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Regular Article |
a Center for Reproductive Biology Research,
c Department of Obstetrics & Gynecology and
b Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2633
ABSTRACT
During spermatogenesis, spermatids synthesize constituent proteins present in mature spermatozoa; however, little information exists on the molecular processes involved. In previous studies, this laboratory reported the characterization of rat sperm ß-D-galactosidase. In this paper, we report the localization of this enzyme along with its biosynthesis and processing. An antibody against rat luminal fluid ß-D-galactosidase was used to immunolocalize the enzyme in the testis and in epididymal spermatozoa. We found that ß-D-galactosidase is localized within the acrosomal cap of spermatids and in the acrosome and cytoplasmic droplet of epididymal spermatozoa. A combination of germ cell radiolabeling, immunoprecipitation, SDS-PAGE, and autoradiography revealed that spermatids produce two forms of ß-D-galactosidase, 90 and 88 kDa. During pulse-chase analysis, a 56-kDa form appeared. Treatment of ß-D-galactosidase immunoprecipitates from testicular spermatozoa with N-glycanase or Endo H revealed that both the 90- and 88-kDa forms become a 70-kDa polypeptide on SDS-PAGE. Since Endo H or N-glycanase treatment provided similar results, the presence of extensive N-linked high mannose/hybrid-type glycans on these proteins is indicated. Treatment of the 56-kDa form of ß-D-galactosidase with Endo H or N-glycanase resulted in the appearance of 52- and 50-kDa forms, respectively. This result suggests that the 56-kDa form contains N-linked high mannose/hybrid as well as complex oligosaccharides. During epididymal maturation, the 90-kDa form of ß-D-galactosidase persists in caput epididymal spermatozoa and is gradually converted to a major 74-kDa form in cauda spermatozoa. In addition to the 90- to 74-kDa forms, cauda spermatozoa show a 56- to 52-kDa form on Western immunoblots. Since only the high-molecular weight forms of ß-D-galactosidase are present on immunoblots of isolated sperm heads, we suggest that they are acrosomal in origin and that the 56-kDa form, which is processed to 52 kDa in cauda spermatozoa, is associated with the cytoplasmic droplet.
epididymis, sperm, sperm maturation, spermatid, spermatogenesis
INTRODUCTION
Glycosidic enzymes typically function within the acidic milieu of the lysosome. These exo-enzymes function in intracellular digestion of specific terminal glycosyl residues from glycoproteins and glycolipids. In the male reproductive tract, lysosomal enzymes are also present in epididymal luminal fluid. One such enzyme is ß-D-galactosidase (E.C. 3.2.1.23) characterized in previous studies [1–4]. Cultured epithelial cells isolated from the caput and cauda epididymidis secrete 61% and 70% of total ß-D-galactosidase per day, respectively. These values reflect the high levels of enzyme present in the epididymal luminal fluid [2, 3]. Further characterization of the luminal fluid ß-D-galactosidase activity revealed an intriguing property: whereas the enzyme activity was optimum at acidic pH (3.5) when using a synthetic (PNP) substrate, as is expected for an acid hydrolase, a neutral pH optimum (6.6) occurred with a [3H]-galactose-labeled glycoprotein substrate [4]. This result indicated that this enzyme may be active in the neutral environment of the epididymal lumen.
In addition to its abundance in luminal fluid, ß-D-galactosidase activity is associated with specific subcellular structures of mammalian spermatozoa, namely, the cytoplasmic droplet [1, 5] and the acrosome [1, 6, 7]. However, enzyme activity in purified cytoplasmic droplets is low relative to intact spermatozoa [1], which suggests that the majority of the sperm enzyme is localized in the acrosome. ß-D-Galactosidase activity in the rat sperm acrosome has been shown to be a soluble, readily released element of enriched acrosomal membrane fractions [1, 6], and it has been shown to have a pH-dependent preference similar to that of luminal fluid ß-D-galactosidase, although it has a different molecular weight [1]. Using an antibody against mouse liver lysosomal ß-D-galactosidase, Western blots of epididymal spermatozoa showed that the predominant form of ß-D-galactosidase gradually shifts from 82 to 80 kDa as spermatozoa migrate from the caput to the cauda [1]. When the luminal fluid (97- and 84-kDa forms) or sperm forms of ß-D-galactosidase are de-N-glycosylated, the resultant protein is resolved at the position of 70 kDa [1]. This demonstrates that the luminal fluid and acrosomal forms of ß-D-galactosidase are differentially glycosylated.
Our understanding of acrosomal enzyme biosynthesis is extremely limited. For instance, it is unclear whether acrosomal enzymes represent testis-specific isozymes. Since testicular germ cells are not amenable to long-term culture and because relatively few proteins have been isolated and characterized, studies of biosynthesis and transport of specific acrosomal proteins have relied on ultrastructural analysis. Like lysosome formation, assembly of the acrosome during spermiogenesis involves transport of glycoproteins from the Golgi apparatus [8] via coated vesicles [9]. On the other hand, there is some evidence that some antigens may not use the typical lysosomal Golgi pathway to arrive at the acrosome [10–12].
Given the limited information on acrosomal enzyme biosynthesis, the following study was undertaken in order to investigate the biosynthesis and processing of one sperm enzyme, ß-D-galactosidase. In this study, we have used immunohistochemical techniques to localize ß-D-galactosidase in the rat testis and cauda epididymal spermatozoa. In addition, we report the biosynthesis and processing of the enzyme during spermiogenesis and sperm maturation. Our results indicate that 1) ß-D-galactosidase is localized within the acrosome and cytoplasmic droplet of spermatozoa; 2) spermatids synthesize two N-glycosylated forms of ß-D-galactosidase, 90- and 88-kDa forms, that are partially processed to an N-glycosylated 56-kDa form during short cultures; 3) despite this conversion, the 90-kDa form of ß-D-galactosidase persists in caput epididymal spermatozoa and is gradually converted to a major 74-kDa form in cauda spermatozoa; and 4) only the 90- to 74-kDa forms are associated with isolated sperm heads. These results allow us to suggest that the higher molecular weight forms are acrosomal in origin and that the 56-kDa form, which is processed to a 52-kDa form in cauda spermatozoa, is associated with the cytoplasmic droplet.
MATERIALS AND METHODS
Materials
The adult male (300–325 g) and retired breeder Sprague-Dawley rats used in this study were obtained from Sasco (Omaha, NE). An adult female New Zealand White rabbit (3–4 pounds) was used for antibody production. Para-aminophenyl ß-D-thiogalactopyranoside agarose, cyanogen bromide-activated Sepharose 4B, DNAase 1 (type II), and trypsin type III were obtained from Sigma Chemical Co. (St. Louis, MO). All electrophoretic chemicals, including marker standards, were from Bio-Rad Laboratories (Richmond, CA). Fluorescein-linked goat anti-rabbit immunoglobulin (IgG) was obtained from EY Laboratories, Inc. (San Mateo, CA). Vectashield fluorescence mounting media was from Vector Laboratories (Burlingame, CA). Serotec reagents were used for immunohistochemistry using alkaline phosphatase-linked secondary antibody (Harlan Bioproducts for Science, Inc., Indianapolis, IN). Enhanced chemiluminescence (ECL) using HRP-conjugated anti-rabbit IgG was from Amersham (Arlington Heights, IL). The Staput cell separation equipment was obtained from Johns Scientific (Toronto, ON, Canada). Collagenase (CLS-1) was from Worthington Biochemicals (Freehold, NJ). Media for culturing of germ cells was derived from the protocol of Grootegoed et al. [13]. Briefly, the media contained minimum essential medium (MEM) powder (GIBCO, Grand Island, NY) plus 0.4% BSA, nonessential amino acids, 105 units penicillin/L, 100 mg streptomycin/L, 1 mg fungizone/L, 5 mM lactate, and 0.022 g NaHCO3/100 ml plus leucine, lysine, and methionine. Enriched Krebs-Ringer bicarbonate media was prepared on the day of use as described in O'Brien [14]. 35S-Methionine (1175 Ci/mmol) was purchased from NEN Research Products (Boston, MA). N-glycanase (E.C. 3.5.1.52) and recombinant endoglycosidase H (Endo H; E.C. 3.2.1.96) were from Genzyme (Boston, MA). Immobilized Protein G-Sepharose 4FF was from Pharmacia LKB Biotechnology, Inc. (Piscataway, NJ). Homogeneous preparations of ß-D-galactosidase (97-kDa form) were prepared as described [4]. All other chemicals were obtained commercially and were of the highest purity available. Kodak Biomax MR film (Rochester, NY) and intensifying screens were used for autoradiography.
Preparation of Antiserum Against Rat Epididymal Luminal Fluid ß-D-Galactosidase
A female virgin New Zealand White rabbit was immunized with purified epididymal luminal fluid ß-D-galactosidase according to Tulsiani et al. [15]. Blood was collected by cardiac puncture after 3–4 immunizations (on Day 87) and serum was then prepared. Fifteen microliters of immune serum immunoprecipitated 91% of ß-D-galactosidase activity from luminal fluid and spermatozoa extracts containing 0.1 units of the enzyme activity.
Preparation of Affinity-Purified Anti-ß-D-Galactosidase IgG
The 
-globulin (IgG fraction) from the preimmune and immune serum was prepared by affinity chromatography on a column of Protein G-Sepharose 4FF using the manufacturer's protocol. The eluted IgG fraction was dialyzed extensively in buffer A (20 mM Tris-HCl [pH 7.5], 0.15 M NaCl, 0.02% NaN3), concentrated in an Amicon unit under 30–40 psi nitrogen. The concentrated IgG (3–4 mg protein/ml) was aliquoted and stored frozen at -80°C until use.
Immobilization of ß-D-Galactosidase and Preparation of Monospecific Anti-ß-D-Galactosidase IgG
Rat cauda luminal fluid, prepared as described in Skudlarek et al. [16], was used for purification of ß-D-galactosidase using an affinity column of p-aminophenyl ß-D-thiogalactopyranoside agarose, as described by Tulsiani et al. [4]. The purified enzyme (~1.22 mg protein) was coupled to 0.3 g of cyanogen bromide-activated Sepharose 4B according to the manufacturer's instructions.
Monospecific IgG was prepared from rabbit anti-ß-D-galactosidase serum. Briefly, the antiserum was centrifuged at 130 000 x g for 30 min at 4°C, and the clear supernatant was brought to loading buffer condition (buffer A). All other steps were carried out at 0–4°C. The sample was applied to the immobilized ß-D-galactosidase column at a flow rate of 1–2 ml/h. The column was then washed with the above buffer until the column fractions showed negligible absorbance at 280 nm. The bound IgG was eluted with 7 M urea in buffer A, concentrated in an Amicon unit as above, and stored frozen at -80°C until used.
Preparation of Preabsorbed (Negative Control) IgG
A 50-fold excess of purified ß-D-galactosidase was used to complex -globulin from the monospecific anti-ß-D-galactosidase IgG preparation. The mixture was maintained at 4°C, and the precipitated complex was removed by centrifugation at 100 000 x g for 30 min. The supernatant was recovered, and the protein concentration was adjusted as desired.
Enzyme Assay
Samples were assayed for ß-D-galactosidase activity using 5 mM of p-nitrophenyl-ß-D-galactoside substrate in a standard incubation mixture (0.5 ml total volume) containing 0.2% Triton X-100 and 10 mM citrate buffer (pH 3.5). Following addition of the extract, the assay mixtures were incubated for 60 min at 37°C, and the process was stopped by the addition of an alkaline buffer adjusted to pH 10.7 [17]. The release of p-nitrophenol was measured by reading the spectroscopic absorbance at 400 nm. One unit of enzyme activity catalyzes the release of 1 µmol p-nitrophenol/h.
Tissue Preparation
Rats were anesthetized by intraperitoneal injection of 0.8 ml Nembutal (50 mg/ml stock solution). They were then perfused (through an abdominal aortic cannula) with saline containing 1000 IU heparin/100 ml until the testes and epididymides were cleared of blood, as described by Forssmann et al. [18]. The organs were then immediately fixed (through the same cannula) by perfusion with Perfix solution (4% paraformaldehyde, 2% trichloroacetic acid, 2% zinc chloride, 20% [v/v] isopropyl alcohol) that had been made the same day. Fixation proceeded for approximately 15 min, whereupon the tissues were excised, cut into pieces (3–4 mm wide), and immersed in Perfix at 4°C for an additional 6 h. Following a standard ethanol dehydration and clearance through xylene, the tissues were embedded in paraffin and sectioned in 5–6-µm slides on poly-L-lysine-coated slides.
Immunohistochemistry
Rehydration and immunostaining were performed as directed by the Serotec protocol. The sections were first treated with Serotec block solution for 30 min. All treatments were performed at room temperature in a humid, sealed container. Primary antibody (monospecific IgG, preabsorbed IgG, or preimmune IgG) was diluted in Tris-buffered saline (TBS) to a concentration of 9 µg protein/ml and applied to testis sections for 60–65 min. This and each subsequent step was followed with three washes (3-5 min) in TBS. The secondary linked antibody solution was then added for 30 min, and the alkaline phosphatase-conjugated anti-biotin antibody was applied for 30 min and revealed with the Vectastain alkaline phosphatase substrate kit (SK 52000), which was applied for 3–4 min. Blocking endogenous alkaline phosphatase with levamisole or omission of primary antibody was also performed. The slides were then counterstained with hematoxylin for ~30 sec, rinsed in water, dehydrated, cleared in xylene, and mounted with permount. The slides were then examined using light microscopy.
Indirect Immunofluorescence
Rats were killed by CO2 asphyxiation. The caudae were minced in PBS containing 0.1% BSA, shaken for 10–15 min, and filtered through 12 layers of cheesecloth. The filtrate was centrifuged at 400 x g for 10 min at room temperature, and the pelleted spermatozoa were resuspended in approximately 0.5 ml PBS containing 0.1% BSA. The sperm suspension was placed onto poly-L-lysine-coated coverslips (100 µl each) and allowed to attach for 15 min in a humid, sealed container. Alternatively, a sperm suspension was fixed in 4% paraformaldehyde on ice, spun at 400 x g for 5 min, resuspended in PBS containing 1% BSA, and allowed to attach to coverslips for 40 min. All further incubations also occurred in this container. Some coverslips were then placed in methanol at -20°C for 15 min for permeabilization. These were then washed twice in PBS (containing 0.1% BSA) for 3 min each time, and all coverslips were incubated with primary antibody (monospecific anti-ß-D-galactosidase IgG), preabsorbed IgG, or preimmune IgG at a concentration of 7–9 µg protein/ml) for 60 min. These were washed as described above and incubated with secondary antibody (fluorescein-linked goat anti-rabbit IgG) at a 1:100 dilution (in the dark) for 60 min. Following washing as described above, the coverslips were mounted with Vectashield fluorescence mounting media and were viewed using fluorescence optics. The number of cells examined in each sample exceeded 100, and the experiments were repeated at least five times.
Spermatid Isolation and Culture
Mixed testicular germ cells were isolated from two 8-wk-old rats or one adult rat by sequential enzymatic digestion exactly as described by O'Brien [14]. Approximately 2.5–5.0 x 108 cells were separated in the Staput unit gravity sedimentation chamber on a gradient of 2%–4% BSA, as described by O'Brien [14]. After sedimentation, fractions (10 ml) were collected. The ~100 fractions were screened by phase microscopy, and the spermatid-enriched fractions were pooled. For immunoblotting, mixed or purified spermatids were washed three times by centrifuging (as described above) in ice-cold PBS; the pellets were then frozen until time of extraction.
For culture, the spermatids were washed into modified MEM media, as described by Grootegoed et al. [13]. For radiolabeling with L-35S-methionine, the cold methionine concentration was diminished to 10 µM (10% of normal concentration). In this media, round spermatids and mixed spermatids could be cultured for at least 16 h while viabilities of greater than 90% were maintained using trypan-blue assessment.
Radiolabeling of Spermatids
L-35S-Methionine was supplemented to culture media at 120–150 µCi/ml. Cells were cultured for 4 or 8 h as described in each figure. After the appropriate interval, cells were centrifuged at 400 x g and washed three times with nonradioactive media. The pelleted cells were either frozen at -80°C for subsequent immunoprecipitation or recultured for pulse-chase studies in nonradioactive media. For pulse-chase studies, after subsequent culturing, cells were washed as above. The pelleted cells were overlaid with 600 µl of 0.1 M Tris-HCl buffer (pH 7.5) containing 0.15 M NaCl and were frozen.
Radioimmune Precipitation and SDS-PAGE
Radiolabeled ß-D-galactosidase was immunoprecipitated as described previously [19]. Briefly, the cell pellets were thawed and brought to 0.05% Triton X-100 with a protease inhibitor cocktail containing chymostatin, benzamidine, antipain, aprotinin, and leupeptin [20]. The mixture was sonicated (3 x 5 sec on ice) in a Fisher sonicator (model 300, Fisher Scientific) set at speed 40, brought up to a concentration of 1% Triton X-100 and 0.1% SDS, and centrifuged at 105 000 x g for 40 min at 4°C. Samples were incubated with 250 µg of the affinity-purified anti-ß-D-galactosidase IgG or preimmune IgG and 108 µl epididymal ß-D-galactosidase (carrier enzyme), as previously described [19]. The antibody-antigen mixture was incubated on ice for 2 h; this was followed by microcentrifugation. The precipitate (antigen-antibody complex) was washed five times in 1 ml 0.1 M Tris-HC1 (pH 7.5), 0.15 M NaCl, and 1% SDS by suspension and centrifugation. The washed pellets were boiled in Laemmli buffer (containing 4 M urea) for 5 min, and the soluble polypeptides were resolved by electrophoresis in 7% polyacrylamide gel (SDS-PAGE system) according to the method of Laemmli [21]. Gels were exposed to Biomax MR film, which was processed automatically after 1–3 wk. In some instances, intensifying screens were used.
Extraction of Spermatozoa and Spermatids
Spermatozoa, isolated as previously described [1], or spermatids in PBS containing 25 mM benzamidine were centrifuged at 600 x g for 10 min, and in some cases, they were frozen at -80°C until time of use. Sperm and spermatid pellets were resuspended in extraction buffer containing 0.1 M Tris-HCl (pH 8.5), 1% Triton X-100, 0.3 M NaCl, and 25 mM benzamidine. The suspensions were sonicated three times for 10 sec each time using a probe sonifier, as described above. They were then centrifuged at 120 000 x g for 20 min. The resuspension, sonification, and centrifugation were repeated two more times. Pooled extracts were then dialyzed exhaustively against 10 mM Tris (pH 7.5), 0.15 M NaCl, and 25 mM benzamidine. Aliquots of the dialyzed sample were assayed for PNP-ß-D-galactosidase activity, and they were also used in immunoprecipitation studies.
Immunoprecipitation and SDS-PAGE
The dialyzed sample containing 2.0 units of PNP-ß-D-galactosidase activity in 0.2–1.0 ml was mixed with 250 µg of affinity-purified immune or preimmune IgG, and the mixture was incubated at 37°C for 30 min and then overnight at 4°C. Following these incubations, the mixture was centrifuged at ~8000 x g for 10 min. The supernatant was saved for enzyme assays. The pellets were washed three times in 10 mM Tris-HCl (pH 7.3), 0.15 M NaCl, and 0.1% SDS, and then the washed antigen-antibody complex was boiled in 4 M urea/Laemmli buffer for 5 min, and the soluble sample was electrophoresed on a 7% polyacrylamide gel (SDS-PAGE), as described by Laemmli [21].
Electrotransfer and Immunoblotting
Resolved polypeptides were transferred to nitrocellulose paper at 100 mA overnight followed by a period of 2–3 h at 300 mA, according to the method of Towbin et al. [22]. Immunodetection was performed using the Amersham ECL protocol, essentially as described in Abou-Haila et al. [23].
Isolation and Extraction of Sperm Heads
Sperm heads were prepared from cauda spermatozoa, essentially as described in Tulsiani et al. [24]. Briefly, pelleted spermatozoa were resuspended in PBS (containing 50 µg trypsin/ml) for 5 min at 25°C and were then centrifuged (80 000 x g for 45 min) on a discontinuous sucrose gradient. The sperm heads were recovered from the bottom of the tubes, resuspended in PBS, and examined by light microscopy.
For biochemical analysis, the sperm head pellet was extracted by suspending in 0.1 M Tris-HCl (pH 8.5), 1% Triton X-100, 0.3 M NaCl, and 25 mM benzamidine, as described above. Immunoblotting and SDS-PAGE were executed, as described above, using primary antibody at a concentration of 1.8 µg protein/ml.
Enzyme Treatments
N-Glycanase and Endo H were used according to manufacturer's instructions, as described previously [4].
RESULTS
ß-D-Galactosidase Immunolocalization in the Testis and Epididymal Spermatozoa
In order to determine the localization of ß-D-galactosidase in germ cells, paraffin sections of adult rat testis were used for immunolocalization studies. At stages XII–XIII of the seminiferous epithelium cycle (Fig. 1a), comprising the basally located zygotene spermatocytes, diplotene spermatocytes, and bundles of elongated spermatids, immunostaining appears to be distributed in the acrosome as well as in the spermatid cytoplasm. The early round spermatids in stage II–III (Fig. 1b) are immunonegative in contrast to the elongated spermatids seen on the same section. The appearance of a punctate immunostain corresponding to the acrosomal granule in stage IV–V (Fig. 1c) and crescent-shaped acrosomal cap in stage VII (Fig. 1d) are also depicted, demonstrating the gradual accumulation of the antigen within the acrosomic system. The immunostaining is also present in the residual bodies and cytoplasmic droplets of late condensing spermatids immediately prior to spermiation (Fig. 1e). Note (in Fig. 1, b and e) that the interstitium is also immunopositive. Large round cells, which may correspond to macrophages, were strongly immunopositive in the interstitial fluid. The only clearly immunonegative region of the interstitium compartment corresponds to the intratesticular blood vessel endothelium. Immunostaining was not seen when primary antibody was omitted (not shown), when preimmune was used (not shown), or when preabsorbed IgG fraction was used (Fig. 1f).
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Cauda epididymal spermatozoa were examined by indirect immunofluorescence to determine the localization of ß-D-galactosidase within these cells. The thin, crescent-shaped staining of the anterior portion of the spermatozoa head corresponding to the acrosome occurred only following permeabilization (Fig. 2, a–f). In this case, the majority of spermatozoa or detached spermatozoa heads exhibited this pattern. Following permeabilization, the cytoplasmic droplet was also strongly immunopositive (Fig. 2, a and b). The acrosome of nonpermeabilized or paraformaldehyde-fixed spermatozoa were essentially immunonegative, showing an occasional random light tail fluorescence (Fig. 2, g–l). Both preabsorbed and preimmune IgG treatments were nonfluorescent (data not shown).
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Synthesis of ß-D-Galactosidase by Spermatids
Previous studies have indicated the presence of several molecular forms of ß-D-galactosidase in rat spermatozoa and in epididymal fluid [1]. In an attempt to establish the changes in the various molecular forms of ß-D-galactosidase during synthesis, purified spermatids were metabolically labeled and subjected to pulse-chase analysis. As seen in Figure 3a, immunoprecipitation of radiolabeled ß-D-galactosidase revealed two prominent bands of 90 and 88 kDa. These two forms were present in equal amounts in pulse periods from 20 min to 16 h (data not shown). In an attempt to determine whether these forms are glycolytically processed, immune precipitates were treated with Endo H (Fig. 3b) or N-glycanase (Fig. 3c). Both treatments revealed a reduction in apparent molecular weight of both 90- and 88-kDa forms to a 70-kDa form, thus indicating substantial N-glycosylation. Because the glycan portions of the 90- and 88-kDa forms are equally sensitive to both enzymes, the two forms appear to contain only high mannose/hybrid-type N-linked oligosaccharides.
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We next attempted to determine the relationship between the 90- and 88-kDa forms with pulse-chase studies. Figure 4a represents a typical 4-h pulse and 12-h chase. During the chase period, the 90- and 88-kDa forms became less intense, and a 56-kDa form appeared. Treatment of the chase products with Endo H (Fig. 4b) showed a shift in molecular weight of the 90- and 88-kDa forms to 70 kDa; additionally, a reduction in the apparent molecular weight of the 56-kDa form (to 52 kDa) was apparent (other bands present on this blot were nonspecific). Treatment with N-glycanase (Fig. 4c) resulted in a shift of the 90- and 88-kDa products to 70 kDa and a shift of the 56-kDa form to 50 kDa.
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Molecular Forms of ß-D-Galactosidase in Spermatids and Epididymal Spermatozoa
We next sought to determine the molecular weight of sperm ß-D-galactosidase to investigate whether this enzyme undergoes changes during sperm development and maturation. Extracts of spermatids and epididymal spermatozoa were immunoprecipitated, electrophoresed on SDS-PAGE, and used for Western blot analysis. As seen in Figure 5, immunoblots clearly showed major differences in the molecular forms of ß-D-galactosidase present in spermatids and epididymal spermatozoa. In spermatids, the major forms were 90 and 88 kDa. These forms also eluted from a mixed testicular germ cell extract applied to a p-aminophenyl ß-D-thiogalactopyranoside agarose column (data not shown). The major forms in caput spermatozoa were 97 and 90 kDa. In the corpus, the predominant forms were 90 and 80 kDa. In the cauda, this shifted to a less abundant 90-kDa form and predominant 80- and 74-kDa forms. Similar results were obtained using whole immune IgG (not shown) and affinity-purified anti-ß-D-galactosidase IgG.
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In order to determine whether these forms were acrosomal (rather than lysosomal or cytoplasmic droplet-associated), molecular forms present in isolated sperm heads and cauda spermatozoa were compared using Western blot analysis. As seen in Figure 6, in addition to the 90- and 74-kDa forms, strong 56–52-kDa band(s) in cauda spermatozoa were revealed by these techniques; these bands were not visible in the immunoprecipitation technique shown in Figure 5. These lower molecular weight bands, also present in caput and corpus spermatozoa (data not shown), are not present in isolated sperm heads, although this immunoblot was repeatedly stripped and reprobed with increased concentrations of IgG. Therefore, it is most likely that the 90- to 74-kDa forms of ß-D-galactosidase are acrosomal, whereas the 56- to 52-kDa forms are associated with the cytoplasmic droplet.
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DISCUSSION
These studies demonstrate the presence of ß-D-galactosidase within the acrosome (Figs. 1 and 2) and cytoplasmic droplet (Fig. 2) of epididymal and testicular sperm, a result that is in agreement with the previous assessment of ß-D-galactosidase activity associated with these structures [1, 6]. The presence of immunoreactive ß-D-galactosidase in the cytoplasmic droplet was not surprising, since purified cytoplasmic droplets are known to contain acid hydrolases, including ß-D-galactosidase [1, 5, 25]. Acrosomal localization of spermatozoa ß-D-galactosidase has also been documented in the rabbit by Nizolajczyk and O'Rand [7]. In addition, the enzyme was reported on the rabbit sperm surface over the acrosomal and postacrosomal regions and was suggested to have a potential role in sperm-zona binding [7].
Based upon its stage-specific immunolocalization (Fig. 1), ß-D-galactosidase is expressed in round spermatids beginning with stage IV of the seminiferous epithelium cycle; this expression is consistent with the coordinated expression of some acrosomal proteins during development of the acrosome [6, 8, 26–29]. The immunohistochemical results in the testis do not show positive immunoreactivity in spermatocytes. However, additional studies suggest that ß-D-galactosidase is expressed in isolated pachytene spermatocytes (unpublished data). The latter result is consistent with spermatocyte expression of other lysosomal enzymes [6, 30–32]. The differences in immunoreactivity may be due to fixation or immunostaining techniques. In order to determine whether sperm ß-D-galactosidase displays molecular properties similar to lysosomal ß-D-galactosidase, metabolic labeling, immune precipitation, and autoradiography were executed with isolated round spermatids; the germ cells were shown to actively import glycoproteins into the developing acrosome [8]. Biosynthetic labeling of spermatids revealed the presence of two major forms of ß-D-galactosidase, forms which resolved at 90 and 88 kDa (Fig. 3a). These two forms were present in labeling windows of 15 min to 8 h (following this period, immune precipitation became technically beset with nonspecific proteins). The 90- and 88-kDa forms were also apparent on nonradioactive immunoprecipitates (Fig. 5). These forms appear to be related, since they both generated a single 70-kDa band when digested with N-glycanase or Endo H. This suggests that the two forms are differentially N-glycosylated forms of a common polypeptide backbone of 70 kDa, as reported by Skudlarek et al. [1]. Furthermore, the results indicate that the two forms contain N-linked high mannose/hybrid-type glycans.
Typically, biosynthesis of lysosomal enzymes involves the proteolytic processing from an enzymatically inactive precursor to an enzymatically active product. In the case of epididymal lysosomal ß-D-galactosidase, the precursors, 87 kDa and 84 kDa, were replaced by a 63-kDa form during pulse-chase studies [2]. Thus, we suspect that the 90- and 88-kDa germ cell forms of ß-D-galactosidase may be precursors, based on their molecular weight. To determine whether these forms were converted to other forms of ß-D-galactosidase in the spermatid stage, pulse-chase analysis was performed. Metabolic labeling followed by the washing of the cells into cold media resulted in the appearance of a 56-kDa form of ß-D-galactosidase in conjunction with the regression of the 90- to 88-kDa forms (Fig. 4a). It is therefore likely that the 56-kDa form of ß-D-galactosidase is a metabolic byproduct of the 90- to 80-kDa forms. However, unlike the 90- and 88-kDa forms of ß-D-galactosidase, which contain only Endo H-sensitive N-linked high-mannose/hybrid-type glycans, the 56-kDa form appears to have differential sensitivity to N-glycanase and Endo H. This result suggests that the 56-kDa form possesses N-linked high mannose/hybrid-type as well as complex-type glycans (Fig. 4, b and c). This evidence raises questions regarding the relationship between the 90- to 88-kDa and 56-kDa forms. However, given the pulse-chase data, it seems likely that the forms are related. Additional studies will be necessary to establish the relationship between the acrosomal and cytoplasmic droplet-associated forms of ß-D-galactosidase.
The apparent modification of acrosomal ß-D-galactosidase during epididymal maturation was suggested by the authors of an earlier study on rat sperm ß-D-galactosidase [1]. In that study, using an antibody against mouse liver ß-D-galactosidase, the apparent change in molecular mass was from 82 to 80 kDa when comparing caput to cauda spermatozoa. This study confirms the processing of ß-D-galactosidase into lower molecular weight forms during epididymal maturation (Fig. 5). However, the present study suggests that the epididymal changes of ß-D-galactosidase are more dramatic: from the 97- and 90-kDa forms in the caput epididymidis to the 90- and 80-kDa forms in the corpus, followed by less abundant 90- and 80-kDa proteins and predominant 74-kDa forms in cauda spermatozoa. However, it is important to point out that the 97- and 84-kDa forms of ß-D-galactosidase are present in the caput and cauda epididymal luminal fluid, respectively. Thus, the 97-kDa form in caput spermatozoa and the 84-kDa form in cauda spermatozoa are likely luminal fluid contaminants, since spermatids do not synthesize a 97-kDa form of ß-D-galactosidase during metabolic labeling studies (Fig. 5, a–c), nor is the 97-kDa form present in spermatid immunoblots (Fig. 3). Nevertheless, the maturational changes of ß-D-galactosidase are still obvious even after omitting the 97- and 84-kDa forms from the model. A possible explanation for the discrepancy in the epididymal processing of ß-D-galactosidase forms reported earlier [1] is the generation of a more effective antibody against rat epididymal luminal fluid ß-D-galactosidase in the present study. Comparison of these two antibodies (IgG titrations of ß-D-galactosidase activity) demonstrates that the antibody against luminal fluid ß-D-galactosidase is much more effective than the anti-mouse liver ß-D-galactosidase antibody in immunoprecipitating luminal fluid and sperm ß-D-galactosidase activity (data not shown). In addition, the chemiluminescence performed in this study gives a much stronger signal on Western blots than does the immunoperoxidase method described in the previous study. These tools together provide a much clearer vision of the maturational changes of sperm ß-D-galactosidase. Intra-acrosomal processing of other antigens during sperm maturation has been noted; among these are acrosin [33] and acrogranin [34]. We speculate that this alteration involves intra-acrosomal processing and activation of acrosomal ß-D-galactosidase, as evident by the rise in epididymal spermatozoa enzyme activity [1]. In a similar study, Hancock et al. [30] immune-precipitated 
-L-fucosidase from metabolically labeled rat germ cells and found no evidence of processing until passage through the epididymis.
Our hypothesis of separate isozymes of ß-D-galactosidase (one form of which is 90–74 kDa) associated with the sperm head and another (56–52 kDa) associated with the cytoplasmic droplet was supported when immunoblots of intact spermatozoa versus isolated sperm heads were compared. Since only the 90- to 74-kDa forms were present in the isolated sperm heads, it is reasonable to suggest that these forms are acrosomal and that the 90- to 88-kDa forms of ß-D-galactosidase synthesized by testicular spermatids are localized in the acrosome. At this time it is not known whether ß-D-galactosidase is associated with specific subcellular structures, such as the Golgi/trans-Golgi network saccules depicted ultrastructurally within the cytoplasmic droplet by Oko et al. [35]. The lower molecular weight of the cytoplasmic droplet-associated form of ß-D-galactosidase is comparable to that of lysosomal ß-D-galactosidase, which has a monomer molecular weight of 63 kDa in epididymal epithelial cells [2], as well as the 58-kDa form of rabbit testis ß-D-galactosidase [7]. The functional significance of the presence of multiple isoforms of beta-galactosidase in the male reproductive tract is not known at the present time. Although the secreted forms of the glycohydrolase in the epididymal fluid have been shown to be optimally active at the neutral pH when two glycoprotein substrates are used [4], the substrate specificity, pH-optimum, and enzymatic activity of the acrosomal forms of the enzyme are not known. Further studies will be required to characterize these isoforms of ß-galactosidase.
In summary, these data describe the biosynthesis, processing, and localization of rat sperm ß-D-galactosidase. The data strongly suggest that spermatids synthesize N-glycosylated isozymes of ß-D-galactosidase that localize to the acrosome and the cytoplasmic droplet.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the demonstration of germ cell isolation by Dr. Deborah O'Brien. We are grateful to Drs. Lynn Matrisian, Gary Olson, and Thomas Oeltman for critical reading of the manuscript, to Dr. Jean-Jacques Lareyre for assistance in the preparation of the graphics, and to Mrs. Loreita Little for expert secretarial assistance.
FOOTNOTES
First decision: 9 November 1999.
1 Supported by National Institutes of Health grants HD25869, HD03820, and HD05797 and by an American Fellowship to C.A.C. from the American Association of University Women Educational Foundation. 
2 Correspondence: Marie-Claire Orgebin-Crist, Center for Reproductive Biology Research, Vanderbilt University School of Medicine, Rm. C3306 MCN, 1161 21st Ave. S., Nashville, TN 37232-2633. FAX: 615 343 7797; m-c.orgebin-crist{at}mcmail.vanderbilt.edu
Accepted: April 26, 2000.
Received: October 7, 1999.
REFERENCES
-D-mannosidase: similarities to sperm plasma membrane -D-mannosidase. Biochem J 1993; 290:427–436.-fucosidase. Biol Reprod 1993; 48:1228–1238.[Abstract]- and ß-subunit gene expression in the rat testis and epididymis. Mol Reprod Dev 1997; 46:227–242.[CrossRef][Medline]This article has been cited by other articles:
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  K. J. Johnson, A. Zecevic, and E. J. Kwon Protocadherin {alpha}3 Acts at Sites Distinct from Classic Cadherins in Rat Testis and Sperm Biol Reprod, February 1, 2004; 70(2): 303 - 312. [Abstract] [Full Text] [PDF]
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