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Gamete Biology |
Hormones/Growth/Development Research Group,3 Ottawa Health Research Institute, Ottawa, Ontario K1Y 4E9, Canada
Departments of Obstetrics and Gynecology,4
and
Biochemistry/Microbiology/Immunology,5 University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada
Environmental and Occupational Toxicology Division,6 Health Canada, Ottawa, Ontario K1A 0L2, Canada
Department of Anatomy and Cell Biology,7 McGill University, Montreal, Quebec H3A 2B2, Canada
Department of Anatomy, Faculty of Science,8 Mahidol University, Bangkok, Thailand
Laboratory Medicine,9 Anatomical Pathology, Ottawa Hospital, Civic Campus, Ottawa, Ontario K1Y 4E9, Canada
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, fertilization, gamete biology, male reproductive tract, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, we demonstrated that AS-A also exists on the surface of mature mouse sperm retrieved from the cauda epididymis and the vas deferens, uterine mouse sperm that have undergone in vivo capacitation [8], and ejaculated pig sperm [9]. Masking sperm surface AS-A by treating live sperm with anti-AS-A immunoglobulin (Ig)G or Fab results in inhibition of sperm-zona pellucida (ZP) binding, indicating that sperm surface AS-A participates in this binding process. Purified AS-A can bind directly to the ZP [8, 9]. AS-A binds to SGG with high affinity (Kd = 8.9 nM) in the absence of a detergent or saposin B, although this binding does not result in SGG desulfation [10]. The high affinity between the two molecules may also explain their colocalization in the sperm head [8, 11]. Because SGG is also engaged in ZP binding, AS-A and SGG may act together as complexes in this binding process. Accumulated evidence suggests that SGG is synthesized in spermatogenic cells and then targeted to their plasma membranes. The level of SGG remains stable during spermiogenesis, sperm maturation, and the initial phase of sperm capacitation [12–14]. However, despite the existence of AS-A transcripts in spermatogenic cells [15] and AS-A glycoprotein in the sperm acrosome [6], it has been unclear whether acrosomal AS-A trafficks to the plasma membrane of spermatogenic cells and testicular sperm or whether AS-A is present in the epididymal fluid and adsorbed onto the sperm surface during epididymal transit. Here, we present evidence favoring the latter hypothesis, which suggests that acquisition of AS-A onto the sperm plasma membrane may be part of the sperm maturation process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Anti-AS-A IgG antiserum was produced in our laboratory against human liver AS-A purified as described previously [16] (a gift from Dr. A. Fluharty, University of California, Los Angeles, CA). Details of this antibody production, and purification of its IgG fraction, and preparation of preimmune rabbit serum (PRS) IgG were published previously [8]. Affinity-purified anti-AS-A IgG was then prepared as previously described [17].
Polyclonal rabbit anti-SGG IgG antiserum monospecific to SGG and its analog SGC was produced in our laboratory. Anti-SGG IgG/Fab and its affinity-purified fractions and the corresponding PRS IgG/Fab were prepared as described previously [11].
Light Microscopic Immunocytochemistry of AS-A on Mouse Testis, Epididymis, and Vas Deferens Sections
CD-1 male mice (
10 wk old) were used for all studies. Mice (n = 5) were perfused with Bouin solution in situ through the left ventricle. The testis, epididymis, and vas deferens were removed in one piece and further fixed in Bouin solution for an additional 2 h. Tissues were then embedded in paraffin for sectioning according to standard procedures. Sections (4 µm thick) were deparaffinized and rehydrated through decreasing concentrations of ethanol (100% to 70%). The tissues were treated with 1% hydrogen peroxide in 70% ethanol and subsequently with 1% lithium carbonate in 70% ethanol to quench endogenous peroxidase and to remove residual picric acid, respectively. The tissues were then treated with 300 mM glycine to neutralize residual formaldehyde. The antigen was retrieved by microwaving the tissues (which were immersed in 0.01 M citrate buffer, pH 6.0) for 3 min at the highest power and an additional 7 min at low power. To block nonspecific binding, the tissues were incubated for 15 min at room temperature with 10% normal goat serum in Tris-buffered saline (TBS). The tissues were then reacted (90 min, 25°C) with affinity-purified anti-AS-A IgG. Following successive washing with 0.1% Tween in TBS, the slide was examined for antigen-antibody interaction using the Vectastain ABC Elite kit (Vector Laboratories, Burlingame, CA). This process involved treatment of the slide with biotinylated secondary antibody (goat anti-rabbit IgG, 30 min, room temperature), followed by incubation with avidin-biotin-horseradish peroxidase complex and detection by a reaction with a peroxidase substrate, diaminobenzidine. Concentrations of chemicals and conditions for these treatments were as described by their manufacturers. The brown product of the peroxidase reaction signified AS-A localization. Tissues treated with 10 µg/ml PRS IgG served as negative controls.
Collection of Sperm from the Testis, Caput and Corpus Epididymis, and Cauda Epididymis and Vas Deferens and of Fluid from the Cauda Epididymis and Vas Deferens
To collect testicular sperm, decapsulated testes were first minced into 1- to 2-mm pieces in Kreb Ringer bicarbonate medium buffered with Hepes (KRB-Hepes: 119.4 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM sodium lactate, 1 mM sodium pyruvate, 5.6 mM glucose, 28 µM phenol red, 4 mM NaHCO3, and 21 mM Hepes, pH 7.4), supplemented with 0.3% BSA (KRB-Hepes-BSA), and containing 1 mg/ml DNaseI (Sigma, St. Louis, MO). Protease inhibitor cocktail (PIC; Roche Diagnostics, Mannheim, Germany) was also added to the medium at a dose recommended by the manufacturer (i.e., 1 tablet per 10 ml of the medium). Large fragments and clumps of Leydig and Sertoli cells were pelleted (30 x g, 25°C, 5 min). The supernatant (2 ml), containing loose testicular cells, testicular sperm, and red blood cells, was layered on top of a 2-ml 45% Percoll solution in KRB-Hepes. The tube was centrifuged (600 x g, 25°C, 5 min) to pellet red blood cells, which were then carefully removed using a Prot/Elec Pipet Tip (BioRad Laboratories, Hercules, CA) without disturbing the testicular cells and testicular sperm that were sedimented at the interface between the loaded medium and the 45% Percoll solution. The tube was further centrifuged (1000 x g, 25°C, 30 min) to selectively pellet testicular sperm. After removing the top fluid layers, the pelleted testicular sperm were washed (350 x g, 25°C, 10 min) once in KRB-Hepes and directly used for immunoblotting or resuspended in KRB-Hepes-BSA for indirect immunofluorescence (IIF) or for Alexa-430 AS-A binding experiments.
To collect caput and corpus epididymal sperm, the caput and corpus epididymis was cut once longitudinally and then immersed in KRB-BSA. Caput and corpus epididymal sperm were allowed to swim out into the medium by incubating the tissue at 37°C with gentle shaking for 15 min. At the end of the incubation, the tissue was removed from the tube, and the cell suspension was centrifuged at 45 x g to pellet red blood cells, tissue debris, and cell aggregates. The supernatant containing mainly caput and corpus epididymal sperm was transferred into a new tube and centrifuged at 284 x g for 10 min. The pelleted caput and corpus epididymal sperm were washed once in KRB-BSA by centrifugation (284 x g, 10 min) and subject to IIF and flow cytometry.
Sperm and fluid were also collected from the vas deferens and cauda epididymis. Collection was first made at the vas deferens by inserting PE10 polyethylene tubing sleeved over a 25-ga needle, which was attached to a 1-ml tuberculin syringe, into the tubule. Following fluid aspiration, the vas deferens tubule was washed twice with PIC-supplemented KRB-Hepes, and all washes were combined with the original fluid. To collect fluid from the cauda epididymis, the organ was longitudinally cut once with a sharp razor blade and gently squeezed with flat-end tweezers. The thick fluid that oozed from the organ was collected and mixed with KRB-Hepes-PIC. Fluid from both the vas deferens and cauda epididymis was combined and centrifuged (500 x g, 25°C, 10 min) to pellet sperm. Caudal epididymal and vas deferens sperm were washed once in KRB-Hepes-PIC and resuspended in KRB-Hepes-BSA for IIF. A peripheral plasma membrane protein extract was also prepared from washed caudal epididymal and vas deferens sperm by treatment with a sucrose solution (320 mM) containing 1 mM EDTA and 1 mM ATP (AES) [8] and used for immunoblotting. The collected supernatant was further centrifuged in a Microfuge 18 (Beckman Coulter, Palo Alto, CA) (18 000 x g, 4°C, 10 min) to remove all sperm and particulates. The sperm-free fluid was then used for immunoblotting.
IIF for AS-A and SGG and Flow Cytometry of AS-A on Live Testicular Sperm, Caput and Corpus Epididymal Sperm, and Caudal Epididymal and Vas Deferens Sperm
Live testicular sperm, caput and corpus epididymal sperm, and caudal epididymal and vas deferens sperm were separately incubated (5% CO2, 37°C, 30 min) with 10 µg/ml anti-AS-A IgG or affinity-purified anti-AS-A IgG (see [8, 17] for purification and use in immunofluorescence) or with 10 µg/ml affinity-purified anti-SGG IgG [11], all in KRB-Hepes-BSA. Sperm were then washed in the same medium and incubated with 25 µg/ml goat anti-rabbit IgG conjugated with the Alexa 488 fluorochrome (Molecular Probes, Eugene, OR). Caudal epididymal and vas deferens sperm treated with PRS IgG in place of primary antibody served as controls. To assess cell viability during the staining process, propidium iodide (Clontech, Palo Alto, CA) was added to the cell suspension at a final concentration of 0.5 µg/ml 5 min before the the secondary antibody incubation period ended. Sperm were then washed twice in KRB-Hepes-BSA, resuspended in the same medium, mounted onto a slide in PBS/glycerol (1:1, v/v), topped with a cover slip, and viewed under a Zeiss IM35 epifluorescence microscope (Carl Zeiss Canada Ltd., Toronto, ON, Canada).
Live testicular sperm, caput and corpus epididymal sperm, and caudal epididymal and vas deferens sperm that were subjected to immunofluorescent staining (but without propidium iodide incubation) were analyzed by flow cytometry to assess individual fluorescence intensity. After extensive washes to remove unbound fluorescent secondary antibody, the sperm were filtered through a 70-µm mesh (Tube-top Filicon; DAKO Diagnostics Canada, Mississauga, ON, Canada) immediately before analysis on a Coulter Epics Profile II Flow Cytometer (Beckman Coulter, Fullerton, CA). Samples were excited by an argon ion laser at 488 nm, and the emission fluorescence was monitored at 525 ± 20 nm band pass. Sperm cells were gated from debris using their unique properties of forward and side light scattering. Data from at least 5000 events were collected, and relative AS-A staining intensity was determined using FCS Express Software (De Novo Software, Orangeville, ON, Canada).
Reverse Transcription Polymerase Chain Reaction of AS-A from Cauda Epididymis and Vas Deferens and from Testes and Nucleotide Sequencing of the Product
Total RNAs from cauda epididymis and vas deferens and from testes were separately extracted using Trizol reagent (Gibco BRL, Burlington, ON, Canada), following the manufacturer's protocol. Using RETROscript First-Strand Synthesis Kit for reverse transcription polymerase chain reaction (RT-PCR) (Ambion, Austin, TX), total RNAs (1 µg, determined from A260) were reverse-transcribed into first-strand cDNAs in a 20-µl reaction mixture. Subsequently, 2 µl of this reaction mixture containing the first-strand cDNA was used as a template for PCR. The primers for amplification of AS-A were as follows: F-ASA: 5'-atggggtctttgctgttcgg-3' (nucleotides 566–587; numbering as described for human AS-A sequence [18]); R-ASA: 5'-ttctggtaaggtggcatcggac-3' (complementary to nucleotides 2412–2434). Amplification reactions were carried out in a Mastercycler (Brinkmann Instruments, Mississauga, ON, Canada). These reactions were initiated with 10-min denaturation at 94°C, followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 45 sec, and extension at 72°C for 45 sec, and ending with further extension at 72°C for 10 min. RT-PCR products were then resolved by electrophoresis in a 2% agarose gel containing ethidium bromide (0.5 µg/ml) followed by visualization under an ultraviolet lamp (UV Transilluminator, Gel Doc 1000; BioRad). RT-PCR experiments were repeated three times using RNA samples prepared on different days from three different animals. Negative controls were performed in all experiments by omitting the RT step.
The RT-PCR product of AS-A from cauda epididymis and vas deferens was purified by agarose gel electrophoresis. The product band was excised and purified using a Concert Rapid Gel Extraction System (Gibco BRL, Gaithersberg, MD). Approximately 100 ng of the purified PCR product of AS-A was used for nucleotide sequencing, which was performed by the chain termination method using an automated capillary electrophoresis and fluorometric detection system (ABI Prism 3100 Genetic Analyzer; Applied Bioystems, Foster City, CA).
Immunoblotting of Sperm and Fluid Collected from Cauda Epididymis and Vas Deferens with Anti-AS-A
Epididymal fluid collected from the cauda epididymis and vas deferens and the peripheral plasma membrane protein extract of caudal epididymal and vas deferens sperm were subjected to SDS-PAGE (gel: 12% acrylamide, 0.75 mm thick) [19], followed by electroblotting onto nitrocellulose membrane [20]. The blot was incubated with anti-AS-A antiserum followed by horseradish peroxidase (HRP)-conjugated secondary antibody (Bio-Rad). Conditions of the incubation for antibody-antigen interaction were as described previously [8]. The AS-A signals on the nitrocellulose blot were captured by a Typhoon 8600 Variable Mode Imager (Molecular Dynamics, Sunnyvale, CA) in the chemiluminescence mode, using Typhoon Scanner Control 1.0.
Quantification of AS-A in the Caudal Epididymal and Vas Deferens Fluid
AS-A purified from the pig sperm surface extract to a single band on SDS-polyacrylamide gels [9] was used as a standard. Caudal epididymal and vas deferens fluid containing 6 µg of total proteins or AS-A standard (1–15 ng, as quantified using Protein Assay Solution; Bio-Rad) in 100 µl of 100 mM sodium carbonate, pH 9.6, was allowed to attach to the bottom surface of the wells of a Limbro/Titerteck 96-well polystyrene plate (ICN Biomedical, Aurora, OH) overnight at 4°C. The attached proteins were blocked with 1% BSA in TBS at 25°C for 1 h followed by three washes in TBS and 0.05% Tween 20. The protein wells were then incubated with 100 µl of anti-AS-A antiserum (1:1600 dilution in TBS) at 25°C for 1 h. Following successive washing in TBS and 0.05% Tween 20, 100 µl of HRP-goat anti-rabbit IgG (1:3000 dilution in TBS) was added to the wells for incubation at 25°C for 30 min. After washing thoroughly with TBS and 0.05% Tween 20, 100 µl of an HRP substrate, o-phenylenediamine dihydrochloride (0.05% in 100 mM sodium citrate, pH 5.0), and H2O2 (1.5 µl/ ml of the substrate solution) were added to the wells, which were then incubated at 25°C until the yellow color developed. The reaction was then stopped with 50 µl of 30% H2SO4, and the color intensity was measured at A490.
Binding of Alexa-430 AS-A onto the Caudal Epididymal Sperm
Alexa-430 AS-A and Alexa-430 ovalbumin were prepared by conjugating AS-A and ovalbumin, respectively, with the Alexa 430 fluorochrome (Molecular Probes) as instructed by the manufacturer. Caudal epididymal sperm (1.2 x 106) were incubated (37°C, 5% CO2, 1 h) with various concentrations (0–60 nM) of Alexa-430 AS-A or with 60 nM Alexa-430 ovalbumin in 600 µl of KRB-BSA. At the end of the incubation, sperm were washed free from the unbound protein by centrifugation (600 x g, 25°C, 10 min) in KRB-BSA. The washed sperm pellet was resuspended in 600 µl of KRB-Hepes, and 100-µl aliquots (200 000 sperm) were placed in each well of a Costar black 96-well microtiter plate (Corning, Corning, NY) for measurement of fluorescence intensity using a Spectramax GeminiXS spectrofluorometer (Molecular Devices) at the excitation and emission wavelengths of 425 and 520 nm, respectively. The amount of AS-A bound per sperm was then calculated from the Alexa-430 AS-A standard curve. The data obtained were analyzed for the Kd value of AS-A-ZP binding by Scatchard plotting using Grafit 4.0 software for Windows (Erithacus Software, Surrey, U.K.). In an alternative experiment, 600 nM AS-A was included with 60 nM Alexa-430 AS-A for sperm incubation. Measurement of Alexa-430 AS-A binding to sperm was then performed as described above.
To investigate the roles of SGG and mannose phosphate receptors on the sperm surface in AS-A binding, 200 000 caudal epididymal sperm in 150 µl KRB-Hepes were pretreated (37°C, 5% CO2, 30 min) with various concentrations of anti-SGG Fab (0–10 µM), PRS-Fab (0–10µM), or mannose 6-phosphate (0–5 mM) before incubation with 63 nM Alexa-430 AS-A following the procedure described above. Data were expressed as percentages of the control fluorescence intensity values (i.e., from incubations of Alexa-430 AS-A with sperm without any competitors) for each of the competitor concentrations. Differences in the levels of AS-A binding to sperm in the control incubations (no competitors added) and in those with competitors included were analyzed by ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To elucidate how AS-A, generally known as a lysosomal enzyme, targets to the surface of mature sperm [8, 9], immunocytochemistry of mouse testis and epididymis sections and IIF of live mouse sperm were performed using affinity-purified anti-AS-A IgG antibody. All experiments, performed with tissues and sperm collected from five different mice, gave consistent results. In testes, AS-A immunocytochemical staining was observed in the Sertoli cell cytoplasm, appearing from the base of the seminiferous epithelium toward the supranuclear region, which was stained with higher intensity and showed globular patterns. This finding suggests that the enzyme compartmentalized into cytoplasmic organelles, presumably lysosomes (Fig. 1a). Early residual bodies did not show any reaction, in contrast to late residual bodies, which were intensely stained (Fig. 1b).
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Immunocytochemical reaction for AS-A was also observed in spermatids. Staining was intense at the acrosomal cap of round spermatids and at the mature expanded acrosome of elongating spermatids (Fig. 1, a and b). In contrast, AS-A staining was not observed in primary spermatocytes or spermatogonia or in the lumen (Fig. 1, a and b). Tissue sections that were exposed to PRS IgG in place of anti-AS-A IgG showed no staining (Fig. 1c). All of these results indicated that the immunocytochemical staining observed in Sertoli cell organelles, presumably lysosomes, and spermatid acrosomal machinery was specific to AS-A.
IIF of live sperm revealed that the majority of testicular spermatozoa (80%) showed no staining on the cell surface (Fig. 2A). These sperm also did not stain for propidium iodide, indicating that their plasma membrane was intact. A histogram frequency of AS-A staining intensity, based on flow cytometry analysis, showed a major peak of testicular sperm (80%), with background fluorescence intensity similar to that of the negative control cells, i.e., mature sperm (caudal epididymal and vas deferens sperm) that were subjected to PRS IgG and Alexa-488-conjugated secondary antibody (Fig. 3A). The results therefore suggest that live intact testicular sperm are devoid of AS-A on the surface. In contrast, the remaining 20% of testicular sperm were stained with propidium iodide, indicating that their surface membranes were damaged (Fig. 3A). These propidium iodide-positive sperm also showed Alexa-488 staining, although without any consistent patterns. Because these sperm presumably were no longer viable, this fluorescein isothiocyanate staining with inconsistent patterns may reflect nonspecific binding of the primary and/or secondary antibodies to sperm.
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The absence of AS-A staining on testicular mouse spermatozoa might be due to the masking of the surface of these sperm. To test this possibility, IIF of SGG, a male germ cell surface sulfoglycolipid [11–14], was performed with live testicular sperm and mature sperm from cauda epididymis and vas deferens. Results shown in Figure 4 (A and B) indicated that both testicular and mature sperm exposed to affinity purified anti-SGG IgG possessed specific fluorescent staining patterns, i.e., at the convex ridge of the sperm head and the posterior head region. In contrast, when PRS IgG was used in place of affinity purified anti-SGG IgG for sperm incubation, no fluorescent staining was observed on both sperm types (Fig. 4C). Therefore, it was apparent that SGG could be detected by IIF on the surface of both testicular sperm and mature sperm, this argued against the possibility that the testicular sperm surface was masked.
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Immunocytochemical results revealed the presence of AS-A in the epididymis. In the initial segment, reaction was restricted to narrow and apical cells (as identified by their shape and location [21]) (Fig. 1d). In the caput, corpus and cauda regions, clear cells (as identified by their possession of numerous vacuoles [21]) were reactive (see an example in the corpus, Fig. 1e). In the caudal region, a much greater number of cells reacted with anti-AS-A. The majority of reactive cells often were grouped together, appearing as positively stained strips (Fig. 1f). These cells spanned from the basal membrane to the apical membrane, showed a brush border at a high magnification and were the majority of the epididymal epithelial cells, and therefore they were identified as principal cells [21]. The AS-A immunoreaction in principal cells was over the entire cytoplasm, appearing as network patterns (Fig. 1f, inset). In the vas deferens, most principal cells surrounding the lumen were reactive for AS-A, although the intensity of the staining was not as high as for principal cells in the cauda epididymis (Fig. 1g). However, the staining of AS-A in the lumen in both the cauda epididymis and vas deferens was not readily apparent (see example in Fig. 1, f and g). It is possible that the amount of AS-A secreted into lumen may be small and was therefore washed out from the sections during the processing of the tissue sections.
To further demonstrate that the cauda epididymis and vas deferens were involved in AS-A biosynthesis, RT-PCR was performed on these tissues using AS-A primers synthesized based on sequences in the 5' and 3' regions of testis AS-A cDNA sequence. RT-PCR products of the same size (1868 base pairs) were obtained from both testis and cauda epididymis and vas deferens RNA (Fig. 5). Nucleotide sequencing revealed 100% identity between these two RT-PCR products, with 100% match to the mouse testis AS-A sequence previously described [22]. These results indicate that the sequence of the AS-A transcript synthesized in the epididymal epithelial cells is identical to that from testis.
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Although AS-A reaction was not evident in the lumen of the cauda epididymis and vas deferens, possibly because of the small amount, 95% of live sperm retrieved from these tissues showed positive immunofluorescent staining in the head region (Fig. 2C). Caudal epididymal and vas deferens sperm were collected without any tissue mincing, and >95% of these sperm were viable, as indicated by their ability to exclude propidium iodide. Therefore, AS-A staining observed on these sperm heads, which consistently demonstrated two distinct patterns, should represent AS-A existence on intact sperm in the cauda epididymis and vas deferens. The major staining pattern (80% of total sperm) showed AS-A signals at both the acrosomal ridge and the head posterior region (Fig. 2C), the same sites where SGG is localized (Fig. 4) [11]. In the remaining sperm (20%), AS-A immunofluorescent staining was found only at the acrosomal ridge. The intensity of the AS-A fluorescent signal was variable in the population of caudal epididymal and vas deferens sperm. Flow cytometry, performed on two different experimental days, showed one major peak (histogram frequency) of AS-A staining intensity, encompassing 70%–80% of caudal epididymal and vas deferens sperm. AS-A staining intensity of the major population of these sperm was 8–10 times higher than the corresponding background fluorescence observed in sperm collected also from the cauda epididymis and vas deferens and subjected to PRS IgG. Approximately 20%–30% of caudal epididymal and vas deferens sperm showed higher intensity of AS-A staining than did the major peak population (Fig. 3, A and B). Because the majority of caudal epididymal and vas deferens sperm were viable, the trailing of the sperm population showing higher AS-A staining intensity indicated inherent heterogeneity of sperm quality. Corroborating the immunocytochemistry results, which revealed AS-A expression only in endocytotic clear cells in the corpus (Fig. 1e) and caput (data not shown) epididymis, was the absence of AS-A fluorescent staining in the majority (70%) of caput and corpus epididymal sperm (Figs. 2B and 3B). However, another 30% of caput and corpus epidiymal sperm possessed AS-A staining with an intensity similar to that of the major peak of caudal epididymal and vas deferens sperm (Fig. 3B). Fluorescent staining of AS-A in caput and corpus epididymal sperm was localized mainly to the postacrosomal region of the sperm head (Fig. 2B). This population of caput and corpus epididymal sperm may have come from the epididymis area just above the beginning of the cauda epididymis. AS-A, secreted from principal cells in the cauda region, may diffuse into the lumen of this epididymis area. The transit sperm in this border area may also be more mature than sperm in the proximal region of the caput epididymis and may be capable of capturing AS-A in the epididymal fluid onto their surface. All of these results indicate that AS-A is present on the surfaces of all caudal epididymal and vas deferens sperm and in a minor population of caput epididymal sperm, in addition to its intracellular existence (i.e., in the acrosome; Fig. 1, a and b).
To verify whether AS-A was present in the caudal epididymal and vas deferens lumen, fluid from these reproductive tract regions was collected. Care was taken to cause the least amount of damage to the epithelial cells during collection, which could have resulted in the release of intracellular AS-A into the fluid. Immunoblotting revealed the presence of AS-A in the caudal epididymal and vas deferens fluid (Fig. 6). A single band of AS-A was observed, possessing the same molecular mass (68 kDa) as that of the AS-A present in the mouse sperm surface extract [8]. AS-A present in the fluid retrieved from the cauda epididymis and vas deferens was quantified by ELISA at 1.28 ng/µg of total fluid proteins (an average of two analyses with <4% difference).
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Binding of Alexa-430 AS-A onto Caudal Epididymal Sperm
AS-A has high affinity (Kd = 8.9 nM) for SGG monolayers [10], which may explain how AS-A in the caudal epididymal fluid is deposited onto the epididymal sperm surface, provided that SGG on intact sperm has similar affinity to AS-A. Figure 7 shows that Alexa-430 AS-A bound to caudal epididymal sperm in a concentration-dependent manner (Fig. 7A, open circles). At 60 nM, the binding of Alexa-430 AS-A to sperm was approaching saturation (Fig. 7A). However, binding of Alexa-430 ovalbumin to caudal epididymal sperm was at a very low level (2% of that observed for Alexa-430 AS-A throughout all concentrations used; Fig. 7A, closed triangles, inset right). The binding of sperm to Alexa-430 ovalbumin was then considered nonspecific, and the values obtained were used for subtraction from the fluorescence values of Alexa-430 AS-A binding to sperm prior to kinetic analysis (Fig. 7B). Fluorescence microscopy revealed that Alexa-430 AS-A bound to the sperm head at the convex ridge and the postacrosomal region (Fig. 7A, insert left). Both of these sites contain SGG (Fig. 4) [11]. When 600 nM unlabeled AS-A was included with 60 nM Alexa-430 AS-A for sperm incubation, binding of Alexa-430 AS-A to sperm was abolished to the background level, implicating specificity of the binding. Analysis of the Alexa-430 AS-A-sperm binding curve revealed Kd of 46.0 ± 15.0 nM AS-A (Fig. 7B), a value within the same range as that of AS-A binding to SGG monolayers [10]. The high SD of this Kd value may reflect inherent heterogeneity of sperm quality. When testicular sperm were used in place of caudal epididymal sperm for incubation with Alexa-430 AS-A, binding of the enzyme to the sperm surface was not observed (data not shown). Possibly, the sperm surface needed to be modified as part of the continuing sperm maturation process during migration through the epididymis to gain the AS-A binding ability. This Alexa-430 AS-A binding to sperm was dependent on surface SGG molecules. Sperm treated with anti-SGG Fab showed a dramatic decrease in Alexa-430 AS-A binding, compared with untreated sperm (Table 1). In contrast, sperm treated with PRS Fab did not show any decrease in Alexa-430 AS-A binding (Table 1). The results strongly suggest that deposition of AS-A onto the sperm surface was via its affinity to SGG on the sperm plasma membrane. Because 
-mannosidase, an acidic glycohydrolase present in the epididymal fluid, deposits onto sperm via interaction with mannose-6-phosphate receptor on the sperm surface [23] and because phosphorylation has been identified on the mannose residues of testis AS-A N-linked oligosaccharides [24], AS-A deposition onto the sperm surface may also be through its interaction with mannose-6-phosphate receptor. To test this hypothesis, mannose-6-phosphate (having affinity for mannose-6-phosphate receptor) was included in Alexa-430 AS-A-sperm coincubates. Mannose-6-phosphate, even at 1 mM, did not inhibit Alexa-430 AS-A binding to sperm, indicating that deposition of Alexa-430 AS-A onto sperm was not through the mannose-6-phosphate receptor mechanism.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunocytochemical staining also revealed the presence of AS-A in Sertoli cells. In the majority of these cells, the globular body-like staining patterns of AS-A, regionalized mainly in the supranuclear region, might reflect lysosomal localization of the enzyme. This postulation is supported by previous reports of the presence of AS-A in lysosomes of Sertoli cells [25] and other somatic cells [18, 26–30] (Fig. 1a). Late residual bodies present in Sertoli cells of certain stages of the spermatogenic cycle were also stained intensely with anti-AS-A (Fig. 1b), although early residual bodies showed negative staining. Because the late residual bodies eventually fuse with the Sertoli cell lysosomes [31], the results suggest that AS-A present in the late residual bodies is derived from Sertoli cell lysosomes following the fusion of one with the other. This result for AS-A is similar to that described for ß-hexosaminidase A [32]. AS-A in Sertoli cells does not appear to be secreted, because the enzyme was not found in the medium of primary culture of mouse Sertoli cells (N. Tanphaichitr, unpublished results).
Results from IIF and flow cytometry analyses for AS-A on live testicular sperm, caput and corpus epididymal sperm, and caudal epididymal and vas deferens sperm (Figs. 2 and 3) indicate that AS-A is acquired by the sperm surface during sperm transit through the epididymis. A few lines of evidence indicate that principal cells from the cauda epididymis and vas deferens are the source of this sperm surface AS-A. Immunocytochemistry results revealed AS-A staining in strips of principal cells in the cauda epididymis (Fig. 1f) and most of the principal cells in the vas deferens (Fig. 1g). Principal cells are recognized for their secretory activities [21, 33]. The network patterns of AS-A staining in principal cells (Fig. 1f, inset), resembling the endoplasmic reticulum and Golgi apparatus [34], suggest that AS-A is synthesized in principal cells. Similar staining patterns have been noted in principal cells for SGP-2, immobilin, and other known secreted proteins of the epididymis [35]. The presence of the AS-A transcript in the epididymis as shown by RT-PCR (Fig. 5) also supports this interpretation. Immunoblotting confirmed the presence of AS-A in the epididymal fluid (Fig. 6). These results strongly suggest that AS-A is secreted by epididymal epithelial cells into the lumen.
Although immunocytochemistry also revealed the presence of AS-A in the initial segment, caput, and corpus and cauda epididymis, the enzyme was restricted to apical cells, narrow cells, and clear cells (Fig. 1, d–f). As demonstrated for other lysosomal enzymes, immunocytochemical reaction and electron microscopic immunogold labeling in these cell types appear to represent the targeting of these enzymes from the Golgi apparatus via small vesicles directly to their lysosomes, which were abundant in these cells [21, 31, 36, 37]. This may also be the case for AS-A. Alternatively, because narrow cells, apical cells, and clear cells are known for their endocytotic activities [21, 33], AS-A present in these cells may have originated from transit sperm with damaged acrosomes through the endocytosis mechanism. AS-A in these cells may function in desulfation of SGG, endocytosed from remnant membrane vesicles in the epididymal fluid. These vesicles could originate from the shedding of excess plasma membranes of round spermatids during their differentiation to testicular spermatozoa and/or from plasma membranes of damaged sperm in transit through the initial segment and the proximal region of the caput epididymis.
Once secreted in the fluid, AS-A may readily be deposited onto the caudal epididymal and vas deferens sperm surface. This proposal is supported by the observation that AS-A in the epididymal fluid and that on the sperm surface have the same molecular mass (68 kDa) (Fig. 6). Deposition of AS-A onto the mature sperm surface occurred through its high affinity for surface SGG (Kd = 46 nM) (Fig. 7 and Table 1). However, deposition was not dependent on mannose-6-phosphate receptor, which is present on the sperm surface [38]; millimolar concentrations of mannose-6-phosphate did not inhibit Alexa-430 AS-A binding to intact caudal epididymal sperm (Table 1). The interaction between AS-A and mannose-6-phosphate receptor on the sperm surface may be of a low affinity, making it impossible for mannose-6-phosphate to compete with AS-A binding to sperm surface SGG, which occurred with high affinity.
ELISA results indicated that AS-A is a minor protein in the caudal epididymal fluid (i.e., 0.13% by weight of total proteins in the caudal epididymal and vas deferens fluid). This result and the strong implication, presented herein, that sperm surface AS-A originates from the epididymal fluid explain why AS-A exists in very small amounts on the surface of mature mouse sperm (1 pmole/106 sperm [8]). The physiological significance of sperm surface AS-A, however, has been demonstrated. AS-A remains on the anterior head surface of uterine mouse sperm and ejaculated pig sperm and is involved in sperm-egg interaction both in vitro and in vivo [8, 9]. The very small amount of AS-A in the epididymal fluid explains why immunocytochemistry could not detect AS-A in this entity. The molar ratio of AS-A to SGG is very low in mature sperm (SGG is about 600 pmoles/106 sperm [39], and AS-A is 1 pmole/106 sperm). This low molar ratio may be essential in maintaining the integrity of the sperm plasma membrane. When purified AS-A at a nonphysiological high concentration was added to capacitated sperm, the sperm developed a premature acrosome reaction [10]. The wide distribution of free SGG molecules (AS-A unbound) may be beneficial for interaction between AS-A-SGG complexes and the ZP glycans. AS-A, being a peripheral plasma membrane protein [8], may first bind to the ZP glycans and anchor them next to the sperm surface for additional interaction with the galactosylsulfate head groups of SGG molecules, which are extended only a short distance from the sperm plasma membrane bilayers. Carbohydrate-carbohydrate interaction between the sugar head groups of glycolipids with other glycans has been well documented and may also be the basis of SGG-ZP glycan interaction [40, 41]. Although this type of interaction is not as strong as protein-carbohydrate or protein-protein interactions, it can be compensated by availability of multiple glycolipid molecules within the glycan-binding domain. Restricted secretion of AS-A by the epididymis may thus regulate the low molar ratio of AS-A to SGG with a beneficial consequence of better binding of mature sperm to the ZP. As observed with other molecules involved in egg interaction (e.g., P26H [42], -mannosidase [43], and DE protein [44, 45]), acquisition of AS-A by the sperm surface constitutes part of sperm maturation by providing sperm with increasing ZP-binding ability.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Nongnuj Tanphaichitr, Ottawa Health Research Institute, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada. FAX: 613 761-5365; ntanphaichitr{at}ohri.ca
Received: 13 August 2002.
First decision: 12 September 2002.
Accepted: 21 May 2003.
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—), caput epididymal (—
—), and caudal epididymal and vas deferens (—
—) mouse sperm analyzed by flow cytometry. Sperm exposed to PRS IgG served as a negative control (—
—). A) Comparison between testicular sperm and caudal epididymal and vas deferens sperm. B) Comparison between caput epididymal sperm and caudal epididymal and vas deferens sperm
) was measured spectrofluorometrically. Sperm incubated with 60 nM Alexa-430 ovalbumin (