Effect of swainsonine on rat epididymal glycosidases M. D. Skudlarek and M.-C. Orgebin-Crist Center for Reproductive Biology Research, Vanderbilt School of Medicine, Nashville, Tennessee 37232, U.S.A.

Summary. Each epididymis of control and swainsonine-fed rats (5 \g=m\g/mldrinking water) was divided into 5 segments, and tissue, spermatozoa and sperm-free supernatants were prepared from each segment. When levels of 3 lysosomal glycosi- dases and total protein were determined, the proximal cauda contained the greatest concentration of each glycosidase. The specific-activity profile for \g=b\-glucuronidase and \g=b\-galactosidasewas similar in swainsonine-fed and control rats. However, the concentration of \g=a\-d-mannosidasein tissue of all segments was significantly greater in swainsonine-fed rats than in age-matched controls. Enzyme activity for \g=a\-d-manno- sidase after swainsonine treatment was significantly greater in spermatozoa from the caput, than in spermatozoa from the corpus and the cauda epididymidis. Since the \g=a\-d-mannosidaseactivity was optimal at pH 4\m=.\5and studies with highly specific anti- body to lysosomal \g=a\-d-mannosidaseimmunoprecipitated all of the \g=a\-d-mannosidase present in detergent extracts of epididymal tissue, spermatozoa, and sperm-free super- natant, the enzyme studied is of lysosomal origin. Keywords: epididymis; glycosidases; swainsonine; rat

Introduction

Swainsonine is an found in the plants Swainsona canescens, spotted , and the fungus Rhizoctonia leguminicola. Animals ingesting these plants develop an a-mannosido- sis-like condition (Dorling et ai, 1978; Colgate et ai, 1979; Molyneux & James, 1982; Schneider et ai, 1983). It has also been reported that locoweed affects the testis and the reproductive tract in the ram and that cytoplasmic vacuolation occurs in Sertoli cells, germ cells, and epithelial cells lining the epididymal duct (James & Van Kampen, 1971; Molyneux & James, 1982). In general, in-vivo feeding with swainsonine increases lysosomal a-D-mannosidase levels, but there are differences between species and between tissues within a species. For example, in-vivo feeding with swainso¬ nine increases a-D-mannosidase concentration in rat liver, brain and kidney, in pig liver and brain, but not in pig kidney (Tulsiani & Touster, 1983a; Tulsiani et ai, 1984; Novikoff, et ai, 1985). The epididymis is a rich source of glycosidases (Conchie et ai, 1959; Kemp & Killian, 1978; Skudlarek & Swank, 1981; Chapman & Killian, 1984; Skudlarek & Orgebin-Crist, 1986) and epididymal epithelial cells in culture release a-mannosidase, ß-glucuronidase and ß-galactosidase (Skudlarek & Orgebin-Crist, 1986). The present study examines the effect of swainsonine on these 3 epididymal glycosidases.

Materials and Methods

Materials. Swainsonine isolated from Rhizoctonia leguminicola was provided by Dr H. P. Broquist of Vanderbilt University. Polyclonal antibody (IgG fraction) to lysosomal a-D-mannosidase antibody was supplied by Dr R. P. Tulsiani and Dr O. Touster of Vanderbilt University. This antibody has been shown to be specific for lysosomal a-D-mannosidase (Novikoff et ai, 1985). The 4-methylumbelliferyl-ß-D-galactoside, -ß-D-glucuronide, and

Downloaded from Bioscientifica.com at 09/26/2021 10:41:18AM via free access - -D-mannoside were from Sigma (St Louis, MO, U.S.A.). Staphylococcus aureus cells (IgGSorb) were obtained from the Enzyme Center, Inc. (Maiden, MA, U.S.A.). The cells were washed (Moreman & Touster, 1985) and suspended in 20 mM-Tris-HCl buffer, pH 7-5 containing 1 % Triton X-100 + 0-5 M-NaCl (10% suspension).

Animals. Adult Sprague-Dawley rats (~300g body weight) were purchased from Sasco, Omaha, NE, U.S.A. Animals were killed by C02 asphyxiation.

Swainsonine treatment. It has previously been shown that in-vivo feeding of swainsonine in the drinking water at 5 pg/ml increased levels of lysosomal a-D-mannosidase in rat liver and brain after 4 weeks (Tulsiani & Touster, 1983a). Male rats were therefore given swainsonine (5 pg/ml) in their drinking water continuously for 4 weeks.

Epididymal minces. The epididymis was separated into 5 segments, i.e. proximal and distal caput, corpus, and proximal and distal cauda. Each segment was minced at 4°C in 5 volumes of Hank's balanced salt solution (Gibco, + Grand Island, NY, U.S.A.) without Ca2+ or Mg2 Epididymal tissue, spermatozoa and sperm-free supernatants were prepared as follows. After swirling the minces. for 5 min and letting them settle, the supernatant solution containing spermatozoa was aspirated and 5 volumes of the above Hank's solution were added. This washing procedure was repeated 3 times. The 4 supernatant solutions were combined and centrifuged for 10 min at 200 g. The sperm-free supernatant and the sperm pellet, after 2 washes in Hanks, were frozen at 20°C until assay. After two final washings, the epididymal tissue was frozen as above. Less than 1-5% of the total —sperm number contaminated the tissue, and ~3% of the sperm pellet had epithelial cell contamination.

Assays. Samples were homogenized in Tris-buffered saline (0-25 M-NaCl, 5 mM-Tris, 0-3% Triton, pH 7-4) for 10 sec at 4°C with a glass, power-driven Potter-Elvehjem tissue grinder, centrifuged at 40 000 g for 20 min, and supernatant solutions were assayed. In immunoprecipitation studies, detergent extracts were first prepared by homogenizing in 0-25M-NaCl, 5 mM-Tris (pH 7-4), 1% Triton, 1% sodium deoxycholate and centrifuging at 1000000g for 1 h. Activities of a-mannosidase (EC3.2.1.24), ß-glucuronidase (EC3.2.3.31), and ß-galactosidase (EC 3.2.1.23) were determined at pH 3-5-4-6 by fluorometric analysis using 4-methylumbelliferyl derivatives of substrates: 4-mu-a-D-mannoside, 4-mu-ß-D-glucuronide or 4-mu-ß-D-galactoside, respectively, as substrate (Brandt et ai, 1975; Opheim & Touster, 1978). Substrate buffers of pH 30-80 for a-D-mannosidase are in the legend to Fig. 3. One unit of enzyme activity hydrolyses 1 pmol substrate/h at 37°C. Protein was measured by the method of Lowry et ai (1951).

Immunoprecipitation with lysosomal a-D-mannosidase antibody. IgGSorb (200 µ of a 10% suspension) was washed 3 times with buffer (0-25 M-NaCl, 5 mM-Tris, pH 7-5), antibody (titre = 2-5 µ /unit) spécifie for lysosomal a-D-mannosidase was added (Novikoff et ai, 1985), incubated for 60 min at room temperature with occasional mixing, centrifuged, and washed 4 times with the above buffer. The detergent extract containing 0-4 units ofantigen was added to the antibody-adsorbed IgGSorb and the mixture was kept at room temperature for 60 min with occasional mixing. After incubation, the samples were centrifuged, and the supernatant solution was assayed for a-D-mannosidase activity and protein.

Results

Glycosidase activity in the epididymis After in-vivo feeding with swainsonine (5 µg/ml), a-D-mannosidase specific activity in the tissue homogenate was significantly higher than in controls in all epididymal segments, except the distal cauda, while protein content remained unchanged (Fig. 1). When the homogenate was fractionated into tissue, fluid and sperm pellet, the increase in epididymal mannosidase activity occurred in the tissue of all segments, but not in the released fluid (Fig. 2d). Mannosidase activity after swainsonine treatment was higher in spermatozoa from the caput, but not from the corpus and the cauda epididymidis. Swainsonine had no apparent effect on the concentration and distribution of ß-glucuronidase and ß-galactosidase (Figs 2b, c). There were regional differences in the localization of the 3 glycosidases in the epididymal segments of the control and swainsonine-treated rats (Fig. 2). The highest specific activity for ß-glucuronidase, ß-galactosidase, and -mannosidase was in the proximal cauda segment. We calculated that this segment also contained >40% of total epididymal enzyme activity. A differential distribution of the enzymes was also observed. Most ß-glucuronidase was found in the tissue (64-87% of total enzyme activity), while most ß-galactosidase was found in the sperm-free supernatant (50-84%). Mannosidase was found in both the tissue and sperm-free supernatant.

Downloaded from Bioscientifica.com at 09/26/2021 10:41:18AM via free access Proximal caput Distal caput Corpus Proximal cauda Distal cauda

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Fig. 1. -D-Mannosidase activity in the epididymis of normal ( ) and swainsonine-fed (D) rats. Swainsonine (5 µg/ml) was administered in the drinking water for 4 weeks. Each of 5 epididymal segments was homogenized and assayed for enzyme activity (units/mg cell protein) using 4-methylumbelliferyl mannosidase as substrate. Values are the mean ( + s.e.) of duplicates in 3 separate experiments. *P < 005 compared with value for normal rats.

Epididymal a-D-mannosidase activity at different H values To demonstrate the nature of a-D-mannosidase reported here, we studied enzymic activity over a wide pH range in the Triton-deoxycholate detergent epididymal extract obtained from the control and swainsonine-treated rats. The results presented in Fig. 3 show that the detergent extract from control and swainsonine-treated rats has an acidic pH optimun of 4-5, a result suggesting that the enzyme studied here is of lysosomal origin. This was confirmed by immunoprecipitation studies using highly specific polyclonal antibody to rat epididymal mannosidase. This antibody has been shown to cross-react only with the lysosomal form of a-D-mannosidase (Novikoff et ai, 1985). The results presented in Fig. 3 clearly indicate that all of the a-D-mannosidase activity present in the detergent extract of the epididymal tissue was immunoprecipitated with the antibody. Although the epididymis from rats treated with swainsonine contains twice as much enzyme as that of control rats, the pH optimum and immunological property of the enzyme from the two sources remain the same. When the homogenate was fractionated into tissue, fluid, and spermatozoa, similar pH optima and quantitative immunoprecipitation of the enzyme were obtained. These results indicate that the a-D-mannosidase activity in all three fractions of the epididymis is of lysosomal origin.

Discussion

Spermatozoa gradually acquire motility and fertilizing ability as they pass through the epididymis. Some changes associated with those functions are dependent on the epididymal environment (Orgebin-Crist et ai, 1981). These studies have shown that spermatozoa, as they pass through the epididymis, exhibit an altered cell surface glycoprotein pattern which appears to influence sperm-egg interaction. It is possible that these changes result from the addition/alteration of the carbohydrate of moieties by the action of glycosidases and/or glycosyltransferases. Glycosidases have been identified in the epididymis (Conchie et ai, 1959) and in isolated rat epididymal epithelial cells (Kemp & Killian, 1978; Chapman & Killian, 1984; Skudlarek & Orgebin-Crist, 1986). The rat epididymis contains specific activity concentrations of glycosidases up to 90-fold greater than in other tissues (Skudlarek & Swank, 1981). There are also large regional

Downloaded from Bioscientifica.com at 09/26/2021 10:41:18AM via free access Fig. 2. Glycosidase activity in 5 epididymal regions. Tissue, spermatozoa and sperm-free super¬ natant were prepared as described in the 'Materials and Methods'. Values are the mean ( + s.e.) of 7 experiments. *P < 0-05 for swainsonsine values (H) compared with controls (^). differences in the concentraction of glycosidases in the epididymis. In the present studies, the greatest specific activity of ß-glucuronidase, ß-galactosidase and -mannosidase was in the proximal cauda segment. This segment also contains >40% of the total content of these enzymes in the epididymis. Our results confirm previous, histochemical studies showing a higher ß-glucuronidase enzyme activity in the distal corpus and proximal cauda than in the caput (Hayashi, 1967; Moniem & Glover, 1972). Epididymal glycosidases appear to be localized in different compartments. ß-Galactosidase is readily released from minced tissue, suggesting that it is localized extracellularly or in an easily releasable organellar form. Conversely, little ß-glucuronidase is released, suggesting that it has a tighter association with membranes or is in an organelle which is not easily released. This is in agreement with studies of other tissues in which a high proportion (up to 40%) of total ß-glucuronidase is sequestered in the endoplasmic reticulum associated with a specific protein, egasyn (Lusis & Paigen, 1977). Glycosidase release from minced tissue in the present study should

Downloaded from Bioscientifica.com at 09/26/2021 10:41:18AM via free access Fig. 3. Effect of pH on epididymal -D-mannosidase activity. Epididymal extracts were measured for enzyme activity using 4-methylumbelliferyl-a-D-mannoside as substrate. Buffers were 0-1 M-sodium citrate (pH 3-0-3-6), 0-1 M-sodium (pH 40-60) and 0-1 M-potassium phosphate (pH 6-0-8-0). Epididymal extracts were from normal rats (·, A) and swainsonine- treated rats ( , ) before (·, O) and after ( , ) immunoprecipitation with lysosomal a-D- mannosidase antibody. not be equated with secretion by the epithelial cells, since epididymal tissue contains several cell types and the released fluid may be contaminated by the content of damaged cells. However, we have shown that both ß-glucuronidase and ß-galactosidase are synthesized in higher molecular weight precursor forms by cultured rat epididymal epithelial cells, processed to mature enzyme, and released into the extracellular medium (Skudlarek & Orgebin-Crist, 1986). These epididymal glycosidases may be involved in the modification of the sperm surface glycoproteins during epididymal transit. Several mannosidases, namely the lysosomal, cytosolic, endoplasmic reticulum, and 3 Golgi forms have been purified and characterized from the rat liver (Opheim & Touster, 1978; Tabas & Kornfeld, 1979; Tulsiani et ai, 1977, 1982). However, in rat epididymal tissue, only lysosomal a-D- mannosidase, with a pH optimum of 4-5 (Snaith & Levvy, 1969), and cytosolic a-D-mannosidase with a neutral pH optimum (Snaith, 1977) have been identified. In bovine tissue, the acidic enzyme activity was highest in seminal plasma and the epididymis where the activity seems to be mainly in secretory form; the neutral a-mannosidase activity was high in epididymal homogenate and in epididymal and ejaculated spermatozoa (Jauhiainen & Vanha-Perttula, 1987). In the present study, the cytosolic form of -D-mannosidase was undetectable. However, Co2 + is required for cytosolic a-mannosidase activity (March & Gourlay, 1971; Phillips et ai, 1976; Shoup & Touster, 1976; Dutta & Majumder, 1984) and we may have been unable to detect cytosolic a-D-mannosidase + (Fig. 3) because of the absence of Co2 in our assay mixture. Nevertheless, immunoprecipitation of all of the enzymic activity with an antibody specific for the lysosomal form of the enzyme clearly indicates that the form we are studying in rat epididymal tissue is of lysosomal origin. Swainsonine, a plant toxin, has been shown to be a potent inhibitor in vitro of lysosomal -D-mannosidase in several tissues (Dorling et ai, 1980; Tulsiani et ai, 1982; Kang & Elbein, 1983),

Downloaded from Bioscientifica.com at 09/26/2021 10:41:18AM via free access including the epididymis (Jauhiainen & Vanha-Perttula, 1987), and of Golgi mannosidase II in liver (Tulsiani et ai, 1982). However, when it is administered to rats and pigs, it causes an increase in levels of the lysosomal form of -D-mannosidase and a decrease in levels of Golgi mannosidase II in liver (Tulsiani & Touster, 1983a; Tulsiani et ai, 1984). We now find that, in the epididymis, as in liver of swainsonine-fed rats, the lysosomal form of -D-mannosidase is increased. Therefore, the epididymis is affected by in-vivo feeding with swainsonine. Swainsonine has also been shown to alter the oligosaccharide structure of secreted glyco- proteins (Arumugham & Tänzer, 1983; Gross et ai, 1983; Lodish & Kong, 1984; Yeo et ai, 1985) via the inhibition of Golgi mannosidase II (Elbein et ai, 1981; Tulsiani et ai, 1982; Arumugham & Tanzer, 1983; Gross et ai, 1983; Tulsiani & Touster, 1983b). We have failed to detect mannosidase II in the epididymis using enzymic analyses and immunoprecipitation with a highly specific polyclonal antibody (Tulsiani et ai, 1977; Novikoff et ai, 1983) to rat liver Golgi mannosidase II (unpublished results). Likewise, mannosidase II could not be detected in rat, pig and sheep brain (Tulsiani et ai, 1988), but a novel -D-mannosidase was discovered in rat brain (Tulsiani & Touster, 1985). In spite of the absence of mannosidase II in pig and sheep brain, swainsonine induced synthesis of abnormal TV-linked glycoproteins (Tulsiani et ai, 1988). These findings suggest that a swainsonine sensitive processing enzyme distinct from mannosidase II is present in the brain of these animals and in epididymal tissue and stress the need to study the pathway of glycoprotein synthesis in a variety of tissues. Future investigations will be directed towards identifying mannosidase(s) and other processing enzymes involved in the modification and maturation of TV-linked glycoproteins in the epididymis.

We thank Dr H. P. Broquist for supplying the swainsonine used in these studies and to Dr D. R. P. Tulsiani and Dr O. Touster for providing antibodies (IgG fraction) to epididymal -D-mannosidase and rat liver Golgi mannosidase II.

References

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