP. Sloddart RW: A post-embedding avidin-biotin peroxidase system to demon­ strate the light and electron microscopic local­ ization of lectin binding sites in rat kidney tubules. Histochem J 1986:18:371-379.

69 Bennett G, Lehlond CP: Biosynthesis of glyco­ proteins present in plasma membrane. Ivsosomes and secretory materials, as visualized by radioautography. Histochem J 1977:9:393. 70 Schulte BA. Spicer SS: Histochemical methods for characterizing secretory and cell surface sialo-glycoconjugates. J Histochem Cytochem 1985;33:427-438. 71 Schulte BA. Rao KPP. Kreutner A. Thontopo'ulos GN. Spicer SS: Histochemical examination of glycoconjugates of epithelial cells in the human fallopian tube. Lab Invest 1985:52:207-219. 72 Dingle JT, Dean RT. Sly W: Lysosomes in Biology and Pathology. Amsterdam. Elsevier. 1984. vol 7. p 479. ’ 73 Pappu AS. Adhikari HR. Vakil UK. Fatterpaker P. Sreenivasan A. Bachhawat BK: Char­ acterization of lysosomal membrane from rat liver. Indian J Biochcm Biophvs 1978:15: 89-94. 74 Lewis V. Green SA. Marsh M. Vihko P. Helenius A . Mellman I: Glycoproteins of the lysosomal membrane. J Cell Biol 1985:100: 1839-1847. 75 Chen JW. Murphy TL. Willingham MC. Pastan 1. August JT: Identification of two lyso­ somal membrane glycoproteins. J Cell Biol 1985:101:85-95. 76 Henning R: Possible functions of carbohy­ drates in lysosomal membranes. Biochem Soc Trans 1977:5:62. 77 Chan L. Wong YC: Complex carbohydrate his­ tochemistry of the lateral prostate and seminal vesicle of the guinea pig. Acta Anat 1991:142: 326-333. 78 Bloom W. Fawcett DW: A Textbook of Histol­ ogy. ed 11. Saunders. Philadelphia. 1986. pp 641-678. 79 Spicer SS. Baron DA. Sato A. Schutle BA: Variability of cell surface glycoconjugates. Relation to differences in cell function. J Histo­ chem Cytochem 1981:29:994-1002. 80 Luft JH: The structure and properties of the cell surface coat. Int Rev Cytol 1976:45: 291-382. 81 Dannon D. Goldstein L, Marikovsky Y. Skutelskv E: Use of cationized ferritin as a label of negative charges on cell surfaces. J Ullrastruct Res 1972:38:500-510. 82 Gonnella PA. Neutra MR: Glycoconjugates distribution and mobility in apical membranes of absorptive cells of suckling rat ileum in vivo. Anat Rec 1985:213:520-528. 83 Pipan N. Pscnicnik M: The carbohydrates of secretory granules and the glycocalyx in devel­ oping mucoid cells. Cell Tissue Res 1985:242: 437-443. 84 Roth J. Taatjes DJ: Glycocalyx heterogeneity of rat kidney urinary tubule: Demonstration with a lectin-gold technique specific for sialic acid. Eur J Ceil Biol 1985:39:449-457.

85 Brown D. Orci L: Interactions of lectins with specific cell types in toad urinary bladder. J Histochem Cytochem 1986:34:1057—1062. 86 Tanintura F. Morioka H, Tachibana M: Glyco­ conjugates in the glycocalyx of the guinea pig middle ear mucosa as revealed by postembed­ ding staining with lectin gold complexes. Acta Histochem Cytochem 1987:20:315-320. 87 Takagi M. Saito 1. Kuwata F. Otsuka K: Spe­ cific binding of peanut agglutinin and soybean agglutinin to chondroitinasc ABC digested car­ tilage proteoglycans: Histochemical. ultrastructural cylochemical. and biochemical char­ acterization. Histochem J 1988:20:88-98. 88 Chan L. Wong YC: Ultrastructural localization of proteoglycans by cationic dyes in the epithe­ lial-stromal interface of the guinea pig lateral prostate. Prostate 1989:14:147-162. 89 Chan L. Wong YC: Cytochemieal character­ ization of cuprolinic blue-stained proteoglycans in the epithelial-stromal interface of the guinea pig lateral prostate. Prostate 1989:14:133-145. 90 Peters BP. Goldstein 1.1: The use of fluores­ cein-conjugated Bandeiraea shnpUdfotm B4isolectin as a histochemical reagent for the detection of cr-£)-galactopyranosyl groups. Exp Cell Res 1979:120:321-334. 91 Lee MC. Damjanov I: Anatomic distribution of lectin-binding sites in mouse testis and epi­ didymis. Differentiation 1984:27:74-81. 92 Gallagher BC: Basal laminar thinning in branching morphogenesis of the chick lung as demonstrated by lectin probes. .1 Embryol Exp Morphol 1986:94:173-188. 93 Velasco A. I lidalgo J. Muller M. Garcia-1 lerdugo G: Ultrastructural demonstration of lec­ tin binding sites in the Golgi apparatus of rat epiphyseal chondrocytes. Histochemistry 1988: 89:177-184. 94 Vorbrodt AW. Lossinsky AS. Wisniewski HM: Enzyme cytochemistry of blood-brain barrier disturbances. Acta Neuropalhol 1983:8:43-57. 95 Kreutzberg GW: Enzyme cytochemistry of the brain capillaries. Acta Neuropalhol Suppl 1983:8:35-41. 96 Vorbrodt AW. Lossinsky AS. Wisniewski IIM: Ultrastructural studies of concanavalin A receptors and 5 -nucleotidase localization in normal and injured mouse cerebral micro-vas­ culature. Acta Neuropalhol 1984:63:210-217. 97 Dornand .1. Bonnafous JC. Mani JC: Effect of Con A and other lectins on pure 5-nucleotidase isolated from lymphocytic plasma membranes. Biochem Biophvs Res Common 1978:82: 685-692. 98 Williamson FA. Morre DJ. Shcn-Millcr J: Inhi­ bition of 5'-nudeotides by concanavalin A: Evidence for localization on the outer surface of the plasma membrane. Cell Tissue Res 1976:170:477-484.

40

Chan/Wong

Glycoconjugates of the Lateral Prostate

Downloaded by: King's College London 137.73.144.138 - 10/13/2018 1:10:44 AM

55 Slot JW. Geuze HJ: Immunoeléctron micro­ scopic exploration of the Golgi complex. J I lislochem Cytochcm 1983:31:1049-1059. 56 Wong YC. Tam CC: Effect of Gossypol Acetic Acid on Thiamine Pyrophosphatase Activity of the Guinea Pig Seminal Vesicles. Proc 3rd Int Congr Cell Biol. Tokyo 1984. p 1250. 57 Wong YC. Tam CC: A structural and cytochemical study of the effects of gossypol on the epithelial cells of the guinea pig seminal vesi­ cle. J Reprod Fertil 1988:84:659-668. 58 Castle JD . Jamieson JD. Palade GE: Secretion granules of the rabbit parotid gland: Isolation, subfractionation and characterization of the membrane and content subfractions. J Cell Biol 1975:64:182-210. 59 Castle JD. Palade GE: Secretion granules of the rabbit parotid: Selective removal of secre­ tory contaminations from granule membranes. .1 Cell Biol 1978:76:323-340. 60 Lewis DS. MacDonald RJ, Kronquist KE. Ronzio RA: Purification and partial character­ ization of an integral membrane glycoprotein from zymogen granules of dog pancreas. FEBS Lett 1977:76:115-120. 61 Ronzio RA. Kranquist KE. Lewis DS. Mac­ Donald RJ. Mohrlok Si I. O'Donnell II: Gly­ coprotein synthesis in the adult rat pancreas. IV Subcellular distribution of membrane gly­ coproteins. Biochim Biophvs Acta 1978:508: 65-84. 62 Havinga JR. Strous GJAM. Poorl C: Biosyn­ thesis of the major glycoprotein associated with zymogen-granule membranes in the pancreas. Eur J Biochcm 1983:133:449-454. 63 Buckley K. Kelly RB: Identification of a trans­ membrane glycoprotein specific for secretory vesicles of neural and endocrine cells. J Cell Biol 1985:100:1284-1294. 64 Roda LG. Slater F.P. Hogue ARA: Glycopro­ teins and glvcopeptides in the chromaffin granule membrane. Neurochem Res 1980:5: 1243-1250. 65 Warchol JB. 1lerbert DC. Rcnnels EG: Glyco­ proteins in membranes of secretory granules of the anterior pituitary gland. Histochemistry 1976:46:139-146. 66 Feigenson ME. Schnebli HP. Baggioline M: Demonstration of Hein-binding sites on the outer face of azurophil and specific granules of rabbit polymorphonuclear leukocytes. J Cell Biol 1975:66:183-188. 67 Meyer Dl. Burger MM: The chromaffin gran­ ule surface localization of carbohydrate on the cytoplasmic surface of an intracellular organ­ elle. Biochim Biophvs Acta 1976:443:428-436. 68 Neiss WF: Ultracytochemistry of intracellu­ lar membrane glycoconjugates. Adv Anal Embryol Cell liiol 1986:99.