INTRODUCTION

Mammalian spermatozoa undergo many changes as they move through the male and female reproductive tracts, ultimately delivering their haploid genome to the egg. As spermatozoa traverse the epididymis, they become progressively motile and capable of binding to the egg zona pellucida (1). Coincidentally, various secreted epididymal proteins bind to spermatozoa followed by a complement of accessory gland-derived proteins (2). In the female reproductive tract, several interactions between the spermatozoon and its environment are also evident, including: 1) oviductal epithelial cell binding, 2) development of the competence to fertilize an egg, known as capacitation, 3) induction of an exocytotic event known as the acrosome reaction, 4) hyperactivation of motility, 5) cumulus mass and zona pellucida binding and penetration, and 6) egg plasma membrane binding and fusion. Many of these interactions appear to show relative or absolute species specificity, suggesting sperm-specific adhesion and signaling molecules.

Antibodies can serve as powerful reagents for the identification of sperm-specific proteins. Auto- and allo-antisera, as well as monoclonal antibodies, have been raised against sperm cells from several species, and these antisera often recognize proteins that appear to participate in sperm and egg interactions (3, 4, 5). Some of these antigens, such as rabbit sperm autoantigen, hyaluronidase, and fertilin, have been characterized at the molecular level (6, 7, 8).

To identify novel sperm-specific membrane proteins, we produced antiserum to guinea pig sperm membranes in female guinea pigs based on the hypothesis that only male-specific proteins would stimulate an immune response. The antiserum was then used to identify cDNA clones from an enriched spermatogenic cell cDNA expression library.

Using this approach, we isolated a cDNA clone that predicted a novel glycoprotein related to a family of cell adhesion molecules originally found in the liver (9). The protein (named sperad), first expressed by the haploid spermatid, was localized to the periacrosomal plasma membrane of mature spermatozoa, suggesting a role in cell-cell interaction. Although many members of this large family display homotypic adhesion properties, the expression of sperad in Sf9 cells did not result in cell aggregation. Therefore, sperad is likely to function in heterotypic cell interactions. Since the glycoprotein also contains a proline-rich intracellular domain, a feature different from other members of this family, unique intracellular signaling may also represent a function of this plasma membrane protein.

MATERIALS AND METHODS

Guinea pig caudal epididymal spermatozoa were washed in a solution containing 4 mM HEPES, 140 mM NaCl, 4 mM KCl, 10 mM glucose, 2 mM MgCl₂, 100 µM EGTA, pH 7.4, as described by Hardy et al. (10). Spermatozoa were allowed to acrosome react in 4 mM HEPES, 140 mM NaCl, 4 mM KCl, 10 mM glucose, 10 mM CaCl₂, pH 7.4, containing 20 µg/ml A23187 for 10 min at 37 °C; a protease inhibitor mixture (1 mM diisopropyl fluorophosphate, 10 mM EDTA, 0.1 mM leupeptin, and 10 µM E-64) was then added. The cells were cooled on ice (5 min) and centrifuged at 400 × g, 5 min, 4 °C. The supernatant solution was recovered, and the sperm pellet was washed in 10 volumes of the buffer containing protease inhibitors, centrifuged at 400 × g, 5 min, and the solution pooled with the first supernatant solution. The combined solution was centrifuged at 12,000 × g, 15 min, 4 °C to remove cell debris, and the resulting supernatant fraction further centrifuged at 100,000 × g, 1.5 h, 4 °C to recover membrane vesicles. This membrane pellet was washed twice with 10 mM HEPES, pH 7.4, 500 mM NaCl, 10 mM KCl, 10 mM EDTA, and stored in 20 mM HEPES, 150 mM NaCl, pH 7.4, at 20 °C.

A second membrane fraction was prepared by resuspending the 400 × g pellet of acrosome reacted spermatozoa in 5 volumes of buffer containing protease inhibitors, followed by cavitation of the suspension at 700 p.s.i., 20 min, 2-4 °C. The cavitate was centrifuged at 30,000 × g, 30 min, 4 °C to remove cell debris. This pellet was washed, centrifuged again, and the two supernatant fractions combined. The resultant solution was centrifuged at 260,000 × g, 45 min, 4 °C to recover the membrane material. The pellet was resuspended in 10 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM EDTA, 10 mM benzamidine, 10% sucrose, overlaid on a 30/40% sucrose step gradient in the same buffer, and centrifuged at 200,000 × g, 30 min, 4 °C. The membranes at the 10/30% interface were collected, washed with the high salt buffer, and stored as described above.

Antisera were produced by intradermal immunization of female guinea pigs with 700 µg of sperm membrane protein in Freund's complete adjuvant. The animals were boosted once (intramuscular) with 550 µg of sperm membrane protein in Freund's incomplete adjuvant. In each case, 90% of the immunogen was derived from membranes isolated from the acrosome reaction supernatant solution.

Antisera specific for sperad were produced against protein expressed in Escherichia coli BL-21(DE3) using the pRSET vector (InVitrogen). Two constructs were expressed, representing the mature protein (Ala³⁷-Val³³⁰) and the immunoglobulin domains (Ala³⁷-Asn²⁵⁵). The expressed proteins were isolated from bacterial lysates by Ni²⁺- iminodiacetic acid affinity chromatography and preparative electrophoresis. Rabbits were immunized and boosted once with 100 µg of protein/animal.

Total RNA was prepared using the guanidinium thiocyanate method (11). Poly(A)⁺ RNA was isolated with oligo(dT)-cellulose (12).

An enriched spermatogenic cell population was prepared according to Arboleda and Gerton (13). Poly(A)⁺ RNA from this material was used to synthesize a cDNA library in ZAPII according to the manufacturer's directions (Stratagene). Fusion protein expression was induced with 10 mM isopropyl--D-thiogalactopyranoside saturated nitrocellulose filters. The filters were rinsed with TBST¹ (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 0.05% NaN₃ twice, and nonspecific protein binding sites blocked by overnight incubation in TBST containing 0.05% NaN₃ + 5% nonfat milk. The filters were rinsed with TBST (two 1-liter washes), and probed with 1:5000 sperm membrane antiserum diluted in TBST (2 h). Following washing with TBST (four 1-liter washes), the filters were incubated with 1:5000 horseradish peroxidase conjugated rabbit anti-guinea pig IgG (Zymed) in TBST (1 h). The filters were then washed with TBST (two 1-liter washes) and peroxidase activity detected with enhanced chemiluminescence (Amersham). Phage clones were rescued as pBluescript plasmids and sequenced. Identified partial length clone inserts were random-primed labeled with [-³²P]dCTP and used to reprobe the library to identify complete clones. Plaque hybridization was at 42 °C overnight, followed by washing in 0.1 × SSC, 0.2% SDS, 0.1% Na₄P₂O₇ (1 liter at 23 °C, three 1-liter washes at 65 °C). (1 × SSC is 150 mM NaCl, 15 mM sodium citrate.) Full-length clones were sequenced on both strands, and sequences were analyzed using DNASTAR software.

Total RNA (25 µg) from various guinea pig tissues and an enriched spermatogenic cell fraction was electrophoresed through 1% formaldehyde agarose gels and blotted overnight to nylon membranes (14). Blots were hybridized with an [-³²P]dCTP-labeled polymerase chain reaction product (950 base pairs corresponding to nucleotides C²⁵⁹-C¹²¹⁰) as described above for library screening. Blots were washed in 2 × SSC, 0.1% SDS (10 min), and 0.5 × SSC, 0.1% SDS (two 20-min washes) at 65 °C.

Spermatozoa, washed as described above, were further washed with 0.1 M NaP_i, pH 7.2. The sperm pellet was fixed in 10 volumes of 0.1 M NaP_i, pH 7.2 containing 4% paraformaldehyde, 1% glutaraldehyde (30 min, 23 °C). The fixation was quenched with 0.05 volumes of 1.0 M Tris-HCl, pH 6.8 (1 h). The fixed spermatozoa were washed twice with Dulbecco's PBS, spotted onto slides, air dried, and rinsed briefly in water. Spermatozoa were permeabilized in Dulbecco's PBS, 10% normal goat serum, 1 mM EDTA, 0.2% Triton X-100 (10 min, 23 °C). Slides were then washed in 500 ml of Dulbecco's PBS (10 min). Following permeabilization, nonspecific protein binding sites were blocked with Dulbecco's PBS, 10% normal goat serum, 1 mM EDTA (20 min). The slides were then incubated with anti-sperad (Ala³⁷-Val³³⁰) or preimmune serum, 1:500 in blocking solution (2 h). After washing as above, the slides were incubated in the dark with Texas Red-conjugated goat anti-rabbit IgG (Vector) diluted 1:1000 in blocking solution (1 h). Following washing, the slides were mounted in Fluoromount G (Fisher).

Spermatozoa were fixed as for indirect immunofluorescence. The fixed sperm pellet was processed for frozen sectioning as described by Tokuyasu (15). Sections were treated as for indirect immunofluorescence with the following changes. The antibody incubation times were reduced to 30 min using a 1:200 dilution of primary antibody, and a 1:100 dilution of 10 nm gold conjugated goat anti-rabbit IgG was used for detection.

Paraffin-embedded sections of guinea pig testis (Novagen) were dewaxed, rehydrated, and post-fixed in 4% paraformaldehyde/Dulbecco's PBS (30 min). After rinsing with PBS, endogenous peroxidase activity was inactivated with 0.3% H₂O₂ in methanol (30 min). The sections were washed with PBS, then permeabilized, and blocked as described for indirect immunofluorescence. The primary antibody incubation was as above using a 1:4000 dilution. Bound antibody was detected with the Vectastain Elite ABC kit and diaminobenzidine substrate (Vector). Sections were counterstained with hematoxylin.

Protein concentration was measured with bicinchoninic acid (Pierce) using bovine serum albumin as a standard (16). Denatured samples (1% SDS, 1% -mercaptoethanol, 100 °C for 5 min) were diluted 10-fold into 50 mM NaP_i, 0.2 mM EDTA, 1% Triton X-100, pH 8.0, and deglycosylated using N-glycosidase F (Boehringer Mannheim) at 20 units/mg protein. SDS-polyacrylamide gel electrophoresis was done according to the method of Laemmli (17). Electrophoretically separated samples were transferred to nitrocellulose membranes by Western blotting (50 V, 1 h) (18). Blots were probed with primary antibody in TBST (1 h). After washing with TBST (four changes over 30 min), the blots were probed with horseradish peroxidase-conjugated secondary antibody, washed with TBST (two changes over 30 min), and bound secondary antibody detected with enhanced chemiluminescence.

RESULTS AND DISCUSSION

Several proteins of isolated guinea pig sperm membranes were detected with the antisera produced in female guinea pigs (Fig. 1). The membrane proteins ranged in size from M_r 28,000 to M_r 120,000. No guinea pig sperm membrane proteins were detected with the preimmune serum. That the identified proteins were components of the membrane fraction was also indicated by the absence of detectable immunoreactive components in the soluble fraction released during the acrosome reaction. Some of these proteins were glycoproteins as determined by enzymatic deglycosylation with N-glycosidase F. Treatment with N-glycosidase F did not change the number of immunoreactive proteins, indicating that N-linked oligosaccharides were not solely responsible for the antigenicity.

An enriched spermatogenic cell cDNA expression library prepared from four animals was screened with the antiserum to identify clones encoding the sperm membrane proteins. Five clones were detected, four of which represented the same novel transcript. Subsequent screening with this cDNA insert identified two closely related full-length cDNAs of 1.51 and 1.56 kb (Fig. 2). The clones were full-length as suggested by three observations. First, the predicted initiating methionine represented a strong Kozak consensus sequence (19). Second, an apparent signal peptide followed the predicted start methionine (20). Finally, all three reading frames 5 to the predicted initiating methionine contained a stop codon, demonstrating that no longer open reading frame was present. Each clone encoded a protein consisting of a putative signal peptide, two immunoglobulin-like domains (one variable and one constant based on conserved sequence characteristics), a transmembrane segment, and a repetitive, proline-rich domain (Fig. 4B) (21). The predicted mature proteins had a calculated molecular mass of 32.2 or 33.3 kDa in the absence of glycosylation. Both clones contained three conserved potential N-linked glycosylation sites in the immunoglobulin domains with an additional site in the 1.51-kb clone. It is likely, therefore, that the repetitive proline-rich domain is within the cytoplasmic compartment of the cell.

Several nucleotide differences were spread throughout the sequences. Thus, the two sequences may represent either alleles of the same gene or two closely related genes with the same expression pattern. It is unlikely that the two sequences are splice variants of one gene based on the conserved gene structure of homologous proteins (see below).

That the cDNAs were expressed only in the testis was determined by Northern blot analysis of various guinea pig tissues and an enriched spermatogenic cell sample (Fig. 3). The samples were probed with a polymerase chain reaction product (950 base pairs) corresponding to the coding sequence of the mature protein. A signal of approximately 1.8 kb was detected in the testis and spermatogenic cell RNA samples. No signal was detected in brain, heart, kidney, liver, ovary, or spleen. The broad hybridizing band suggested the presence of each of the two similar mRNA species within a single testis. Using oligonucleotide primers, which flanked the proline-rich repeat region, each cDNA was detected in a single testis by reverse transcriptase-polymerase chain reaction (data not shown).

Comparison of the clones to the GenBank^TM data base indicated that they were novel transcripts related to the biliary glycoprotein family of cell adhesion molecules. The sequence similarity to the biliary glycoproteins was highest in the signal peptide, and gradually decreased through the immunoglobulin domains until the sequences completely diverged carboxyl-terminal to the transmembrane segment (Fig. 4A). This site of sequence divergence corresponded to the end of an exon in the conserved biliary glycoprotein gene structure of rat, mouse, and human (22, 23, 24). At the nucleotide level, additional sequence similarity between the 3-untranslated region of the guinea pig clones (nucleotides 1182-1407 of the 1.56-kb clone) and the biliary glycoprotein intracellular domain encoding exons 7-9 was detected. This sequence was not translated in the encoded guinea pig proteins, since the sperm membrane antiserum recognized the proline-rich region expressed as a glutathione S-transferase fusion protein in bacteria (data not shown). In addition, it is unlikely that a protein containing these biliary glycoprotein intracellular domain exon sequences is produced in guinea pig based on two observations. First, the guinea pig sequence contained a single nucleotide insertion (nucleotide 1228 of the 1.56-kb clone), which would produce a frameshift in the potential biliary glycoprotein reading frame. Second, a nucleotide substitution (nucleotide 1280 of the 1.56-kb clone) produces an in-frame stop codon in the potential biliary glycoprotein reading frame. These changes are probably the result of the elimination of this nucleotide sequence from the protein coding sequence in the guinea pig.

The biliary glycoproteins are members of the carcinoembryonic antigen family, a subdivision of the immunoglobulin superfamily (9). These glycoproteins are widely distributed in epithelial, endothelial, and myeloid lineage cells with high levels of expression found in the liver, intestine, and granulocytes (25). The predicted domain organization of the biliary glycoproteins has been determined in mouse, rat, and human from cloning of the corresponding mRNAs (26, 27, 28). In each case, these glycoproteins possess a signal peptide, an immunoglobulin V-like N-domain, a variable number of immunoglobulin C2-like domains (0-3), a transmembrane segment, and an intracellular domain. Each of the immunoglobulin type domains corresponds to a single exon while the intracellular domain of the biliary glycoproteins is encoded by three exons in the conserved gene structure. Several forms of biliary glycoproteins are produced in all three species. This variation arises as the result of alternative splicing of the exons encoding the C2-like immunoglobulin domains and the intracellular domain producing a variable number of C2-like domains, and either a short (approximately 10 amino acids) or long (approximately 73 amino acids) intracellular domain.

The function of the biliary glycoproteins has been addressed in expression studies, where it has been concluded that they act as cell adhesion proteins, at least in vitro (26, 29). Although these glycoproteins have been suggested to principally mediate homotypic adhesion, heterotypic adhesion to other members of the carcinoembryonic antigen family has been observed (30, 31). Characterization of the adhesive activity of biliary glycoproteins has shown dependence on both calcium and physiological temperatures; the immunoglobulin variable type domain at the N terminus appears to be the functional binding domain (29, 32). Due to the homology of the guinea pig protein to these cell adhesion molecules and its specific cellular location (see below), we have designated it sperad.

The intracellular domain of sperad is proline-rich containing an extended region where every third amino acid is a proline residue. This proline-rich motif is predicted to form a polyproline II helix consisting of three residues per turn (33). The majority of this region comprises the short repetitive sequence PPQPEQ. This would result in one side of the three-sided polyproline II helix possessing a negative charge approximately every 19 Å along the helix axis at physiological pH. Polyproline II helices are known to mediate low affinity (micromolar) binding interactions between proteins (33). In many cases, the binding of polyproline II helices involves the SH3 or WWP domains that are often found in signal-transducing proteins (34, 35). In the case of sperad, its facile extraction with detergent suggests that it does not interact with cytoskeletal components, and no binding protein has yet been identified.

To identify which sperm membrane protein was represented by sperad, we generated a specific polyclonal antibody to a hexahistidine-tagged protein representing the mature sperad protein expressed in bacteria (anti-sperad: Ala³⁷-Val³⁷⁰). Immunoblots of guinea pig sperm membranes using this antiserum identified three proteins of M_r 55,000, 36,000, and 28,000 (Fig. 5). This electrophoretic profile was constant throughout the epididymis as well as through in vitro capacitation and the acrosome reaction. All three of these proteins corresponded to proteins detected with the sperm membrane antiserum as determined by immunoprecipitation with anti-sperad, and subsequent detection of these proteins on immunoblots with the sperm membrane antiserum. We also generated a separate polyclonal antibody to a protein representing the two immunoglobulin domains of sperad expressed in bacteria (anti-sperad ECD: Ala³⁷-Asn²⁵⁵). This antiserum also recognized the same set of proteins. In addition, antibody microaffinity purified from the guinea pig sperm membrane antiserum with the M_r 55,000 sperm membrane protein also recognized all three proteins. The sperm membrane antiserum recognized only the full-length protein and not the truncated immunoglobulin domain protein expressed by bacteria. Thus, based on these observations, the M_r 36,000 and 28,000 proteins probably represent degradation products of the M_r 55,000 protein.

Lane

Deglycosylation of the M_r 55,000 protein with N-glycosidase F resulted in a shift to M_r 44,000 (Fig. 5). The mobilities of the M_r 36,000 and 28,000 components were not affected by N-glycosidase F, suggesting that they do not contain N-linked oligosaccharides. When the full-length clone (Ala³⁷-Val³³⁰) was expressed in bacteria, the isolated fusion protein had a M_r = 44,000 rather than the calculated 32,000. The agreement between the size of the protein expressed in bacteria and the size of the deglycosylated M_r 55,000 membrane protein confirmed that the clones were complete.

Indirect immunofluorescence of fixed, permeabilized guinea pig spermatozoa with anti-sperad localized the protein specifically over the acrosome (Fig. 6). No signal was obtained with acrosome intact spermatozoa using preimmune serum, or using the specific antiserum without a permeabilization step. The predominant epitope recognized by the anti-sperad antibody is the proline-rich repeat as determined by immunoblotting of sperad protein fragments expressed in bacteria (data not shown). Therefore, the inability to detect sperad without permeabilization was further evidence that the proline-rich region was intracellular. Following the ionophore induced acrosome reaction, little or no immunofluorescence remained associated with the sperm cells.

The acrosomal location of sperad suggested that it was associated either with the plasma membrane or the outer acrosomal membrane. Immunoelectron microscopy of caudal epididymal spermatozoa with the anti-sperad antibody demonstrated labeling of the periacrosomal plasma membrane (Fig. 7). There was no difference in labeling pattern between the regions of the membrane involved or not involved in sperm-sperm association. No specific labeling of any other subcellular structure was observed. The preimmune serum did not detectably bind to any structure. The localization together with the electrophoretic mobility of sperad suggest that it may represent the previously described sperm antigens AH-30 and/or PM 52/35 (36, 37).

The finding that sperad was related to cell adhesion molecules, which are primarily homotypic, and its localization to the anterior head plasma membrane of spermatozoa led to the hypothesis that sperad had a role in sperm-sperm association. Sperad was expressed in Sf9 cells to assay for homotypic cell adhesion. Approximately 70-80% of the insect cells expressed sperad as determined by immunofluorescence. The expressed protein was glycosylated, M_r 52,000/50,000 doublet, and immunoreactivity was associated with the plasma membrane suggesting sperad was exposed on the cell surface. No aggregation of these cells was seen in either the absence or presence of calcium. The overexpressing Sf9 cells also did not aggregate with guinea pig spermatozoa. These observations suggest that a role for sperad in sperm-sperm association is unlikely.

To further investigate potential sites of sperad adhesion, we examined the developmental stage of sperad expression during spermatogenesis. With testicular cross-sections, anti-sperad immunoreactivity was detected only in developing sperm cells near or in the seminiferous tubule lumen (Fig. 8). Some seminiferous tubules were not stained at all, probably because of asynchronous development. This pattern of immunoreactivity suggested that sperad expression was limited to late spermatid and mature sperm cells. Therefore, it is unlikely that sperad is important in adhesion to the seminiferous epithelium.

What is the function of sperad in spermatozoa? A role for sperad in mature guinea pig spermatozoa is inferred, since it is specifically expressed only late in spermatogenesis and on mature sperm cells. This post-meiotic expression pattern is shared by many other sperm proteins involved in interaction of the mature spermatozoon with the reproductive system (38, 39, 40). In addition, the similarity to other cell adhesion proteins strongly suggests that sperad is involved in the binding of spermatozoa to another cell. Since sperad is lost during the acrosome reaction, a role in mediating adhesion at the egg plasma membrane is excluded. In addition to this interaction, spermatozoa interact with other reproductive tract components such as epithelial cells of the epididymis, uterus, and oviduct, as well as the cumulus oophorus and zona pellucida associated with the egg.