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Biology of Reproduction 62, 1201-1208 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

A Monoclonal Antibody to Human SP-10 Inhibits In Vitro the Binding of Human Sperm to Hamster Oolemma but Not to Human Zona Pellucida

Toshio Hamatania, Kiyoo Tanabe1,a, Kiyoshi Kameib, Nozomi Sakaia, Yurie Yamamotoa, and Yasunori Yoshimuraa

a Department of Obstetrics and Gynecology, School of Medicine, Keio University, Tokyo 160-8582, Japan b Department of Obstetrics and Gynecology, The Saiseikai Central Hospital, Tokyo 108-0073, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SP-10 is a sperm intra-acrosomal protein, specific to the testis, that is believed to play an important role in egg-sperm binding. While the molecular characterization of the SP-10 protein has been clarified, little is yet known of its functional role in fertilization. We therefore established a monoclonal antibody (mAb pep-SP10) against a peptide (pep-SP10) that included the most hydrophilic portion of human SP-10 between the 135th and 149th amino acids. Human SP-10 was found to be localized in the equatorial region of acrosome-reacted sperm by immunofluorescent staining using our mAb pep-SP10. Monoclonal Ab pep-SP10 inhibited sperm-oolemma binding in the zona-free hamster egg penetration test, but it did not inhibit sperm-zona binding in the hemizona assay. Furthermore, we demonstrated that the oolemmal ligands of human SP-10 did not include ß1 integrins, the most promising candidates for oocyte ligands involved in sperm-oolemma binding, based on the findings of a human sperm-cultured cell binding assay using F9 mouse embryonal carcinoma cells and F9-transformed cells lacking ß1 integrins. In conclusion, our present data suggest that human SP-10, expressed on the equatorial region of acrosome-reacted sperm, indeed mediates sperm-oolemma binding in a ß1 integrin-independent manner, but not sperm-zona binding.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SP-10, a sperm intra-acrosomal protein [1], is believed to play a role in egg-sperm binding [2]. The protein is conserved among different mammalian species including humans, baboons, macaques, pigs, foxes [3–8], and mice [9]. In humans, SP-10 is first detected in the developing acrosome of early round spermatids and persists within the acrosome of ejaculated mature sperm [10]. Furthermore, human SP-10 has been demonstrated to be initially exposed on the sperm surface after acrosome reaction by immunofluorescent staining and also by an immunohistochemical assay on the electron microscopic level [1, 10]. SP-10 has been shown to be testis specific in baboons by Northern blot and polymerase chain reaction analyses [11]. SP-10 is probably a substrate for classical trypsin-like endoprotease, most likely acrosin [6, 12]. Proacrosin/acrosin binding to zona pellucida plays a role in maintaining the attachment of acrosome-reacted spermatozoa to the zona pellucida surface [13].

The mammalian fertilization process includes sperm-zona pellucida and sperm-oolemma interactions. SP-10 is one of the molecules implicated in sperm-zona binding, since monoclonal antibodies to SP-10 inhibited secondary tight binding to the zona pellucida in cattle [14]. On the other hand, SP-10 can also reasonably be assumed to play a role in sperm-oolemma binding. Several candidate molecules expressed on the surfaces of both gametes have been proposed to mediate sperm-oolemma interactions during fertilization. Candidate molecules on the sperm surface include galactosyltransferase [15], complement component C3b [16–19], membrane cofactor protein CD46 [16–20], complement component C1q [21, 22], extracellular matrix proteins [23, 24], ß1 integrins [23, 24], and fertilin (formerly known as PH-30) [2, 25, 26]. Candidate molecules on the oolemma surface include IgG Fc{gamma} receptors [15, 27, 28] and integrins such as the C3b receptor integrin {alpha}Mß2 [19], the fibronectin/vitronectin receptor integrin {alpha}vß3 [29], and the laminin receptor integrin {alpha}6ß1 [30, 31]. ß1 integrins, in particular, are presently considered to be the most promising candidate oolemmal ligands [30, 31].

In the present study, we established a new monoclonal antibody against the most hydrophilic portion of SP-10 (mAb pep-SP10) and determined the localization of SP-10 in human acrosome-reacted sperm. In order to examine the involvement of human SP-10 in sperm-zona binding and sperm-oolemma binding, a human hemizona assay (HZA) and the zona-free hamster egg penetration test of human sperm (HEPT) were studied using mAb pep-SP10, respectively. In addition, human sperm-cultured cell binding assays were also performed using F9 mouse embryonal carcinoma cell lines expressing ß1 integrins and F9-transformed cells lacking ß1 integrins, in order to determine whether or not ß1 integrins interact with SP-10.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Establishment of an Original Monoclonal Antibody to Human SP-10

A monoclonal antibody (mAb pep-SP10) was established against a peptide (pep-SP10) spanning amino acids 135–149, the most hydrophilic portion of SP-10 (amino acids: Y+ SGEQP SDEQP SGEHG). BALB/c female mice were immunized with pep-SP10 conjugated with BSA with Freund's complete adjuvant 3 times (once every 2 wk). An ELISA was used to screen the culture supernatants for antibody reactivity to pep-SP10. Then pep-SP10-coated 96-well plates (Iwaki Glasses, Chiba, Japan) were blocked with 0.01 M PBS, containing 0.01% polyoxyethylene-sorbitant monolaurate (Tween 20) solution to decrease the nonspecific binding. Peroxidase-labeled goat anti-mouse IgG (Zymed Laboratories, South San Francisco, CA) was used as a second antibody at a dilution of 1:1000 in PBS containing 0.1% polysorbate and 1% BSA. The substrate 2,2'-azino-bis-3-ethylbenzthiazoline sulfonic acid was used to develop a color reaction. Optical density (A415) was read on a microplate reader (Corona Electric, Ibaragi, Japan). When an elevation of the antibody titer was detectable by ELISA, the spleens of the immunized mice were removed and homogenized. The splenocytes were separated, purified, and fused with a myeloma cell line at the ratio of 5 to 1 in HAT medium (Cosmobio, Tokyo, Japan). The clones were selected by determining the antibody titer based on the ELISA findings on the 5th and 11th day, followed by repeated ultradilution. The IgG isotype of the antibody in the culture supernatant was examined using ELISA employing class-specific second antibodies to mouse IgG (Amersham Pharmacia Biotech, Buckinghamshire, England) and was determined to belong to the IgG1{kappa} immunoglobulin subclass. The conditioned medium was purified by affinity chromatography (Ampure PA kit; Amersham). The protein concentration of the resulting solution was 150 µg/ml. The efficacy of the resulting antibody was then assessed with ELISA. The extent of reaction with the antigen pep-SP10 was determined with 1:500-diluted purified antibody.

Human Semen and Oocyte Samples

All human samples were obtained from volunteers after their informed written consent was obtained. The investigations were approved by and conducted according to the guidelines of the Ethics Committee of Keio University School of Medicine.

Animals

All experiments using hamster eggs and mouse cultured cells were conducted in accordance with the Laboratory Animal Care and Use Committee of Keio University School of Medicine.

Preparation of Human Semen

Semen samples were obtained from volunteers whose ejaculate was considered to be normal based on the WHO criteria [32]. The semen was filtered using a 50-µm-pore nylon membrane filter (Nippon Rikagaku Kikai, Tokyo, Japan) after liquefaction and was centrifuged by discontinuous Percoll (Pharmacia and Upjohn, Kalamazoo, MI) density gradients (20%, 40%, 60%, and 80%) with human tubal fluid (HTF; Life Technologies, Gaithersburg, MD) at 1000 x g for 25 min [33]. The sperm pellet was resuspended in HTF including 3.5% human serum albumin (HSA) and was gently layered on 200 µl of 80% Percoll, followed by centrifugation at 500 x g for 10 min. The sperm layer on 80% Percoll was separated and gently layered beneath HTF with 3.5% HSA, and motile sperm were then allowed to swim up with incubation at 37°C in 5% CO2 in air for 60 min. Approximately 80% of the upper layer containing motile sperm was withdrawn [34]. This sperm suspension was washed again with HTF containing 3.5% HSA and then centrifuged again as described above. The precipitated sperm were resuspended with HTF including 3.5% HSA. Sperm were incubated with calcium ionophore A23187 (5.0 µM; Wako Chemical, Tokyo, Japan) in HTF at 37°C in 5% CO2 in air for 30 min in order to induce sperm acrosome reaction. At least 90% of sperm were motile before and after the acrosome reaction was induced.

Western Blotting Assay

The efficacy and the specificity of mAb pep-SP10 were also assessed with a Western blotting assay. Human sperm proteins were extracted by placing the sperm pellet, which was obtained using the discontinuous Percoll gradient centrifugation procedure, in PBS with 1% Triton X-100 and 0.1% ß-mercaptoethanol (Sigma Chemical, St. Louis, MO) at 4°C for 10 min. After centrifugation at 12 000 x g with the high-speed micro refrigerated centrifuge MRX-150 (Tomy Seiko, Tokyo, Japan) at 4°C for 30 min, the supernatants were analyzed by a Western blotting assay system (Mini-protean2 1D cell and Mini-transblot cell; Bio-Rad Laboratories Japan, Tokyo, Japan). SDS-PAGE gradient gels (10–20%) were loaded with 20 µg of sperm extract and 1.2 µg of recombinant full-length human SP-10 protein (re-hSP-10; courtesy of Dr. J.C. Herr. Department of Cell Biology, University of Virginia, VA) [35]. These sperm proteins were electrophoresed and electrotransferred on polyvinylidene fluoride microporous membranes (Immobilon-P: Millipore, Bedford, MA). Membrane strips were blocked in PBS with 1% Tween 20 and 8% nonfat milk at 4°C overnight, and then were incubated with mAb pep-SP10 (1:5000, 0.03 µg/ml) or SP-10–3 mAbs (including MHS-10; 1:8000, 0.18 µg/ml; Virginia Biotech., Ivy, VA) at room temperature for 60 min. One control was a group incubated with absorbed mAb pep-SP10, whereas another control was a group incubated with a purified mouse monoclonal IgG1 antibody (0.03 µg/ml; clone DAK-GO1; Dako A/S, Glostrup, Denmark). To absorb mAb pep-SP10 (1:5000, 0.03 µg/ml), 20 µl of re-hSP-10 (1.25 mg/ml) was mixed with 430 µl PBS in each sterile Eppendorf (Hamburg, Germany) tube followed by the addition of 50 µl mAb pep-SP10 (1:500, 0.3 µg/ml). The supernatants were removed after incubation at 4°C for 1 h and used as "absorbed mAb pep-SP10." After 5 washes in PBS with 1% Tween 20, peroxidase-linked anti-mouse IgG antibody (Amersham) was used as a secondary antibody on the blots at room temperature for 60 min. The blots were then washed 5 times in PBS with 1% Tween 20 and developed with the ECL plus Western blotting detection system (Amersham).

Indirect Immunofluorescent Staining Using mAb pep-SP10

The sperm suspension prepared with or without induction of the acrosome reaction was treated with 10% normal goat serum for 15 min to prevent nonspecific binding and then incubated with a 1:100 dilution (1.5 µg/ml) of mAb pep-SP10 at 37°C for 60 min. A purified mouse monoclonal IgG1 antibody (1.5 µg/ml; clone DAK-GO1; Dako A/S) was used as a negative control. The sperm specimens were treated with an anti-mouse IgG1 antibody labeled with Cyedye-3 fluorescence (5.0 µg/ml; Chemicon International, Temecula, CA) at room temperature for 60 min. All specimens were examined using an epifluorescence microscope (IMT2-RFL; Olympus Optical, Tokyo, Japan) by the same researcher. At least 20% of the sperm were motile at time of scoring 1000 motile sperm.

Triple-Stain Technique

The sperm suspension prepared with or without induction of the acrosome reaction was treated with an equal volume of trypan blue (Wako) at room temperature for 15 min and then was treated with 3% glutaraldehyde (Wako) with 0.1 M cacodylic acid (Wako) at room temperature for 60 min. Sperm were smeared on glass slides and air dried. The slides were treated with 0.8% Bismarck brown Y solution (pH 1.8; Sigma) at 40°C for 5 min, followed by treatment with 0.8% Rose Bengal with HCl and PBS (Wako), pH 7.4, at 25°C for 60 min; they were then treated with 95% ethanol. The ratio of acrosome-reacted sperm was assessed using a phase-contrast microscope (BH-2; Olympus).

Human Hemizona Binding Assay (HZA) Using mAb pep-SP10

Human unfertilized oocytes were donated from failed in vitro fertilization (IVF) procedures after written informed consent was obtained. The oocytes were bisected by a micromanipulator (IX70-S8F2; Olympus Optical & Narishige, Tokyo, Japan) in HTF as previously described [36, 37], resulting in two identical matching hemizonae (MHZ). MHZ were stored in medium containing 0.05 M (NH4)2SO4, 0.75 M MgCl2, 0.2 M ZnCl2, and 0.01% polyvinylalcohol (pH 7.4) [38] at 4°C. Immediately prior to assay, MHZ were removed from the medium and rinsed 3 times in HTF. A 1:20 dilution of mAb pep-SP10 (7.5 µg/ml) or a control IgG1 antibody (7.5 µg/ml) was added to preincubated sperm as described above. The sperm suspensions were diluted to 5 x 106 motile sperm/ml and then incubated with the MHZ in HTF at 37°C in 5% CO2 in air for 4 h. A hemizona index was calculated after vigorous pipetting by counting the number of sperm adhering to the outer side of the MHZ or penetrating the MHZ using a phase-contrast microscope by the same researcher; the number of sperm adhering to (penetrating) the MHZ with mAb pep-SP10 was then divided by the number of sperm in the control group.

Zona-Free HEPT Using mAb pep-SP10

The sperm suspension obtained after induction of the acrosome reaction was washed twice with HTF containing 3.5% HSA. The sperm were preincubated with various dilutions (1:500 [0.3 µg/ml], 1:100 [1.5 µg/ml], 1:20 [7.5 µg/ml]) of mAb pep-SP10 or a control IgG1 antibody (7.5 µg/ml) in HTF at 37°C in 5% CO2 in air for 60 min. The preincubated sperm were then further incubated in HTF at 37°C in 5% CO2 in air for 4 h with hamster eggs after removal of their zonae treated with 0.1% trypsin in HTF for 3 min as previously described [39]. In a neutralization test of mAb pep-SP10, one control group contained a control IgG1 monoclonal antibody (0.3 µg/ml). Another control was a group incubated in absorbed mAb pep-SP10. To absorb mAb pep-SP10 (1:500, 0.3 µg/ml), 500 µg of pep-SP10 was mixed with 980 µl PBS in a sterile Eppendorf tube followed by the addition of 20 µl mAb pep-SP10 (1:10, 15 µg/ml). The supernatant was removed after incubation at 4°C for 1 h and then was added to the sperm. The main experimental group contained mAb pep-SP10 (1:500, 0.3 µg/ml). The rates of sperm-penetrated zona-free hamster eggs and the number of sperm adhering to or penetrating the eggs were determined on the glass slides with lacmoid staining using a phase-contrast microscope by the same researcher.

Human Sperm-Mouse Cultured Cell Binding Assay Using mAb pep-SP10

F9 mouse embryonal carcinoma cell lines that express ß1 integrins and F9-transformed cell lines, TKO cells, that lack ß1 integrins were used to assess sperm-cell binding. Mouse F9 and TKO cell monolayers [40] were cultured in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 3.7 g/L sodium bicarbonate, and 60 µM ß-mercaptoethanol. For binding experiments, the cells were treated with trypsin-EDTA for approximately 5 min at 37°C, plated in 12-well culture plates (3.8 cm2/well) at 5 x 104 cells per well, and used for binding assay after culturing for 6 h at 37°C in 5% CO2 in air. The human sperm suspension was prepared after induction of the acrosome reaction, followed by incubation with various dilutions (1:200 [0.75 µg/ml], 1:100 [1.5 µg/ml], 1:50 [3.0 µg/ml]) of mAb pep-SP10 or a control IgG1 monoclonal antibody (3.0 µg/ml) for 1 h at 37°C in 5% CO2 in air. Aliquots of 1.0 x 106 sperm were added to each well of cultured cells. The sperm were allowed to bind for 1 h at 37°C in 5% CO2 in air. Cells were washed three times with HTF. After the addition of trypan blue, the number of sperm bound to each cell was determined within 30 min of the washing with an inverted phase-contrast microscope. The same researcher counted the sperm bound to at least 500 cells under each experimental condition.

Statistical Analysis

Data were expressed as the mean ± SEM. Any significant differences in all data were assessed by one-way ANOVA followed by Fisher's least significant difference test. A P value of less than 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Western Blotting Assay

For Western blotting, re-hSP-10 and Triton X-100 extracts of human sperm were subjected to SDS-PAGE, electroblotted, and probed with mAb pep-SP10 and SP-10-3 mAbs. Monoclonal Ab pep-SP10 reacted strongly with re-hSP-10 (Fig. 1, lane A1) at 55 kDa and with SP-10 proteins at 28 and 32 kDa in the sperm extract (Fig. 1, lane A2). SP-10-3 mAbs reacted strongly with re-hSP-10 (Fig. 1, lane B1) at 55 kDa and with SP-10 proteins at 28 and 32 kDa (Fig. 1, lane B2). In this assay, both absorbed mAb pep-SP10 and a control IgG1 monoclonal antibody were used as negative controls. The absorbed mAb pep-SP10 faintly reacted with re-hSP-10 (Fig. 1, lane C1) but not with any sperm extracted proteins (Fig. 1, lane C2). A control IgG1 monoclonal antibody neither recognized re-hSP-10 (Fig. 1, lane C1) nor reacted strongly with any sperm extracted proteins (Fig. 1, lane C2).



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  		FIG. 1. A Western blot of mAb pep-SP10 reactivity with re-hSP-10 and sperm extracted proteins. Each lane of SDS-PAGE gradient gel (10-20%) was loaded with 1.2 µg of re-hSP-10 (lane 1) or 20 µg of sperm extracted protein (lane 2). Lane MW contained prestained molecular weight markers. Lane A was immunostained with mAb pep-SP10 (1:5000), and lane B was probed with SP-10-3 mAbs (1:8000). Lanes C and D were negative controls. Lane C was immunostained with absorbed mAb pep-SP10. To absorb mAb pep-SP10, mAb pep-SP10 (1:5000) was preincubated with re-hSP-10 (0.5 mg/ml). Lane D was incubated with a control IgG1 monoclonal antibody (0.03 µg/ml)

Localization of SP-10 in Human Sperm

The equatorial segment of the sperm head stained strongly with mAb pep-SP10 (1:100) after induction of the acrosome reaction with calcium ionophore, whereas there was no marked staining when sperm were incubated with a control IgG1 monoclonal antibody under immunofluorescence (Fig. 2). The ratio of sperm whose equatorial regions stained with mAb pep-SP10 increased from 19.9 ± 3.8% to 30.4 ± 6.7% after induction of the acrosome reaction. This was compatible with the results of the triple-stain technique; the ratio of acrosome-reacted sperm likewise increased from 18.6 ± 0.9% to 42.2 ± 12.3% after induction of the acrosome reaction with calcium ionophore.



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  		FIG. 2. Immunofluorescent profiles of acrosome-reacted human sperm using mAb pep-SP10. Micrographs from a phase-contrast microscope (left) a fluorescent microscope (right). A, B) No remarkable staining was seen with a control IgG1 monoclonal antibody (1.5 µg/ml). C, D) Monoclonal Ab pep-SP10 (1:100) reacted strongly around the equator of sperm heads after induction of acrosomal reaction in the fluorescent micrographs (x400). E, F) Higher magnification of C and D (x1000). Published at 75%. Bars = 60 µm in A–D; bars = 10 µm in E and F

Monoclonal Ab pep-SP10 Did Not Inhibit Sperm-Zona Binding in the HZA

Sperm were incubated with mAb pep-SP10 or a control IgG1 monoclonal antibody in HZA, and the number of sperm adhering to or penetrating MHZ was calculated (Fig. 3). The average number of sperm adhering to or penetrating MHZ was 92.1 ± 18.2 in the presence of mAb pep-SP10 (1:20), while it was 88.8 ± 15.0 with a control IgG1 monoclonal antibody (Fig. 3). Consequently no significant difference was observed between the mAb pep-SP10 group and the control group.



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  		FIG. 3. The influence of mAb pep-SP10 in HZA. An oocyte was bisected by a micromanipulator, resulting in two identical MHZ. The number of sperm adhering to the outer side of the MHZ or penetrating the MHZ was counted. The mean values (± SEM) did not significantly differ between the control group and the mAb pep-SP10 group; mAb pep-SP10 (1:20) had no influence on the average number of sperm adhering to or penetrating MHZ in HZA. The hemizona index represented the number of sperm adhering to (penetrating) the MHZ with mAb pep-SP10 divided by the number of sperm in the control group. Hemizona index = 96.4% (n = 23)

Monoclonal Ab pep-SP10 Inhibited Sperm-Oolemma Binding in the Zona-Free HEPT

The sperm were incubated with various dilutions of mAb pep-SP10 or mAb pep-SP10 preincubated with pep-SP10. The rates of sperm-penetrated eggs and the number of sperm adhering to or penetrating zona-free hamster eggs were measured in HEPT (Fig. 4). While the sperm penetration rate in HEPT was 91.8 ± 0.5% with a control IgG1 monoclonal antibody, the rates decreased significantly and dose-dependently after the prior incubation of sperm with mAb pep-SP10 (1:500, 69.2 ± 0.8%; 1:100, 59.6 ± 0.4%; 1:20, 10.9 ± 0.9%). The number of sperm adhering to or penetrating zona-free hamster eggs was 10.80 ± 1.22 with a control IgG1 monoclonal antibody, whereas this number with mAb pep-SP10 also decreased significantly and dose-dependently (1:500, 4.55 ± 0.71; 1:100, 3.55 ± 0.73; 1:20, 3.11 ± 1.00).



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  		FIG. 4. The influence of various dilutions of mAb pep-SP10 in HEPT. The rates of sperm-penetrated zona-free hamster eggs and the number of sperm adhering to or penetrating the eggs were determined. Open bars show the average number of sperm adhering to or penetrating eggs. Solid bars show the ratios of the sperm-penetrated eggs. A significant difference among the mean values (± SEM) that have different superscripts was observed (P < 0.05). Monoclonal Ab pep-SP10 significantly and dose-dependently reduced the sperm penetration rates in HEPT (control, 91.8 ± 0.5% [n = 36]; 1:500, 69.2 ± 0.8% [n = 29]; 1:100, 59.6 ± 0.4% [n = 31]; 1:20, 10.9 ± 0.9% [n = 27]), and also significantly and dose-dependently decreased the number of sperm adhering to or penetrating the zona-free hamster eggs in HEPT (control, 10.80 ± 1.22 [n = 36]; 1:500, 4.55 ± 0.71 [n = 29]; 1:100, 3.55 ± 0.73 [n = 31]; 1:20, 3.11 ± 1.00 [n = 27])

In order to validate that a possible reduction in both the sperm penetration rates and the numbers of sperm adhering to or penetrating the eggs was responsible for mAb pep-SP10, 0.5 mg/ml pep-SP10 was used to absorb mAb pep-SP10 (1:500). This quantity of pep-SP10 was chosen because it demonstrated a maximum absorption of mAb pep-SP10 (1:500) (Fig. 5). When the sperm were incubated with this absorbed mAb pep-SP10 (1:500), the sperm penetration rate in HEPT returned to the control level (a control IgG1 monoclonal antibody, 79.3 ± 0.7%; unabsorbed mAb pep-SP10, 53.6 ± 3.6%; absorbed mAb pep-SP10, 73.6 ± 1.4%) (Fig. 6). Furthermore, the absorbed mAb pep-SP did not decrease the number of sperm adhering to or penetrating the eggs (a control IgG1 monoclonal antibody, 2.79 ± 0.75; unabsorbed mAb pep-SP10, 1.21 ± 0.35; absorbed mAb pep-SP10, 2.17 ± 0.43) (Fig. 6). Both the sperm penetration rates and the number of sperm adhering to or penetrating the eggs did not differ significantly between the absorbed mAb pep-SP10 group and the control IgG1 monoclonal antibody group.



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  		FIG. 5. A titration curve of mAb pep-SP10 (1:500) (solid circles) produced by reactions with various dilutions of pep-SP10. Open circles represent those in the nonimmune mouse sera. The mean values (± SEM) with different superscripts are significantly different (P < 0.05)



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  		FIG. 6. The influence of unabsorbed and absorbed mAb pep-SP10 in HEPT. To absorb mAb pep-SP10, mAb pep-SP10 (1:500) was preincubated with 0.5 mg/ml pep-SP10 at 4°C for 1 h. Open bars show the average number of sperm adhering to or penetrating eggs. Solid bars show the ratios of sperm-penetrated eggs. A significant difference was observed among the mean values (± SEM) that have different superscripts (P < 0.05). Both the sperm penetration rate and the number of sperm adhering to or penetrating the eggs were reduced by unabsorbed mAb pep-SP10 (1:500) (respectively, 53.6 ± 3.6%, 1.21 ± 0.35 [n = 14]), but they did not differ significantly between the control group and the absorbed mAb pep-SP10 (a control IgG1 monoclonal antibody, 79.3 ± 0.7%, 2.79 ± 0.75 [n = 14]; absorbed mAb pep-SP10, 73.6 ± 1.4%, 2.17 ± 0.43 [n = 18])

Monoclonal Ab pep-SP10 Inhibited Sperm-Cell Binding in Human Sperm-Mouse Cultured Cell Binding Assay with Cells Lacking ß1 Integrins

F9 cells, which express ß1 integrins, and TKO cells, which lack ß1 integrins, were used to assess sperm-cell binding. Most of the sperm bound to the cells by the equatorial segment of the sperm head, but few of the sperm bound to the cells by the sperm tail. After vigorous pipetting, the same researcher counted only the motile sperm that tightly bound to the cells by the equatorial segment of the sperm head. The mean number of sperm bound per 100 F9 cells was 10.5 ± 1.1 with a control IgG1 monoclonal antibody, while the numbers decreased significantly and dose-dependently with mAb pep-SP10 (1:200, 6.56 ± 0.83; 1:100, 5.68 ± 1.10; 1:50, 3.88 ± 0.56) (Fig. 7). The number of sperm bound per 100 TKO cells was 5.92 ± 0.91 with a control IgG1 monoclonal antibody, while the number decreased dose-dependently with mAb pep-SP10 (1:200, 3.75 ± 0.66; 1:100, 3.46 ± 0.43; 1:50, 3.12 ± 0.30) (Fig. 7). Furthermore, the number of sperm bound to F9 cells, in comparison with TKO cells, was significantly greater in the control group and in the mAb pep-SP10 1:200 dilution group (Fig. 7).



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  		FIG. 7. The effect of mAb pep-SP10 in the sperm-cultured cell binding assay. The number of sperm bound per 500 cells was determined. Monoclonal Ab pep-SP10 significantly and dose-dependently reduced the number of sperm bound per 100 F9 cells, while it also dose-dependently reduced the number of sperm bound per 100 TKO cells lacking ß1 integrins. Furthermore, the number of sperm bound to TKO cells was significantly less than that bound to F9 cells in each treatment group


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we successfully established a monoclonal antibody against human sperm intra-acrosomal protein SP-10 and thus demonstrated SP-10 to be involved in mediating sperm-oolemma binding but not sperm-zona binding. The monoclonal antibody (mAb pep-SP10) was designed to recognize the most hydrophilic portion of SP-10 (amino acids 135–149). Monoclonal Ab pep-SP10 was found to be an IgG1{kappa} immunoglobulin subclass antibody. Western blotting revealed that mAb pep-SP10 reacted specifically with human SP-10 proteins. It has been shown that re-hSP-10 migrates at 50–55 kDa rather than at the expected size of 29 kDa deduced from the cDNA sequence of human SP-10. This is consistent with previous studies suggesting that slow retardation of re-hSP-10 may be attributable to a high percentage of acidic residues (estimated isoelectric point: 4.75) [1, 35]. SP-10 extracted from ejaculated sperm migrated at 28 and 32 kDa, because SP-10 proteins lose acidic residues and decrease in size during spermatogenesis by endoproteolytic processing of a full-length precursor protein of SP-10 and/or by alternative splicing of the SP-10 gene [3, 12]. SP-10 microheterogeneity is due to the proteolytic activities of several enzymes including a classical trypsin-like endoprotease, most likely acrosin [12].

SP-10 was present at the equatorial region of the sperm head and was shown to be exposed following the acrosome reaction by immunofluorescent staining of human sperm with mAb pep-SP10. In immunofluorescent studies using monoclonal antibodies previously established against SP-10, human sperm permeabilized with Triton X-100 or methanol exhibited not only bar-shaped fluorescence on the equatorial segment of sperm head but also full cap-shaped or faint cap-shaped fluorescence on the sperm head [1, 41]. However, human motile sperm without permeabilization showed hardly any full cap-shaped or faint cap-shaped fluorescence in this study. These findings are compatible with the results from an immunohistochemical study of SP-10 at electron microscopic levels using a previously characterized monoclonal antibody (MHS-10) [1, 10, 42].

Monoclonal Ab pep-SP10 dose-dependently and significantly decreased sperm adherence and penetration to oolemma in the zona-free HEPT of human sperm. The involvement of SP-10 in sperm-oolemma binding is further supported by its localization at the equatorial region. The equatorial region is believed to be the sperm-oolemma fusion site that involves oolemmal microvilli wrapping around the sperm head [43]. However, SP-10 is not involved in sperm-zona binding, because mAb pep-SP10 had no effect on either the adhesion or penetration of sperm to the zona pellucidae in the HZA in the human. As a result, our present data suggest that human SP-10 plays, in part, an important role in adhesion and/or penetration of sperm to oocytes during fertilization, but not in the binding of sperm to zona pellucidae.

The HZA using mAb pep-SP10 is the first human model for study of functional properties of SP-10 in sperm-zona binding. Monoclonal Ab pep-SP10 did not affect sperm-zona binding in humans, but MHS-10 has been shown to inhibit secondary tight sperm-zona binding, thus resulting in sperm detachment from the zona pellucida or incomplete sperm penetration of the zona pellucida in cattle [14, 42]. The antigen of mAb pep-SP10, pep-SP10, contains only 15 amino acid residues; but native SP-10 molecules have multiple MHS-10 epitopes [44]. This may explain why MHS-10 inhibits sperm-zona binding but mAb pep-SP10 does not.

The present study supports the hypothesis that SP-10 plays a role in sperm-oolemma binding; however, the ligands of SP-10 have not yet been clarified. We postulated that integrins might be possible candidates for SP-10 ligands. Integrin subunits {alpha}2, {alpha}4, {alpha}5, {alpha}L, ß1, ß2, and ß7 have been immunolocalized on human oocytes [28, 45], though integrin subunits {alpha}3, {alpha}5, {alpha}6, {alpha}v, ß1, ß3, and ß5 are also expressed on unfertilized mouse oocytes [30, 46]. Integrin {alpha}6ß1, in particular, has been documented to function as a sperm receptor by binding to the mouse sperm inner acrosomal membrane [30, 31]. We thus examined the possibility of binding between pep-SP10 and ß1 integrins, which are expressed abundantly on oolemma. F9 cells express several integrins including {alpha}6ß1 on the surface [40], whereas the ß1 integrin subunit in F9-transformed cells was obliterated by a triple knockout homologous recombination approach (TKO). This generated the TKO cell line, which lacks {alpha}3ß1, {alpha}5ß1, and {alpha}6ß1 on its cell surface [40]; other integrins such as {alpha}vß3, {alpha}vß5, and {alpha}6ß4, as well as other adhesion molecules including laminin and cadherin, remain on the surface of this TKO cell line [40]. The present study demonstrated that the lack of ß1 integrins in TKO cells resulted in a decrease in human sperm binding to cultured cells, similar to results observed with mouse sperm [30]—thus lending further support to the hypothesis that ß1 integrins play a role in sperm-oolemma binding. However, the addition of mAb pep-SP10 resulted in a dose-dependent reduction of human sperm binding to F9 cells and also, unexpectedly, to TKO cells in the sperm-cultured cell binding assay. This implies that SP-10 as well as ß1 integrins mediates sperm-cell binding, and that the oolemmal ligands of SP-10 include unidentified adhesion molecules, other than ß1 integrins, on the surface of TKO cells.

The functional role of SP-10 in sperm-oolemma adhesion is inferred from its molecular sequence characterization. SP-10 amino acid sequences contain a recognizable Ly-6 family motif [47]. The Ly-6 family is serologically and structurally related to cell surface proteins, which are most abundant on peripheral lymphocytes and which are anchored to the plasma membrane through a C-terminal glycosyl-phosphatidylinositol attachment [48]. The Ly-6 family includes complement-binding regulatory protein CD59, urokinase-type plasminogen activator, and snake venom postsynaptic neurotoxin [49–54]. With the exception of SP-10 exon 2, a strong similarity has been noted between the SP-10 and Ly-6 gene structure [7], thus suggesting the involvement of the Ly-6 domain of SP-10 in cell-cell interactions. Some possible mechanisms for this interaction include SP-10 binding to potentially damaging proteins impinging on the sperm head, similar to the protection from complement-mediated damage afforded by CD59 [16, 19, 20, 55, 56], or the localization of enzyme activity necessary for fertilization, similar to the function noted for urokinase-type plasminogen activator receptor [49, 50]. SP-10 exon 2 contains unique repeats of the consensus nucleotide sequence encoding (G/S)E(Q/H)(P/T/A)S [7]; this repetitive motif also exists in pep-SP10. Monoclonal Ab pep-SP10, which recognizes a portion of the domain encoded by SP-10 exon 2, inhibited sperm-egg interactions in the present study. We therefore postulate that the exon 2 region of SP-10 may be involved in sperm-oolemma adhesion.

In conclusion, SP-10 is involved in sperm-oolemma binding but not in sperm-zona binding during fertilization in the human. ß1 integrins on the oolemma surface function as ligands in both human and murine sperm-oolemma binding but do not interact with SP-10 during sperm-oolemma binding. Further studies are called for to determine the oo-lemmal ligands for SP-10 and to precisely elucidate the SP-10 function in fertilization.


   ACKNOWLEDGMENTS
 
The authors wish to thank Dr. S.F. Schick, Dr. L.E. Stephens, Dr. E.A. Almeida, and Dr. C.H. Damsky (Departments of Stomatology and Anatomy, University of California, San Francisco) for the generous gifts of F9 and TKO mouse embryonal carcinoma cells, and to thank Dr. K.L. Klotz and Dr. J.C. Herr (Department of Cell Biology, University of Virginia) for the gift of re-hSP-10 and helpful suggestions. Valuable assistance from Dr. N. Kuji, Dr. A. Suzuki, M. Okazaki, and N. Hida (Department of Obstetrics and Gynecology, School of Medicine, Keio University, Tokyo, Japan) is also appreciated.


   FOOTNOTES
 
First decision: 16 November 1999.

1 Correspondence and current address: Kiyoo Tanabe, Department of Obstetrics and Gynecology, Tokyo Dental College Ichikawa General Hospital, 5-11-13 Sugano, Ichikawa-city, Chiba 272-8513, Japan. FAX: 81 47 325 4456; ktanabe{at}tdc.ac.jp Back

Accepted: December 9, 1999.

Received: October 13, 1999.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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