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a Department of Obstetrics and Gynecology,
b Laboratory of Developmental Biology and Reproduction, Institute of Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, 663-8501, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After acrosomal exocytosis, spermatozoa penetrate through the ZP to fuse with the egg plasma membrane. During this event, the spermatozoa remain bound to the ZP despite the loss of the plasma membrane from the anterior region of the head, exposing the inner acrosomal membrane. Previous studies have indicated that ZP2 functions as a secondary receptor in acrosome-reacted spermatozoa [8–10]. Evidence from one of our prior studies supports this idea [11]. We produced a recombinant protein in Escherichia coli corresponding to amino acid sequence 1 to 198 of a porcine homologue of human ZP2. The recombinant protein was observed to bind to acrosome-reacted spermatozoa from several species in vitro but not to acrosome-intact spermatozoa, suggesting that the N-terminal polypeptide domain of ZP2 functions as a secondary receptor in an interspecies manner.
In this study, we report 1) production of high levels of recombinant human ZP2 protein (rec-hZP2) in E. coli corresponding to amino acid sequence 1–206 of the mature protein; 2) the interaction of rec-hZP2 and human spermatozoa that were induced to acrosome-react with calcium ionophore A23187 in vitro; and 3) the blocking effect of mouse and rabbit antisera, raised against rec-hZP2, on human in vitro fertilization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cDNA encoding the full-length human ZP2 [12] was a gift from Professor Jurrien Dean (NIDDK, Rockville, MD). Polymerase chain reaction (PCR) was employed to construct cDNA corresponding to amino acid residues 1–206 of human ZP2 mature protein. Restriction sites at the 5' and 3' ends were added to facilitate insertion of the PCR product into the expression vector pET21b (Novagen, Madison, WI). Primer sequences were as follows: HZP2Eco:5 ' - G T G A C T T C A G G G A A T T C C A T A G A T - 3' ; H Z P 2HindRC: 5'-ATGTAAGCTTCAGAGACACCATG-3'.
The PCR products were digested with EcoRI and HindIII. After digestion, these products were purified by agarose gel and ligated into pET21b cut with the same enzyme. The resultant expression plasmids, pEThZP2(206), were sequenced using the Sequenase sequence kit (Amersham; now Amersham Pharmacia Biotech, Piscataway, NJ) to verify the insertion of DNA.
Expression of rec-hZP2 in E. coli
E. coli BL21(DE3) was transformed with pEThZP2(206) according to the manufacturer's instructions (Novagen). Single colonies of E. coli harboring pEThZP2(206) were inoculated into 10 ml Luria Broth (LB) medium containing ampicillin at a final concentration of 50 µg/ml, which was then incubated overnight at 37°C. After overnight incubation, the culture was inoculated into 100 ml of LB medium containing ampicillin at the same concentration; incubation was then continued at 37°C for 2 h to reach an optical density (OD)600 of 0.35. Subsequently, isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to the culture medium at a final concentration of 0.4 mM. Cells were further incubated for 2 h and harvested by centrifugation at 5000 x g for 30 min.
Extraction of rec-hZP2
The collected transformants were resuspended in 20 ml of STE buffer (10 mM Tris-HCl, pH 7.9, 100 mM NaCl, 1 mM EDTA) and centrifuged at 3000 x g for 5 min at 4°C. The cell pellet was lysed for 15 min with STE buffer containing lysozyme (200 µg/ml) and 1% (v:v) Triton X-100 (Pierce Chemical Co., Rockford, IL) at 30°C. The mixture was sonicated (six times for 60 sec each with 5 pauses) by a cell disrupter (Misonix, Farmingdale, NY) on ice until the sample was no longer viscous; centrifugation was then performed at 10 000 x g for 20 min at 4°C. The supernatant was discarded; the pellet was washed in binding buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 5 mM imidazole) and then lysed with binding buffer containing 6 M urea for 10 min at 37°C. The soluble fraction was separated by centrifugation at 10 000 x g for 20 min at 4°C, dialyzed against 5 mM ammonium bicarbonate, and then dialyzed against Hepes buffer (20 mM, pH 7.6) for use as rec-hZP2 in this study.
To measure the yield, samples were dialyzed against 5 mM ammonium bicarbonate, lyophilized, and directly weighed.
Western Blot Analysis of Recombinant Proteins
Whole proteins of E. coli (BL21DE3) transformants with pET21b and pEThZP2 after incubation with IPTG were solubilized in an SDS sample buffer (Tefco, Tokyo, Japan). Purified rec-hZP2 was also solubilized in the buffer. The samples were boiled for 10 min, after which 10-µl aliquots per well were run on SDS-PAGE (4–20% gradient gel; Tefco) under reducing conditions for 120 min at 18 mA (constant current) [13]. Proteins were then transferred to polyvinylidene disulfide membranes (Immobilon; Millipore, Bedford, MA) for 90 min at 125 mA (constant current). The membrane was washed (all subsequent washes and incubations were carried out at room temperature [RT]) for 15 min in Tris-buffered saline (TBS; 50 mM trisaminomethane, 200 mM NaCl, pH 7.4) and then cut in half. One strip was stained with Coomassie brilliant blue (CBB). The other strip was blocked in buffer containing 3% (w:v) BSA in TBS for 30 min. To detect the presence of rec-hZP2, the blocked strip was incubated for 30 min with monoclonal antibody Mab5H4, which binds to amino acid residues 50–67 of the porcine homologue of human ZP2 [14, 15]. The strip was then incubated with peroxidase-conjugated goat anti-mouse IgG (1:1000 dilution in TBS; Cappel, West Chester, PA) for 30 min. Color development was carried out using 0.5 mg/ml 4-chloro-1-naphthol and 0.01% (v:v) H2O2 in TBS buffer. Marker-12 (Tefco) protein standards were used.
Sperm Samples and Preparation
Semen was obtained from three fertile donors by masturbation. After at least 30 min was allowed for liquefaction to occur, the spermatozoa were suspended in 10 ml of Biggers, Whitten, and Whittingham (BWW) medium [16] and centrifuged at 800 x g for 3 min. The supernatant was discarded, and the spermatozoa were resuspended with BWW medium containing 0.3% (w:v) BSA. After this washing was repeated twice, motile spermatozoa were isolated by the direct swim-up method and then preincubated in vitro for 3 h (for in vitro fertilization test) and 18 h (for in vitro rec-hZP2-binding test) at 37°C in a humidified atmosphere of 5% CO2 in air.
Assessment of rec-hZP2 Binding in Indirect Immunofluorescent Study
One hundred percent motile spermatozoa were collected from capacitated human spermatozoa by the direct swim-up method. To prepare acrosome-reacted spermatozoa, the spermatozoa were incubated with BWW containing ionophore A23187 (10 µM) for 10 min, centrifuged at 800 x g for 3 min, and resuspended with BWW containing 0.3% (w:v) BSA.
Spermatozoa in which the acrosome reaction had been induced and those in which it had not (1 x 105/200 µl BWW) were incubated with rec-hZP2 (20 µg/ml) in BWW containing 0.3% (w:v) BSA for 2 h (5% CO2 in air at 37°C). Sperm aliquots were centrifuged at 800 x g for 3 min; the pellets were spotted onto 12-well multitest slides (ICN Biomedicals, Costa Mesa, CA), air dried, and fixed with methanol for 10 min for subsequent staining.
The specimen was blocked with PBS containing 3% (w:v) BSA in a humidified box for 1 h at RT (all subsequent incubations were carried out at RT for 1 h). The specimen was then incubated with Mab5H4, followed by a final incubation with a combination of tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG (1:200 dilution in PBS; Cappel) and fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA; 1:200 dilution in PBS; EY Laboratories, San Mateo, CA). After washing, the specimen was examined using a fluorescence microscope (Nikon, Garden City, NY) at x400 and x1000 magnification.
Immunization
Three Japanese White rabbits were injected i.d. with 500 µg each of rec-hZP2, in emulsion with complete Freund's adjuvant, at multiple sites on the back and footpads. After 2 wk, the rabbits were injected with the same antigen in incomplete Freund's adjuvant followed by three additional booster injections i.p. without adjuvant at intervals of 2 days beginning at 2 wk after the second injection. ICR mice were subjected to the same immunization protocol as above; however, the dosage was decreased to 100 µg as opposed to 500 µg of rec-hZP2. Both animals were bled 1 wk after the last booster injection.
ELISA
Antibody production to rec-hZP2 was assessed by ELISA with the cognate peptide. A microtiter plate (Falcon; Becton Dickinson-Vacutainer Systems, Franklin Lakes, NJ) was coated with the peptide in carbonate buffer, pH 9.6 (1 µg peptide per well), and stored at 4°C overnight. After the plate was blocked with PBS containing 1% (w:v) BSA, the antiserum was added to each well (dilution factor from 1:22 x 100 to 1:28 x 100) followed by incubation for 1 h at RT. After three washings with PBS, peroxidase-labeled donkey anti-rabbit IgG or goat anti-mouse IgG (Chemicon International, Temecula, CA) was added to each well as a second antibody. Color development was carried out using 0.2 mg/ml of o-phenylenediamine and 0.01% (v:v) H2O2 in 150 mM citrate-phosphate buffer, pH 5.0.
Immunofluorescent Staining
Human oocytes, obtained with patients' informed consent, were incubated in antisera at a dilution of 1:100 in PBS containing 1% (w:v) BSA for 30 min at RT. After extensive washing with the same buffer, the oocytes were treated with FITC-labeled donkey anti-rabbit IgG antiserum absorbed with mouse, human, and bovine IgG (AP182F; Chemicon) at a dilution of 1:100 at RT for 30 min. After washing, the oocytes were fixed on a glass slide for observation under a UV microscope (FluophotVD; Nikon, Tokyo, Japan).
Inhibition Assay of Human Sperm-Zona Interaction
To examine the blocking effect on binding of human spermatozoa to ZP, unfertilized human oocytes were obtained from clinical in vitro fertilization-embryo transfer programs (with patients' informed consent). Before use in this experiment, oocytes were subjected to extensive pipetting with a fine-bore pipette to remove spermatozoa bound to the ZP. The oocytes were treated with antisera or control sera (1:10) in BWW culture medium containing 0.3% (w:v) BSA for 1 h in 5% CO2 in air. The oocytes, after a brief rinse, were introduced into a drop of the sperm suspension (1 x 106/ml) for insemination. After 15 h, oocytes were washed by repeated pipetting, and the number of spermatozoa bound to or penetrating through the ZP was assessed under a microscope equipped with a Hoffman module (Olympus, Tokyo, Japan).
Statistical Analysis
Means are expressed ± SD. Significance of differences was calculated using Mann Whitney's U-test for unpaired samples.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To produce recombinant proteins in E. coli, cDNA encoding amino acid residues 1–206 of human ZP2 were constructed by PCR for insertion into a pET21b vector (Fig. 1). The cDNA was placed immediately after a T7 promoter. Recombinant proteins were detected in E. coli (BL21DE3) transfected with pEThZP2 by using monoclonal antibody Mab5H4. In Western blot analysis of recombinant proteins in crude E. coli lysates, the largest (26 kDa) and most prominent band via CBB staining was recognized by the monoclonal antibody Mab5H4 (Fig. 2). The recombinant protein was purified by the collection and subsequent lysis of the inclusion bodies. The final yield of rec-hZP2 protein was 80 µg/ml of LB medium. This protein was designated rec-hZP2.
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Binding Capacity of rec-hZP2 to Spermatozoa
Binding of rec-hZP2 to spermatozoa with or without treatment with calcium ionophore A23187 was examined (Fig. 3). Any spermatozoa labeled with TRITC were classified as rec-hZP2-binding spermatozoa. The rec-hZP2 did not bind to any spermatozoa not treated with ionophore, while it bound to spermatozoa treated with ionophore in the proportion of 63% (mean ± SD, 63% ± 15%) in 10 experiments using sperm samples from three different donors. Binding sites for rec-hZP2 were observed in the region from the acrosome to the midpiece. Sperm motility was reduced to 50% by ionophore treatment. Further incubation with rec-hZP2 reduced sperm motility to 10%. The acrosomal status of the spermatozoa was assessed using FITC-conjugated PSA. Any spermatozoa that demonstrated patchy staining or complete loss of PSA staining in the acrosome were classified as "acrosome-reacted" spermatozoa, which were detected in the proportion of 74% (mean ± SD, 74% ± 8.4%) among ionophore-treated samples. Almost all spermatozoa stained with TRITC showed lack of FITC fluorescence.
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The control experiments were carried out by using mouse immunoglobulin light-chain cDNA, which was inserted into a pET21b vector. Recombinant proteins were prepared by the same procedure as for rec-hZP2. No binding of these proteins to spermatozoa was observed before or after treatment with ionophore A23187.
Blocking Effect of Antisera Against rec-hZP2 on Sperm Binding and Penetration into ZP
To verify the role of human ZP2 amino acids 1–206 in secondary sperm binding and penetration into the ZP, antisera against rec-hZP2 were produced in rabbits and mice. The respective ELISA antisera titers were 1:25 600 and 1:12 800. In the immunofluorescent studies, human oocytes were stained using the antisera (1:100 dilution) of rabbits and mice (Fig. 4). The blocking effect of the antisera on human sperm binding and penetration into human ZP was examined at 15 h after insemination. Compared to findings in the control, both antisera significantly blocked human sperm binding and penetration into human ZP (Fig. 5, Table 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant proteins from human ZP2 were produced in E. coli (BL21DE3) by use of expression vector pET21b. The molecular size of rec-hZP2 was 26 kDa, which is identical to the calculated size (25.6 kDa) of the total 232 amino acid residues making up this polypeptide. A number of smaller minor bands were also detected by Mab5H4 as seen in lane 6 of Figure 2. These fragments may have been derived from the intact recombinant protein by proteolytic cleavage. The yield of rec-hZP2 was quite high compared to that in previous studies on the production of recombinant animal ZP proteins in E. coli systems [18, 19].
In our experiment, the acrosome reaction was stimulated by using calcium ionophore A23187, which triggers morphological changes similar to those of the physiologically induced acrosome reaction [20]. We examined the influence of rec-hZP2 on induction of acrosome reaction. Incubation of capacitated sperm with rec-hZP2 did not induce acrosome reaction in spermatozoa. The rec-hZP2 bound only to the ionophore-treated spermatozoa, suggesting that rec-hZP2 bound to acrosome-reacted spermatozoa. Although only calcium ionophore was used for induction of the acrosome reaction in this study, a similar binding reaction was previously observed in pigs. Boar spermatozoa in which the acrosome reaction was induced by native pig ZP demonstrated binding to a recombinant pig ZP protein, composed of a region homologous to rec-hZP2 [21]. The capacity of spermatozoa to react to rec-hZP2 was not identical to that of spermatozoa showing the acrosome reaction as recognized by PSA staining. This means that the release of PSA-binding substances does not completely correlate to the rec-hZP2-binding capacity of spermatozoa. Almost all spermatozoa labeled with rec-hZP2 showed defects of PSA staining in the acrosome, and the proportion (74% ± 8.4%) of acrosome-reacted sperm as assessed by the PSA staining method was a little higher than that of rec-hZP2-labeled spermatozoa (63% ± 15%). These results suggest that spermatozoa could obtain rec-hZP2-binding capacity after the release of lectin-binding substances. This is reasonable considering that the acrosome reaction of spermatozoa is triggered by the primary sperm receptor on ZP and that sperm then bind to the secondary sperm receptor [3, 8, 9]. In view of these physiological conditions, rec-hZP2 labeling could be a more useful way to detect acrosome-reacted spermatozoa than lectin staining. We speculate that the rec-hZP2 binding identifies acrosome-reacted spermatozoa that have obtained the ability to undergo secondary binding to ZP2. When the spermatozoa were incubated with rec-hZP2 for 18 h, rec-hZP2 was detected at the midpiece and the tail of the ionophore-treated spermatozoa (data not shown). The same phenomenon was observed in pigs [11]. A natural conclusion is that the rec-hZP2-binding site migrates from the acrosome through the midpiece to the tail as spermatozoa proceed to penetrate through the ZP. The migration pattern was similar to that of mouse Sp17, which is thought to be a sperm protein for secondary sperm binding to ZP2 [22].
Antisera against rec-hZP2 confirmed the important role of the human ZP2 polypeptide sequence (N-terminal amino acids 1–206). Sperm binding and penetration into the ZP were strongly blocked by both rabbit and mouse antisera. Although the effect of steric hindrance was not completely neglected, these results indicated that rec-hZP2 could be used for the development of contraceptive vaccines in humans. For clinical use of rec-hZP2, the following problems must be resolved: 1) possible side effects including induction of oophoritis and 2) the possibility of autoantibody development. Oophoritis might be prevented by using a peptide without pathogenic T-cell epitopes, and autoantibodies could be produced by linking the B-cell epitope to a promiscuous T-cell epitope [23–25]. We have previously reported the production of autoantibodies against the ZP in rabbits [26]. An 18-mer peptide corresponding to amino acids 50–67 of a rabbit homologue of human ZP2, including the B-cell epitope of Mab5H4, was conjugated with diphtheria toxoid. Rabbits immunized with the antigens produced a strong autoantibody against the ZP without displaying any pathogenic findings of the ovaries, and showed a transient fertility.
In conclusion, the N-terminal amino acids 1–206 of human ZP2 contain a binding site for acrosome-reacted spermatozoa and could be a candidate for a contraceptive vaccine in humans.
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FOOTNOTES |
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Accepted: July 22, 1999.
Received: March 17, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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