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Biology of Reproduction 59, 1288-1295 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Rat Sperm Surface Mannosidase Is First Expressed on the Plasma Membrane of Testicular Germ Cells1

Ben M.J. Pereira3,a, Aïda Abou-Hailab, and Daulat R.P. Tulsiani2,a

a Center for Reproductive Biology Research and Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232–2633 b Laboratoire de Biologie Cellulaire, Université René Descartes, Paris, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In previous publications (Tulsiani et al., Biochem J 1993; 290:427–436 and Tulsiani et al., Dev Biol 1995; 167:584–595), we reported that sperm surface mannosidase is present in rat testis and is modified during spermatogenesis and sperm maturation. The present studies were directed towards examining the origin of {alpha}-D-mannosidase activity present on fertile spermatozoa. Mixed germ cells prepared after sequential enzymatic digestions of rat testis were separated by unit gravity sedimentation using 2–4% linear bovine serum albumin gradient. Fractions enriched in spermatocytes, round spermatids, and condensed/elongated spermatids (> 95% pure cells) were separately pooled and assayed for [3H]Man9-mannosidase activity before (intact) and after lysis with Triton X-100. Interestingly, the cells contained a significant level of {alpha}-D-mannosidase activity. Approximately 70% of the total [3H]Man9-mannosidase activity present in the detergent-solubilized germ cell extract cross-reacted with anti-rat sperm mannosidase, and 25% of the activity cross-reacted with anti-Golgi mannosidase I. This result indicates that most of the mannosidase activity present in the germ cell extract is antigenically similar to the enzyme present on the cauda spermatozoa. Using cell fractionation techniques, we obtained evidence suggesting that the germ cell-associated mannosidase activity is an integral component of the plasma membranes. Taken together, these results indicate that sperm surface mannosidase is first expressed on the testicular germ cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian fertilization is the result of a complex set of molecular events that enable the spermatozoa to recognize and bind to the egg's extracellular coat, the zona pellucida (ZP). Sperm-egg binding is initiated when the spermatozoa first attaches to the zona-intact egg. The reversible interaction results in species-specific irreversible binding of the complementary molecule(s) present on the surface of the opposite gametes [1]. Evidence thus far available strongly suggests that in most species, the receptor molecule(s) present on the surface of the acrosome intact spermatozoa recognizes and binds to the glycan residue (ligand) of the homologous ZP. Thus, sperm-egg interaction is a species-specific carbohydrate-mediated event that depends on the glycan-recognizing enzymes (glycosidases and/or glycosyltransferases) or lectin-like sugar binding proteins on the sperm plasma membrane [2–7].

In previous publications, our group presented evidence for the occurrence of an {alpha}-D-mannosidase activity on the sperm plasma membranes of several species including man [8–10]. The involvement of the sperm enzyme in sperm-egg interaction is suggested by several studies summarized below. 1) The enzyme is an integral membrane component [8, 11]. 2) The enzyme is optimally active at neutral pH, and its catalytic site is oriented towards the sperm surface [8, 12]. This result suggests that the enzyme will be functional at the site of fertilization. 3) The mouse ZP2 and ZP3 contain high mannose/hybrid-type glycan chains [13]. These haptens are believed to be the ligand for the sperm mannosidase. 4) The inclusion of the sperm mannosidase inhibitors or competitive substrate in sperm-egg binding assays in vitro inhibited the number of sperm bound per egg in a dose-dependent manner in the mouse [10] and rat [14]. 5) Finally, the increase in sperm-associated mannosidase during maturation correlates with the fertilizing ability of rat spermatozoa [12]. Taken together, these results are consistent with the suggested receptor-like role for the sperm mannosidase. A polyclonal antibody raised against an isoform of the enzyme purified from rat epididymal luminal fluid cross-reacted with the {alpha}-D-mannosidase activity present in the detergent-solubilized cauda spermatozoa and plasma membranes prepared from rat testis and cauda spermatozoa [15]. The antibody was used to demonstrate that the enzyme is expressed in rat testis in an enzymatically inactive/less active precursor form that undergoes proteolytic processing by a trypsin-like protease in the testis and epididymis to generate an enzymatically active mature form in the epididymal cauda spermatozoa [11]. In this report, we have used purified rat testicular germ cells and cell fractionation techniques to examine the distribution of sperm surface mannosidase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Male Sprague Dawley rats (~400 g BW) were from Sasco (Omaha, NE). The Sta-Put cell separator was made by Johns Scientific (Toronto, ON, Canada). All p-nitrophenyl substrates used for enzyme assays, DNase I (type II), trypsin (type III), and soybean trypsin inhibitor were from Sigma (St. Louis, MO); collagenase (CLS-1) was from Worthington Biochemicals (Freehold, NJ); electrophoretic and electroblotting chemicals including biotinylated molecular weight marker proteins were from Bio-Rad (Richmond, CA). Staphylococcus aureus cells (IgGSorb) were from the Enzyme Center (Malden, MA). Mannose-labeled oligosaccharide ([3H]Man9GlcNAc) was prepared according to Tulsiani et al. [16]. Affinity-purified anti-sperm mannosidase IgG was prepared as described [15]. Highly specific polyclonal antibody (IgG fraction) against rat liver Golgi mannosidase IA was prepared as detailed previously [17]. This antibody has been shown to cross-react with Golgi mannosidase IA and IB. The reagents used for immunostaining were from Vector (Burlingame, CA). All other chemicals were obtained commercially and were of the highest purity available.

Isolation of Testicular Germ Cells

Animals were killed by CO2 asphyxiation. The testis along with the epididymis and fat pad was excised and perfused with enriched Krebs-Ringer bicarbonate medium (EKRB) through the testicular artery. The perfused testis was detunicated, and the seminiferous tubules were dispersed by enzymatic digestions in the presence of 5% CO2:air as described [18]. In brief, the testis was incubated with 60 ml of collagenase solution (1 mg/ml EKRB) for 20 min at 33°C, and the reaction was stopped by washing the exposed seminiferous tubules with EKRB (3 washes). The tubules were then subjected to trypsin (0.5 mg/ml EKRB) and DNase (1 µg/ml EKRB) treatment for 15 min at 33°C. The resulting cell suspension was made homogeneous by gently pipetting for 3–5 min. The reaction was stopped by the addition of trypsin inhibitor (0.5 mg/ml EKRB), and the cells were filtered through a 70-µm nylon mesh. The filtered cells were pelleted by centrifugation at 400 x g for 5 min, and the pelleted cells were washed by suspending in EKRB buffer containing 0.5% BSA and centrifugation as above (2 washes). Finally, the washed cells were suspended in 20–25 ml of EKRB buffer containing 0.5% BSA. An aliquot containing 5–6 x 108 cells was loaded on a Sta-Put sedimentation chamber, and the cells were allowed to sediment by gravity on a 2–4% linear BSA gradient as described [18]. After 2 h and 40 min at 4°C, the separated cells were removed by collecting fractions (10 ml). The cells were sedimented (400 x g for 5 min), and their purity was monitored by phase contrast microscopy. The fractions rich in spermatocytes, round spermatids, and condensed/elongated spermatids were pooled separately and used for further studies.

Preparation of Germ Cell Plasma Membranes

The purified germ cells (spermatocytes, round spermatids, and condensed/elongated spermatids) obtained after unit gravity sedimentation on a linear BSA gradient (see above) were pooled, suspended in 3 ml of PBS containing 25 mM benzamidine, and sonicated (2 x 15 sec) in a Fisher (Pittsburgh, PA) sonicator set at position 40. The disrupted cells were centrifuged at 1000 x g for 10 min at 4°C. The supernatant was removed by aspiration, and the residue was suspended in 3 ml of the above PBS solution, sonicated, and centrifuged as above. The pooled supernatant was layered on top of 45%, 40%, and 30% sucrose (w:v), and the tubes were centrifuged for 2 h at 26 000 rpm in an SW 28 rotor (Beckman Scientific, Palo Alto, CA). The germ cell membranes resolved into three distinct membrane bands designated band I (present on top of 30% sucrose), band II (interphase of 30/40% sucrose), and band III (interphase of 40/45% sucrose). In addition, some membranes separated at the bottom of the tube. The membranes present in the three bands and the pellet were carefully removed, diluted with water and 1 M NaCl (final salt concentration, 0.4 M), and pelleted by high-speed centrifugation (120 000 x g for 30 min). The washed membranes were used for biochemical and morphological studies.

Preparation of Germ Cell Extract

The germ cells were solubilized by suspension in potassium phosphate buffer (10 mM, pH 7.2) containing 0.5% Triton X-100 and 0.25% sodium deoxycholate followed by sonication as above. The cell suspension was centrifuged at 165 000 x g for 30 min. The supernatant was removed by aspiration and designated germ cell extract.

Isolation of Rat Cauda Spermatozoa and Sperm Plasma Membranes

The cauda spermatozoa and the salt-washed sperm plasma membranes were prepared by our published procedure [8].

Electron Microscopy

The pelleted membranes present in band I and band II were fixed for 2 h in 3% glutaraldehyde prepared in 0.1 M sodium cacodylate buffer, pH 7.3. The fixed membranes were rinsed in the buffer alone, postfixed in 1% osmium tetroxide, dehydrated in ethyl alcohol, and embedded in epoxy resin. Thin sections were prepared on copper grids, stained with uranyl acetate and lead citrate, and examined in a Hitachi (Hitachi Instruments, Inc., San Jose, CA) H-800 electron microscope.

Electrophoresis, Electrotransfer, and Western Blot Analysis

Membrane fractions were solubilized in SDS, and the polypeptides were separated by electrophoresis (SDS-PAGE) carried out under reducing conditions by the method of Laemmli [19]. The resolved polypeptides were transferred to a nitrocellulose paper by the method of Towbin et al. [20], as described [21]. Immunoreactive polypeptides were identified using affinity-purified anti-mannosidase IgG as a primary antibody followed by biotinylated secondary antibody as described [15].

Immunoprecipitation Studies

The washed IgGSorb (100 µl) was incubated at room temperature for 60 min with or without antibody to rat liver Golgi mannosidase IA or anti-rat sperm {alpha}-D-mannosidase. The mixture was centrifuged in an Eppendorf (Hamburg, Germany) microfuge for 1 min. The pellet was mixed with a small volume of germ cell extract, and the mixture was kept at room temperature for 60 min with occasional mixing. The supernatant obtained after centrifugation in a microfuge was assayed for [3H]Man9-mannosidase activity.

Enzyme Assays

Alkaline phosphodiesterase I, ß-D-galactosidase, and N-acetyl ß-D-glucosaminidase activities were assayed using p-nitrophenyl (PNP) substrates. The standard assay mixture containing buffer, substrate, Triton X-100 (0.1%, w:v), and sample in a total volume of 0.5 ml was incubated at 37°C as follows: alkaline phosphodiesterase I, PNP-5'-thymidylate (1 mM), and Tris-HCl buffer, pH 9.0 (50 mM); ß-D-galactosidase, PNP-ß-D-galactopyranoside (5 mM), and sodium citrate buffer, pH 3.5 (100 mM); N-acetyl ß-D-glucosaminidase, PNP-N-acetyl ß-D-glucosaminide (5 mM), and sodium acetate buffer, pH 4.5 (100 mM). After incubation for 15–60 min, the reaction was stopped by the addition of 1-ml alkaline buffer [22]. The released p-nitrophenol was quantitated by measuring the absorbance at 400 nm. Enzyme and substrate blanks were run with all assays. One unit of enzyme catalyzes the release of 1 µmol of p-nitrophenol per hour at 37°C.

Oligosaccharide-cleaving mannosidase activity was assayed by our published procedure [23]. Briefly, the incubation mixture (100 µl) contained mannose-labeled oligosaccharide (~4000 cpm of [3H]Man9GlcNAc), sodium cacodylate buffer, pH 6.4 (50 mM), Triton X-100 (0.2%, w:v), and enzyme in the absence or presence of 1-deoxymannojirimycin (d-MM), a potent inhibitor of mannosidase IA and IB [17]. After incubation for 2 h at 37°C, the reaction was stopped by heat treatment at 100°C for 5 min. The released [3H]mannose was separated from the oligosaccharide by gel filtration on a column of Bio-Gel P-2 (Bio-Rad) [23] and was quantitated by scintillation counter. One unit of the activity is the amount of enzyme that catalyzes the release of 1000 cpm of free [3H]mannose per hour at 37°C.

Protein was assayed by the Bio-Rad protein assay according to the manufacturer's protocol using BSA as standard.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Oligosaccharide-Cleaving {alpha}-D-Mannosidase Activity in the Germ Cells

Previous studies from this laboratory have shown the presence of a novel {alpha}-D-mannosidase activity on the plasma membrane fractions prepared from rat testes and spermatozoa from the caput, corpus, and cauda regions of the epididymis [11, 15]. Since the membrane fractions were prepared from the whole testis, it is possible that the membranes originated from the somatic rather than the germ cells. It was, therefore, important to examine the {alpha}-D-mannosidase activity in the purified germ cell preparations. Highly purified cell fractions were prepared after enzymatic dispersion of testicular germ cells followed by unit gravity sedimentation on a linear BSA gradient. The procedure allowed us to separate (Fig. 1) and isolate highly enriched populations (> 95% purity) of spermatocytes, round spermatids, and condensed/elongated spermatids (Fig. 2). The purified germ cell populations as well as spermatozoa prepared from the caput, corpus, and cauda regions of the epididymis were assayed for the oligosaccharide ([3H]Man9GlcNAc)-cleaving mannosidase activity in the presence or absence of Triton X-100. Data from these studies presented in Figure 3 show the presence of Man9GlcNAc-cleaving mannosidase activity in the germ cell fractions and spermatozoa. Inclusion of Triton X-100 had no significant effect on the enzyme activity, a result consistent with our earlier studies suggesting that the active site(s) (substrate cleaving site) of the enzyme is present on the cell surface [8, 12].



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  		FIG. 1. The elution profile of rat testicular germ cells after unit gravity sedimentation on a continuous BSA gradient. Fractions (10 ml) were collected, and the purity of cells was examined using phase contrast microscopy. The fractions were pooled as follows: spermatocytes, fractions 25–56; round spermatids, fractions 65–72; condensed/elongated spermatids, fractions 84–99.



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  		FIG. 2. Microscopic appearance of germ cells prepared from the rat testis. A) Mixed germ cells obtained after enzymatic dispersion of the testis; B) spermatocytes; C) round spermatids; D) condensed/elongated spermatids. Other details are described in Materials and Methods and in the legend to Figure 1. Aliquots of the mixed cells (A) or separated cells (B–D) were photographed using Nomarski differential interference contrast optics. Scale bar = 15.2 µm.



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  		FIG. 3. Quantitation of [3H]Man9-mannosidase activity in the enriched germ cells and spermatozoa from three epididymal regions in the presence (solid bars) or absence (open bars) of Triton X-100. Germ cells: SC, spermatocytes; RS, round spermatids; CES, condensed/elongated spermatids. Sources of spermatozoa: CT, epididymal caput; CS, corpus; CD, cauda. Data reported are the mean of duplicates in three separate experiments, and the vertical bar indicates ± SD.

Characterization of Man9-Mannosidase Activity of the Germ Cells

Since the Man9GlcNAc is a good substrate for the sperm surface mannosidase [8, 15] as well as the {alpha}1,2-mannose-specific processing mannosidases present in the endoplasmic reticulum [24] and Golgi membranes [17, 23, 25], it was of interest to characterize the mannosidase activities present in the germ cell extract. This was done by using anti-Golgi mannosidase I antibody and d-MM, a potent inhibitor of {alpha}1,2-specific mannosidases [17, 26]. In these studies, germ cell extract was incubated with the IgGSorb, which had been preadsorbed with different concentrations of mannosidase I antibody as described [17]. After incubation at room temperature for 60 min and centrifugation in a microfuge, the supernatant was assayed for the Man9GlcNAc-mannosidase activity. Data presented in Figure 4 showed that the antibody immunoprecipitated about 25% of the total mannosidase activity, a result suggesting that some of the Man9GlcNAc-mannosidase activity present in the germ cells is due to the Golgi mannosidase I. This result was confirmed by quantifying the mannosidase activity in the germ cells in the presence and absence of d-MM. The inhibitor showed a reduction of 20–25% activity (data not included). Furthermore, when the germ cell extract was incubated with the sperm-specific mannosidase antibody, only 70% of the Man9GlcNAc-cleaving mannosidase activity was immunoprecipitated. Taken together, these results strongly suggest that approximately 25% of the total Man9-mannosidase activity present in the germ cells is due to the presence of processing Golgi mannosidase I, and most of the remaining activity corresponds to the sperm-specific mannosidase.



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  		FIG. 4. Immunoprecipitation studies using anti-rat liver Golgi mannosidase IA antibody (IgG fraction). Aliquots from the solubilized germ cell extract were added to 100 µl of prewashed IgGSorb to which indicated amounts of the antibody had been preadsorbed. The suspension was kept at room temperature for 60 min and then centrifuged in a microfuge for 1 min. Man9-mannosidase activity remaining in the supernatant was assayed as described in Materials and Methods and plotted against the concentration of antibody. Data reported are the mean of two separate experiments.

Enzyme Activities in the Subcellular Fractions of Germ Cells

The presence of a significant amount of Man9-mannosidase activity in the germ cell extract that cross-reacted with anti-sperm mannosidase antibody prompted us to examine the distribution of the enzyme activity in the germ cell membrane fractions. Since the number of individual germ cells obtained from a single preparation were not sufficient to prepare membranes for complete biochemical and morphological studies, the cells obtained after the unit gravity sedimentation were pooled and used for the preparation of membrane fractions by discontinuous sucrose density gradient centrifugation as described in Materials and Methods. Four distinct membrane fractions obtained by the procedure were analyzed for alkaline phosphodiesterase I, a plasma membrane marker enzyme [9, 27]; Man9-mannosidase; and two acid glycohydrolases (ß-D-galactosidase and N-acetyl glucosaminidase) believed to be acrosomal marker enzymes [28]. Data from these studies when plotted according to de Duve et al. [29] showed that the membranes present in band I had the highest relative specific activity for phosphodiesterase I and Man9-mannosidase activities but low specific activity for the two glycohydrolases (Fig. 5). However, membranes present in bands II and III had higher relative activity for the two glycohydrolases but lower phosphodiesterase I and Man9-mannosidase activities, a result suggesting that band I contained membranes rich in plasma membrane marker enzyme (phosphodiesterase I) and mannosidase activity.



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  		FIG. 5. Distribution of [3H]Man9-mannosidase and marker enzymes in germ cell membranes. Aliquots from sonicated germ cells were saved, and the remaining sample was used for the preparation of germ cell membranes by discontinuous sucrose density gradient centrifugation as described in Materials and Methods. Fractions are indicated as follows: small dotted bars, sonicated germ cells; solid bars, band I; large dotted bars, band II; cross-hatched bars, band III; open bars, pellet. Data reported (mean of three separate experiments) are plotted according to de Duve et al. [29]. All triplicates agreed within 15%. The relative specific activity is the percentage of total activity/percentage of total protein.

Examination of the membranes present in band I and band II by electron microscopy showed that the former fraction contained round vesicles of different size and shape with little or no contamination by the endoplasmic reticulum (Fig. 6A). However, a significant number of vesicles present in band II resembled the structure typical of the endoplasmic reticulum (Fig. 6B). These data, in conjunction with the biochemical studies presented in Figure 5, suggest that the vesicles seen in Figure 6A were germ cell plasma membranes. Moreover, only the Man9-mannosidase activity present in the membranes of band II, but not band I, was sensitive to d-MM. (data not included). Combined, these data allow us to conclude that the membranes in band I were mostly plasma membranes whereas the band II contained ER and Golgi membranes.



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  		FIG. 6. Electron micrographs of rat germ cell membranes. The pooled germ cells were disrupted by sonication, and the membrane fractions were prepared by discontinuous sucrose gradient centrifugation as described in Materials and Methods. A) Membranes present in band I; B) membranes present in band II. The arrows identify the putative endoplasmic reticulum. Scale bar = 0.5 µm.

Immunodetection of {alpha}-D-Mannosidase

Next, we tried to obtain evidence for the presence of molecular forms of mannosidase on the germ cell membranes. This was done by immunoblotting analyses after solubilization and resolution of the salt-washed membranes present in band I and band II, and cauda sperm plasma membranes. Data from these studies presented in Figure 7 show the presence of an immunoreactive band of 115 kDa only in the germ cell membranes present in band I (but not band II) and the cauda sperm plasma membranes. In addition, a prominent immunoreactive band of an apparent molecular mass of 150 kDa was seen in the germ cell plasma membranes (lane 4). This band was less prominent in the membranes of band II (lane 5) and only a minor band in the sperm plasma membranes (lane 6).



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  		FIG. 7. Immunoblot analysis of the rat germ cell membranes present in bands I and II and in the cauda sperm plasma membrane. The salt-washed membranes were prepared as described in Materials and Methods and solubilized in SDS buffer. The solubilized membrane (~60 µg) proteins from germ cell membranes in band I and II, or ~40 µg sperm plasma membrane proteins, were resolved on 7% SDS-PAGE, and the mannosidase was visualized by Western blot analysis using preimmune (lanes 1–3) or immune (lanes 4–6) IgG. Lanes 1 and 4, band I; 2 and 5, band II; 3 and 6, sperm plasma membranes. An immunoreactive band of 115 kDa can be seen in lanes 4 and 6. In addition, a band of higher molecular mass (indicated by arrow) can be seen in lanes 4–6.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mammalian spermatozoon undergoes modifications during spermatogenesis, maturation in the epididymis, and capacitation in the female reproductive tract. Only capacitated spermatozoa are capable of binding the zona-intact egg [1–3, 30]. Many sperm surface proteins have been suggested to have a receptor-like role in binding to a glycan chain(s) on the surface of the zona pellucida (for review see references [2, 3]). The sperm proteins are either the hapten-recognizing enzymes glycosyltransferase [7, 31] and/or glycosidases [2, 8, 10] or lectin-like sugar binding proteins [4, 32–36].

That the sperm mannosidase is present in rat testis was suggested from two earlier studies. First, our initial studies provided evidence for the presence of mannosidase activity in testis, which cross-reacted with the anti-rat epididymal luminal fluid mannosidase, a soluble enzyme shown by us to be an isoform of rat sperm mannosidase [15]. Second, using the Western blotting approach, we showed the presence of precursor forms (apparent molecular mass 125 kDa and 135 kDa) and a mature form (apparent molecular mass 115 kDa) in the testicular plasma membranes [11]. Combined, the studies allowed us to suggest that the {alpha}-D-mannosidase activity present on mature spermatozoa is synthesized in the testis. In the studies presented here, the published procedure of O'Brien [18] was used to prepare highly enriched populations of pachytene spermatocytes, round spermatids, and condensed/elongated spermatids with excellent recovery (Fig. 1). When the purified cell preparations were assayed for Man9-mannosidase and enzyme activity expressed per 106 cells, the spermatocytes routinely contained higher activity than the round spermatids or condensed/elongated spermatids (Fig. 3).

It is important to point out that the oligosaccharide ([3H]Man9GlcNAc) used for the quantitation of the enzyme in Figure 3 is a good substrate for the sperm mannosidase as well as the {alpha},1,2-mannose specific Golgi mannosidase I [17, 24, 25]. Therefore, it was necessary to characterize the mannosidase activity seen in the purified germ cells. This was done in two ways. First, we incubated the detergent-solubilized germ cell extract with anti-rat liver Golgi mannosidase IA, an antibody shown by us to cross-react with Golgi mannosidases IA and IB [17]. The antibody immunoprecipitated nearly 25% of the total Man9-mannosidase activity, a result suggesting the presence of Golgi mannosidase I. This result was confirmed by assaying the enzyme activity in the presence of d-MM. The inclusion of d-MM in the assay mixture caused a reduction of ~25% of the total Man9-mannosidase activity. Since d-MM does not inhibit sperm mannosidase activity [8], it is reasonable to assume that the observed inhibition of the enzyme activity was due to the presence of Golgi mannosidase I.

A significant amount of the mannosidase activity present in the germ cells (~70% of the total Man9-mannosidase activity) cross-reacted with anti-rat sperm mannosidase IgG, a result suggesting that most of the enzyme present on germ cells in Figure 3 was antigenically similar to the sperm mannosidase. Comparison of the enzyme activity in the isolated germ cells and spermatozoa prepared from the caput, corpus, and cauda epididymides showed that the spermatocytes contained higher enzyme activity than the round spermatids or condensed/elongated spermatids. In addition, there was a gradual increase in enzymatic activity as the spermatozoa moved from the proximal to the distal region of the epididymis. The increase in enzyme activity during sperm maturation was reported by our group to be due to the conversion of enzymatically inactive/less active precursor forms to an enzymatically active mature form by proteolysis [11].

Distribution of Man9-mannosidase activity in the germ cell membranes was carried out using marker enzymes of known subcellular localization. In these studies, phosphodiesterase 1 present on the plasma membrane-rich fraction prepared from rat liver [37] and human sperm [9] was used as plasma membrane marker enzyme. In addition, we assayed two acid glycosidase activities believed to be present in the sperm acrosome [28]. Quantification of the Man9-mannosidase and marker enzyme activities in the germ cell membrane fractions showed that Man9-mannosidase, like phosphodiesterase I, had the highest relative activity in the membranes present in band I. The two acid glycohydrolases assayed showed no enrichment in band I, a result suggesting that the lysosomal/acrosomal enzymes were not associated with these membranes (Fig. 5). Examination of the membranes in band I by electron microscopy confirmed the presence of membrane vesicles differing in size and shape (Fig. 6A). The elements characteristic of endoplasmic reticulum and Golgi apparatus were rarely seen in band I but were seen in band II (Fig. 6B). Further confirmation for the association of Man9-mannosidase with the germ cell plasma membranes came after Western blot analysis. In these studies, the salt-washed membranes present in band I and band II, as well as the salt-washed plasma membranes prepared from cauda spermatozoa, were detergent-solubilized and resolved by electrophoresis, and the mannosidase polypeptide was revealed by immunoblotting protocol. This approach detected a 115-kDa mature form of mannosidase in the band I (lane 4, Fig. 7) and cauda sperm plasma membranes (lane 6, Fig. 7). We did not find the precursor forms of 125 kDa and 135 kDa seen in the testicular membranes [11]. However, a band with an apparent molecular mass of 150 kDa that was prominent in the germ cell plasma membranes (lane 4) was only a minor band in the cauda sperm plasma membranes (lane 6). The relationship of the high-molecular mass band to the precursor forms (125 kDa and 135 kDa) and the mature form (115 kDa) of the sperm mannosidase is not known at the present time.

Our failure to immunodetect the precursor forms in the germ cell membranes is probably due to the proteolytic cleavage of these forms during preparation of the germ cells after trypsin digestion of the testis. The protease, when incubated with the purified testicular and caput sperm membranes in vitro, modified the precursor forms to the 115-kDa mature form. Inclusion of benzamidine and aprotinin, known inhibitors of trypsin-like proteases, during in vitro trypsin treatment prevented proteolysis of the precursor forms [11]. These studies are consistent with the proposed proteolysis of the precursor forms during the germ cell separation. It is important to point out that the results presented in this report are the first demonstration that the sperm mannosidase is expressed on the germ cells and that it is an integral component of the plasma membranes. This finding is important and will allow us to examine the biosynthesis and processing of the enzyme using radiolabeling of the antigen in the germ cells in culture. These studies are desirable and will disclose the relationship among the various molecular forms of the sperm mannosidase.


   ACKNOWLEDGMENTS
 
The excellent secretarial assistance of Ms. Danielle Bligny and Ms. Lynne Black is gratefully acknowledged. We are indebted to Dr. Marjorie D. Skudlarek for a critical reading of the manuscript and to Dr. Loren H. Hoffman for the electron micrographs in this manuscript. The electron microscopic studies were performed by the Electron Microscopy Core Laboratory of the Vanderbilt Center for Reproductive Biology Research.


   FOOTNOTES
 
1 Supported in part by grants HD 25869 and HD 34041 from the National Institute of Child Health and Human Development. Back

2 Correspondence. FAX: 615 343 7797. Back

3 Current address: Department of Biosciences and Biotechnology, University of Roorkee, India. Back

Accepted: July 27, 1998.

Received: May 6, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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