Acta histochem. 88, 131- 137 (1990) VEB Gustav Fischer Verlag Jena
Department of Veterinary Anatomy, College of Agriculture and Veterinary Medicine, Nihon University, Fujisawa, Kanagawa, Japan, and Department of Anatomyl), Nagoya City University Medical School, Nagoya, Japan
Glycoconjugate histochemistry of the prostate gland in the pig By AZUMA TSUKISE and KAZUYORI YAMADA I) With 16 Figures (Received October 14, 1989)
Abstract In the porcine prostate, glycoconjugates elaborated and released by the glandular cells have been studied by means of a series of histochemical methods of light microscopy. The employed methods included several routine procedures in combination with digestion by carbohydrate-degrading enzymes and peroxidase-labelled lectin diaminobenzidine procedures. In the glandular tissues, the particular glycoconjugates were shown to be secretory glycoproteins which contained vicinal diol, sulphate, and carboxyl groups and were provided with different saccharide residues such as lXD-mannose, lX-D-glucose, lactose, N-acetyl-D-galactosamine, ~-D-galactose, lX-L-fucose, N-acetyl-D-glucosamine, and N-acetyl-neuraminic acid. These glycoconjugates were, likewise, found to exhibit positive reaction for proteins. The present histochemical results have been discussed with special reference to the histophysiological functions performed by the prostate gland in the pig.
Key words: glycoconjugates, histochemistry, prostate gland, pig.
1. Introduction It has been well known that an ejaculation of the porcine semen is unusually voluminous, as compared with that in other mammalian species of comparable body weights (MANN 1964). The major moiety of such voluminous semen is contributed by secretory substances elaborated and released by the accessory sex glands of the pig. In keeping with this fact, the porcine accessory sex glands are well developed and are known to produce a fraction which is indespensable for the stability of spermatozoa (MANN 1964). In a series of previous histochemical studies on the mammalian prostate glands (FRANKS et al. 1964; K.-H. WROBEL 1972; M. WROBEL 1972; AUMULLER 1973; TSUKISE and YAMADA 1981, 1984; ORGAD et al. 1984; NOGUEIRA et al. 1985; YAMADA 1985), glycoconjugates have been shown to be an important key substance of secretion and to play an essential role for the physiological activity of the glands. In the pig. however, detailed informations are limited concerning the histochemical analysis of glycoconjugates involved in the secretory substances elaborated and released by the epithelial cells lining the prostate gland. In view of these circumstances, the present study; attempts have been made to study glycoconjugates secreted by the epithelial cells of the porcine prostate gland by means of a wealth of current histochemical methods of light microscopy. The results obtained in the present work are believed to be useful for elucidating the natures of the true physiological functions performed by the porcine accessory sex gland.
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2. Materials and methods 6 adult male pigs of Hampshire strain (body weight: 110 to 120 kg) were employed as the donor animals for the prostate glands (external portion). These animals were sacrificed by exsanguination and their prostate glands were dissected out. Tissue pieces from these glands with an approximate size of 0.5 X 1.0 x 1.0 cm3 were immediately fixed at 4 °C orroom temperature for 48 h in 1 of the 3 fixing fluids; Bouin's fluid, 10% formalin containing 2% calcium acetate (LEPPI1968), and 10% formalin in 95% ethanol (McMANUS and MOWRY 1958). The fixed tissues were then dehydrated in graded ethanol series, cleared in benzene, and embedded in paraffin wax. Paraffin sections were cut at a thickness of 6 11m, affixed to glass slides, dried, deparaffinized in xylene, and hydrated through an ethanol series of descending concentrations. One group of the hydrated sections were subsequently stained with the following procedures; haematoxylin-eosin (HE), periodic acid-Schijf[pAS; PEARSE (1968)], aldan blue (AB) pH = 1.0 (LEV and SPICER 1964), AB pH = 2.5 (SPICER et al. 1967), AB (pH = 2.5)-PAS (SPICER et al. 1967), and coupled tetrazonium [TZ; PEARSE (1968)]. Prior to the AB pH - 1.0 procedure, some sections were subjected to sulphation (YAMADA and HOSHINO 1972). Other sections were digested with lX-amylase [Bacillus subtilis; Seikagaku Kogyo Co., Japan; CASSELMAN (1959)], prior to staining with the PAS procedure. In addition, still other sections were subjected to digestion with neuraminidase [Arthrobacter urea/aciens; Marukinshoyu. Japan; SPICER et al. (1967)], prior to being stained with the AB pH = 2.5 procedure. For these 2 types of the enzyme digestion experiments, appropriate controls were performed. The other group of the hydrated sections were reacted with peroxidase (PO)-Iectin (LT)-diaminobenzidine (DAB) procedures (PEARSE 1985). As the lectins of choice, concanavalin A (Con A), peanut agglutinin (PNA), soy bean agglutinin (SBA), Ricinus communis agglutinin-I (RCA-I), Ulex europaeus agglutinin-I (UEA-l), Doliches biflorus agglutinin (DBA), wheat germ agglutinin (WGA), and Limaxflavus agglutinin (LFA) were employed. To substantiate that each lectin specifically binds particular saccharide residues of glycoconjugates, PO-LT with an appropriate monosaccharide-DAB procedures were performed. Furthermore, the activity of endogenous peroxidase was checked by certain tissue sections reacted with DAB only.
3. Results In the prostate gland of the pig, the secretory epithelium consisted of a single layer of low columnar or cuboidal cells (Fig. I). The nucleus of these cells was spherical or oval in shape and situated in the basal cytoplasm (Fig. 1). In the distal cytoplasm of the cells, eosinophilic granules of different sizes were accumulated (Fig. 1). In the epithelium lining the glandular acini, basal cells of varying morphologies were occasionally interposed between the low columnar or cuboidal cells (Fig. 1). If tissue sections of the prostate gland were reacted with PAS, granules of different sizes in the
Fig. I. The glandular epithelium consists of a single layer of low columnar or cuboidal cells containing a basally situated nucleus. HE stained, x 400. Fig. 2. Granules of different sizes within the distal cytoplasm exhibit distinct positive reaction. PAS stained. x 200. Fig. 3. The positive PAS reaction of the majority of the granules remains unchanged. lX-amylase-digested PAS. x 200. Fig. 4. The free surface and granules in the epithelial cells and luminal secretions in the glandular lumen show moderate positive reaction. AB pH = 1.0 stained. x 400. Fig. S. The free surface and granules in the epithelial cells and luminal secretions within the glandular lumen exhibit moderate to strong positive reaction. AB pH = 2.5 stained. x 200. Fig. 6. The AB (pH = 2.5) reaction of glandular epithelial cells is feeble following digestion with neuraminidase. Neuraminidase-digested AB pH = 2.5 stained. X 200. Fig. 7. The free surface, granules of different sizes within glandular epithelial cells, and luminal secretions in the glandular lumen exhibit strong positive reaction. AB (pH = 2.5)-PAS stained. X 100. Fig. 8. The glandular epithelial cells and luminal secretions show positive reaction. TZ stained.
X
400.
Histochemistry of the prostate gland in the pig
Figs. I to 8. Part of the prostate gland of a pig.
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distal cytoplasm of the epithelial cells were found to exhibit distinct positive reaction (Fig. 2). In the lumen of the glandular acini, secretions were visualized which, likewise, showed moderate or weak positive PAS reaction (Fig. 2). Digestion with
Department of Veterinary Anatomy, College of Agriculture and Veterinary Medicine, Nihon University, Fujisawa, Kanagawa, Japan, and Department of Anatomyl), Nagoya City University Medical School, Nagoya, Japan
Glycoconjugate histochemistry of the prostate gland in the pig By AZUMA TSUKISE and KAZUYORI YAMADA I) With 16 Figures (Received October 14, 1989)
Abstract In the porcine prostate, glycoconjugates elaborated and released by the glandular cells have been studied by means of a series of histochemical methods of light microscopy. The employed methods included several routine procedures in combination with digestion by carbohydrate-degrading enzymes and peroxidase-labelled lectin diaminobenzidine procedures. In the glandular tissues, the particular glycoconjugates were shown to be secretory glycoproteins which contained vicinal diol, sulphate, and carboxyl groups and were provided with different saccharide residues such as lXD-mannose, lX-D-glucose, lactose, N-acetyl-D-galactosamine, ~-D-galactose, lX-L-fucose, N-acetyl-D-glucosamine, and N-acetyl-neuraminic acid. These glycoconjugates were, likewise, found to exhibit positive reaction for proteins. The present histochemical results have been discussed with special reference to the histophysiological functions performed by the prostate gland in the pig.
Key words: glycoconjugates, histochemistry, prostate gland, pig.
1. Introduction It has been well known that an ejaculation of the porcine semen is unusually voluminous, as compared with that in other mammalian species of comparable body weights (MANN 1964). The major moiety of such voluminous semen is contributed by secretory substances elaborated and released by the accessory sex glands of the pig. In keeping with this fact, the porcine accessory sex glands are well developed and are known to produce a fraction which is indespensable for the stability of spermatozoa (MANN 1964). In a series of previous histochemical studies on the mammalian prostate glands (FRANKS et al. 1964; K.-H. WROBEL 1972; M. WROBEL 1972; AUMULLER 1973; TSUKISE and YAMADA 1981, 1984; ORGAD et al. 1984; NOGUEIRA et al. 1985; YAMADA 1985), glycoconjugates have been shown to be an important key substance of secretion and to play an essential role for the physiological activity of the glands. In the pig. however, detailed informations are limited concerning the histochemical analysis of glycoconjugates involved in the secretory substances elaborated and released by the epithelial cells lining the prostate gland. In view of these circumstances, the present study; attempts have been made to study glycoconjugates secreted by the epithelial cells of the porcine prostate gland by means of a wealth of current histochemical methods of light microscopy. The results obtained in the present work are believed to be useful for elucidating the natures of the true physiological functions performed by the porcine accessory sex gland.
132
A. TSUKISE and K. YAMADA
2. Materials and methods 6 adult male pigs of Hampshire strain (body weight: 110 to 120 kg) were employed as the donor animals for the prostate glands (external portion). These animals were sacrificed by exsanguination and their prostate glands were dissected out. Tissue pieces from these glands with an approximate size of 0.5 X 1.0 x 1.0 cm3 were immediately fixed at 4 °C orroom temperature for 48 h in 1 of the 3 fixing fluids; Bouin's fluid, 10% formalin containing 2% calcium acetate (LEPPI1968), and 10% formalin in 95% ethanol (McMANUS and MOWRY 1958). The fixed tissues were then dehydrated in graded ethanol series, cleared in benzene, and embedded in paraffin wax. Paraffin sections were cut at a thickness of 6 11m, affixed to glass slides, dried, deparaffinized in xylene, and hydrated through an ethanol series of descending concentrations. One group of the hydrated sections were subsequently stained with the following procedures; haematoxylin-eosin (HE), periodic acid-Schijf[pAS; PEARSE (1968)], aldan blue (AB) pH = 1.0 (LEV and SPICER 1964), AB pH = 2.5 (SPICER et al. 1967), AB (pH = 2.5)-PAS (SPICER et al. 1967), and coupled tetrazonium [TZ; PEARSE (1968)]. Prior to the AB pH - 1.0 procedure, some sections were subjected to sulphation (YAMADA and HOSHINO 1972). Other sections were digested with lX-amylase [Bacillus subtilis; Seikagaku Kogyo Co., Japan; CASSELMAN (1959)], prior to staining with the PAS procedure. In addition, still other sections were subjected to digestion with neuraminidase [Arthrobacter urea/aciens; Marukinshoyu. Japan; SPICER et al. (1967)], prior to being stained with the AB pH = 2.5 procedure. For these 2 types of the enzyme digestion experiments, appropriate controls were performed. The other group of the hydrated sections were reacted with peroxidase (PO)-Iectin (LT)-diaminobenzidine (DAB) procedures (PEARSE 1985). As the lectins of choice, concanavalin A (Con A), peanut agglutinin (PNA), soy bean agglutinin (SBA), Ricinus communis agglutinin-I (RCA-I), Ulex europaeus agglutinin-I (UEA-l), Doliches biflorus agglutinin (DBA), wheat germ agglutinin (WGA), and Limaxflavus agglutinin (LFA) were employed. To substantiate that each lectin specifically binds particular saccharide residues of glycoconjugates, PO-LT with an appropriate monosaccharide-DAB procedures were performed. Furthermore, the activity of endogenous peroxidase was checked by certain tissue sections reacted with DAB only.
3. Results In the prostate gland of the pig, the secretory epithelium consisted of a single layer of low columnar or cuboidal cells (Fig. I). The nucleus of these cells was spherical or oval in shape and situated in the basal cytoplasm (Fig. 1). In the distal cytoplasm of the cells, eosinophilic granules of different sizes were accumulated (Fig. 1). In the epithelium lining the glandular acini, basal cells of varying morphologies were occasionally interposed between the low columnar or cuboidal cells (Fig. 1). If tissue sections of the prostate gland were reacted with PAS, granules of different sizes in the
Fig. I. The glandular epithelium consists of a single layer of low columnar or cuboidal cells containing a basally situated nucleus. HE stained, x 400. Fig. 2. Granules of different sizes within the distal cytoplasm exhibit distinct positive reaction. PAS stained. x 200. Fig. 3. The positive PAS reaction of the majority of the granules remains unchanged. lX-amylase-digested PAS. x 200. Fig. 4. The free surface and granules in the epithelial cells and luminal secretions in the glandular lumen show moderate positive reaction. AB pH = 1.0 stained. x 400. Fig. S. The free surface and granules in the epithelial cells and luminal secretions within the glandular lumen exhibit moderate to strong positive reaction. AB pH = 2.5 stained. x 200. Fig. 6. The AB (pH = 2.5) reaction of glandular epithelial cells is feeble following digestion with neuraminidase. Neuraminidase-digested AB pH = 2.5 stained. X 200. Fig. 7. The free surface, granules of different sizes within glandular epithelial cells, and luminal secretions in the glandular lumen exhibit strong positive reaction. AB (pH = 2.5)-PAS stained. X 100. Fig. 8. The glandular epithelial cells and luminal secretions show positive reaction. TZ stained.
X
400.
Histochemistry of the prostate gland in the pig
Figs. I to 8. Part of the prostate gland of a pig.
133
134
A. TsuKlsE and K. YAMADA
distal cytoplasm of the epithelial cells were found to exhibit distinct positive reaction (Fig. 2). In the lumen of the glandular acini, secretions were visualized which, likewise, showed moderate or weak positive PAS reaction (Fig. 2). Digestion with
