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a Division of Research, Department of Obstetrics and Gynecology, Medical College of Ohio, Toledo, Ohio 43614
b New York Fertility Institute, New York, New York 10028
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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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The utility of a sperm antigen in the development of a contraceptive vaccine is contingent on its involvement in the fertilization process and on tissue specificity. During the past decade, several antigens relevant to fertility have been defined (reviewed in [1]). A few of these also affect fertility in actively immunized animals [1,13]. In these studies, purified/semipurified cognate antigens were used for immunization to examine the effect on fertility. However, to obtain Food and Drug Administration approval and to conduct appropriate multicenter fertility trials in a quality-controlled manner, recombinant or synthetic peptide molecules are required. Complementary DNAs encoding for a few sperm antigens have been cloned and sequenced, and the recombinant proteins or their peptides expressed by some of the cloned cDNAs are being examined for their effect on fertility [14–16].
The fertilization process includes a cascade of events that the spermatozoon must undergo before fusing with the oocyte plasma membrane. One of the key steps in the fertilization cascade is the recognition and binding between the complementary molecules present on the spermatozoon and zona pellucida (ZP) of the oocyte [17]. The sperm-ZP interaction constitutes an important event in the fertilization process, and the molecular sequences involved are the most attractive candidates for the development of a contraceptive vaccine.
On the basis of the above findings, the present study was conducted to investigate the sperm peptide sequence(s) involved in recognition and binding to the complementary molecule of the ZP in humans. This was achieved by screening the FliTrx random phage display library (Invitrogen, San Diego, CA) that has expression of 1.77 x 108 different peptide sequences. A solubilized preparation of human oocyte ZP proteins was used as a probe to obtain reactive clones. The long-term objective of this study was to search for sperm peptide sequence(s) that can find potential applications in the development of a contraceptive vaccine and in the diagnosis and treatment of infertility in humans.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The FliTrx random phage display library used in the present study was obtained from Invitrogen. The library was screened with solubilized human ZP proteins (HZP) according to the manufacturer's protocol. After the fifth round of panning, the eluted cells were streaked on LB plates containing 100 µg/ml ampicillin to select colonies. Potential positive colonies were further confirmed for reactivity with HZP using the Western blot procedure. The colonies were grown and cells were suspended in 100 µl SDS-PAGE buffer (nonreduced), boiled for 10 min, and centrifuged; the supernatant was run in SDS-PAGE (5–15% gradient gel) [18], and the resolved proteins were transferred to nitrocellulose paper for Western blot analysis [19]. The blot was reacted with HZP (20 µg/20 ml) and then with rabbit antibodies to HZP (described below; 10 µg/20 ml); the reacted proteins were localized by incubating the antibody-reacted strips first with alkaline phosphatase-conjugated goat anti-rabbit antibodies (heavy- and light-chain specific; Cooper Biomedical Inc., Malvern, PA) and then with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrate, as described elsewhere [16,20].
Human Oocyte ZP Proteins and Their Antibodies
The human oocytes were obtained from female partners (21–35 yr old) of infertile couples who attended the Infertility Service for in vitro fertilization-embryo transfer [21,22]. These infertile couples had infertility attributed to the male partners, and the female partners were without any ovarian abnormality. Appropriate informed consent and Institutional Review Board approval were obtained for study participation and collection of ova. There were an excess number of left-over mature eggs, cryopreserved in 3.5 M propanediol containing 0.27 sucrose and 5% BSA in PBS until used. These eggs were never exposed to sperm and could have otherwise been discarded. They were rapidly thawed, washed in Ham's F-10 medium, and freed of adhering granulosa cells by incubation with 0.1% hyaluronidase (Sigma Chemical Co., St. Louis, MO) in PBS. The eggs were washed (3 times) in PBS to remove adhering cumulus cells, and the zonae were heat solubilized in sodium carbonate buffer (0.001 M, pH 9.0) at 60°C for 1 h [21,22]. The solubilized HZP were dialyzed against PBS for 48 h at 4°C before use.
Rabbit antiserum was prepared against HZP as described elsewhere [23]. Briefly, sexually mature virgin female rabbits (n = 2) were actively immunized systemically with HZP emulsified with Freund's adjuvant; and after three booster injections, the animals developed high titer antibodies (> 1:5012) against HZP, as examined using ELISA. Preimmune serum and the sera from control animals injected similarly with only PBS, emulsified with Freund's adjuvant, were used as positive controls in various assays.
DNA Isolation and Sequencing
DNA was isolated from the uninduced cells/clones that showed positive reaction with HZP in the Western blot procedure after five cycles of panning. The plasmid DNA was isolated using the simple nucleic acid preparation (S.N.A.P.) miniprep kit (cat. #K1900-01; Invitrogen) and sequenced by dsDNA Cycle Sequencing System (Gibco-BRL, Gaithersburg, MD) [16,20]. Sequencing primers were FliTrx forward and reverse sequencing primers. The 36 base pairs corresponding to the dodecamer peptide sequence were inserted at RsrII/AvaII (5' end) and AvaII (3'-end) of the vector DNA and could be released using AvaII. The search for nucleotide (nt) and amino acid (aa) sequence homology in GenBank, National Biomedical Research Foundation (NBRF), and Swiss sequence banks was performed using Fasta and tFasta search programs [24]. The consensus aa sequence was derived from the various positive peptide sequences by using the Lineup program (Genetics Computer Group, Madison, WI). The peptides were synthesized by solid-phase synthesis using Fmoc chemistry (Biosynthesis Inc., Lewisville, TX). Deprotection was achieved by 20% piperidine in dimethylformamide, and the peptides were cleaved from the resin by 85% trifluoroacetic acid (TFA). The peptides were then precipitated in methyl tert-butyl ether and purified by using reverse-phase HPLC. The fractions eluted with 0.5% TFA in acetonitrile were dried in a speed vacuum, redissolved in water, and lyophilized. All four peptides were water soluble and had > 95% purity level.
Antibodies to Peptide
The peptides were conjugated to tetanus toxoid (TT; Wyeth Laboratories, Radnor, PA) by the method described by Tsong et al. [25], using 1-ethyl-3 (3-dimethylaminopropyl). Antiserum was raised in sexually mature virgin female rabbits of the New Zealand white strain [23], and the antibodies were purified by using a protein A-Sepharose 4B column. The monovalent Fab's were prepared using pepsin by the method of Nisonoff and colleagues [26] or described elsewhere [27]. The Fab's were further immunoaffinity purified by using peptide-BSA-Sepharose 4B immunobeads prepared as described below.
Hemizona Assay
The hemizona assay was performed to examine the effects of peptides and their Fab' antibodies on human sperm-ZP binding [28]. One half of the hemizona pair was experimental (treated with the peptide or Fab's), and the other half of the same oocyte was used as a control (untreated). Two sets of experiments were performed. In the first set, peptide/Fab's were incubated (37°C, 1 h) with sperm (100-µl drop), washed, and then incubated (4 h) with the untreated experimental hemizona. Control sperm were incubated with the medium only. In the second set, peptide/Fab's were incubated (37°C, 1 h) with the experimental hemizona, washed, and then incubated (4 h) with the untreated sperm in a 100-µl drop. Control hemizonae were incubated with the medium only. The number of sperm tightly bound to the outer surface of the hemizona was determined and expressed as the hemizona index: HZI = [Number of sperm bound on experimental hemizona/Number of sperm bound on control hemizona] x 100.
Each sample was tested with 7–12 hemizonae obtained from 3–8 different women in three different experiments performed on different days using sperm from at least three different fertile men. For the immunoadsorption experiments, the Fab's were incubated (4°C, overnight) with the peptide (1:10, w:w) and centrifuged (10 000 rpm, 5 min), and the supernatant was tested in the hemizona assay.
Interaction Between the Peptide and Oocyte ZP
The interaction between synthetic peptide and oocyte ZP proteins was examined by Western blot and immunoprecipitation. For Western blot analysis, the HZP was run in two identical SDS gels (5–15% gradient) and transferred to nitrocellulose membranes. BSA was biotinylated and separated from unbiotinylated protein according to the manufacturer's protocol (ECL protein biotinylation module; Amersham, Piscataway, NJ) [20]. The peptide was conjugated to biotinylated BSA by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as described above. The Western blots were incubated with blocking solution for 2 h at room temperature; then one blot was treated with the peptide-biotinylated BSA conjugate in PBS (20 µg/20 ml) and the second identical blot was treated with biotinylated BSA not conjugated to peptide (20 µg/20 ml). The blots were washed, reacted with streptavidin-horseradish peroxidase (1:1500), washed, incubated with substrate, and exposed to x-ray film for 1–5 min. BSA was used as a control in these experiments.
For analysis using the immunoprecipitation procedure, the peptide was conjugated to BSA as described above, and the peptide-BSA conjugate or the BSA alone was coupled to cyanogen bromide-activated Sepharose 4B (Sigma) [21,29]. HZP was divided into two aliquots of 20 µg protein/50 µl each. One aliquot was incubated with peptide-BSA-Sepharose 4B beads, and the second was incubated with the BSA-Sepharose 4B beads suspended in radioimmunoprecipitation assay buffer (50 mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM PMSF [30]. The reaction mixture was incubated for 2 h at 4°C; unadsorbed proteins were washed off, and the reacted zona protein was eluted from the beads by treating with glycine-HCl (0.1 M, pH 2.8). The eluate was neutralized with solid K2HPO4 to pH 7.4, boiled with nonreduced SDS-sample buffer, and subjected to SDS-PAGE; the gel containing the resolved proteins was stained with silver nitrate and visualized.
Tissue-Specific Expression
To examine tissue-specific expression, the solubilized extracts from 10 different human tissues—namely, testis, kidney, liver, spleen, heart, lung, brain, muscle, ovary, and placenta (Human Protein Medley; Clontech, Palo Alto, CA)—were run in SDS-PAGE and transferred to nitrocellulose membrane for Western blot analysis. The membranes were probed with anti-peptide Fab's/control Fab's (15 µg/10 ml), and the reacted bands were localized as described above [16,20].
Indirect Immunofluorescence Technique
Indirect immunofluorescence technique was performed on methanol-fixed sperm cells to examine the subcellular site at which the peptide sequence was present [31]. Motile sperm cells were collected from semen of fertile men by the swim-up procedure, washed (3 times) with PBS, air dried at room temperature, fixed in methanol, and air dried again. The slides were then rinsed in PBS and blocked with PBS containing 5% BSA for 45 min; next they were reacted with anti-peptide Fab's in PBS (10 µg/100 µl) for 1.5 h at room temperature in a moist chamber. After washing in PBS, fluorescein isothiocyanate-labeled goat anti-rabbit IgG (1:40 dilution; Cappel Labs., Malvern, PA) was added and incubated as above for 1.5 h. The slides were washed, mounted in 90% glycerol in PBS containing sodium azide (0.1%) and 1,4-diazabicyclo(2,2,2)octane (10 mg/ml), and examined.
Capacitation was induced by incubating (6–8 h, 37°C in 5% CO2 and 95% air mixture) the swim-up sperm (10 x 106 sperm/ml) in Ham's F-10 medium containing 5% BSA. The capacitated sperm were washed, methanol fixed, and studied for antibody reactivity as described above.
Statistical Analysis
Significance of differences between treated and control groups in Tables 2 and 3 was analyzed by using unpaired and paired Student's t-test. A P value of < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The peptides were synthesized based on these four sequences, and the purified peptides were examined for their effects on binding of human sperm with human ZP in the hemizona assay. Of these peptides, two, namely YLP12 and Consensus17, caused a significant (P < 0.05) inhibition of sperm binding with ZP (Table 2, Fig. 1b). The effects were concentration dependent, with a stronger inhibition at higher doses. The effect was much stronger with the YLP12 as compared to Consensus17 peptide at 75 mM concentration. The inhibition was apparent only when the hemizonae were treated with the peptides before coincubation with sperm, not vice versa. The other two peptides did not affect binding, and the results were similar to those with BSA control (Table 2). Further studies focused on the YLP12 peptide. Immunization with YLP12 peptide conjugated to TT raised high-titer (> 1:5120, ELISA titer) antibodies against the peptide. The immunoaffinity-purified Fab's against the YLP12 peptide significantly inhibited sperm-zona binding (Table 3, Fig. 1d). Control Fab's from animals immunized with TT alone and with preimmune serum did not affect sperm binding. Immunoadsorption of the anti-YLP12 Fab's with purified peptide completely abolished inhibitory activity. The inhibition was apparent only when sperm were treated with the anti-YLP12 Fab's before coincubation with hemizona, not vice versa.
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To examine the component of the human ZP that is recognized by the YLP12 peptide, Western blot and immunoprecipitation procedures were performed. In Western blotting, the peptide YLP12 conjugated to biotinylated BSA specifically reacted with the ZP3 component of human ZP and not with BSA (Fig. 2, Western blot, lanes Aa and Ab, respectively). The biotinylated BSA not conjugated to peptide did not react with HZP or BSA (Fig. 2, Western blot, lanes Ba and Bb, respectively). In the immunoprecipitation procedure, similar results were obtained. The peptide conjugated to BSA coupled to Sepharose 4B beads pulled out the ZP3 component from HZP and nothing from the BSA solution (Fig. 2, Immunoprecipitation, Aa and Ab, respectively). The BSA-Sepharose 4B beads not conjugated to peptide did not react with HZP or any protein in the HZP preparation or BSA solution (Fig. 2, Immunoprecipitation, Ba and Bb, respectively).
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Anti-YLP12 Fab's specifically recognized a protein band of ~72 ± 2 kDa only in the testis lane in the Western blot procedure involving solubilized extracts of 10 different human tissues (Fig. 3).
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Anti-YLP12 Fab's reacted with the acrosomal region of the methanol-fixed human sperm cell, whether noncapacitated (Fig. 4b) or capacitated (Fig. 4d). Fab's from control or preimmune immunoglobulins did not bind to any region of the sperm cell (Fig. 4f).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When tested in the hemizona assay, only two, YLP12 and Consensus17, significantly inhibited sperm binding, with YLP12 having a stronger effect. The other two, SNR12 and GHR12, did not inhibit sperm binding with hemizona, although these peptide sequences showed relatively stronger binding with HZP in the five cycles of biopanning and Western blotting. In both these screening procedures using solubilized HZP, the complementary linear sequences rather than conformational epitopes of the interacting molecules are involved in recognition and binding. In contrast to the situation with solubilized HZP, in the intact HZP the complementary sequences may be present as conformational epitopes and/or may not be exposed on the surface, thus not recognizable by the linear sequences of these peptides in the hemizona assay. Even if the complementary epitopes present on the intact ZP are accessible to these peptides, they may not be relevant to sperm binding. Some specific sequences present in ZP are involved in sperm binding, and others are not [21,32–34]. Various studies have shown that the ZP of the human oocyte is composed of at least three components, namely, ZP1, ZP2, and ZP3, and the ZP3 molecule acts as a primary receptor for sperm binding in various species of animals [21,32–34]. It is possible that SNR12 and GHR12 sequences react with ZP1 and/or ZP2 instead of ZP3 and thus are unable to inhibit sperm binding in the hemizona assay.
The inhibitory effects of YLP12 and Consensus17 peptides were specific, since 1) the second halves of the zonae (hemizonae) from the same oocyte that were used as controls showed sperm binding; 2) the effects of peptides were apparent only when they were preincubated with hemizonae instead of sperm before insemination; and 3) two other same-sized (12-mer) peptides (SNR12 and GHR12) and BSA did not affect binding at the same concentration. Also, the inhibitory effect of YLP12 was further confirmed with use of its monovalent Fab' antibodies, which are devoid of nonspecific binding Fc portion. Immunoadsorption of the anti-YLP12 Fab's with the peptide abolished the inhibitory effect, and the control Fab's from preimmune serum and control animals immunized with the TT alone did not affect sperm binding, indicating specificity of the inhibitory effects. None of the peptides or the Fab's caused agglutination of sperm or any apparent deleterious effect on motility (percent or progressive) of the spermatozoa.
The YLP12 peptide specifically recognized the ZP3 component of the ZP of the oocyte in Western blot and immunoprecipitation procedures. The binding with ZP3 and inhibition of sperm-zona binding by the peptide indicate that the YLP12 sequence may be a complementary receptor sequence on the sperm surface that is involved in recognition/binding to the ZP of the oocyte. The interaction of ZP3 with the peptide also indicates that the recognition/binding may involve protein-protein interaction, recognizing the linear sequences of the complementary molecules of two interacting cells (sperm/oocyte), as is becoming apparent from recent studies using recombinant ZP and sperm proteins [35].
The specific binding of the Fab's with the ~72 ± 2-kDa protein band only in the testis extract indicates the testis-specific expression of the protein having the YLP12 aa sequence. In addition to involvement in the fertilization process, tissue specificity is an important criterion for the selection of an antigen as a candidate for contraceptive vaccine development. The binding of the Fab's with the acrosomal region of the noncapacitated as well as the capacitated sperm cell in the indirect immunofluorescence technique indicates that the ~72 ± 2-kDa protein with YLP12 sequence is present in the acrosomal subcellular region of the sperm cell and is not lost during capacitation.
In conclusion, by using the phage display technique we have identified a testis-specific novel peptide sequence, present in the acrosomal region of the human sperm cell, that is involved in recognition/binding to the ZP3 component of the ZP of human oocytes. Besides helping to provide further insight into molecular mechanisms underlying sperm-zona interaction, the sequence may find applications in contraceptive vaccine development and in diagnosis and treatment of male infertility mediated through sperm dysfunction.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH grant HD24425 to R.K.N. No funds from this grant were used for human IVF and for studies involving human oocyte zona pellucida. 
2 Correspondence: Rajesh K. Naz, Division of Research, Health Education Building Rm. 211, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806. FAX: 419 383 4473; rnaz{at}mco.edu
Accepted: September 15, 1999.
Received: July 21, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and its receptors type I and type II in human oocytes. Mol Reprod Dev 1997; 47:127–133.[CrossRef][Medline]This article has been cited by other articles:
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  A.S. Samuel and R.K. Naz Isolation of human single chain variable fragment antibodies against specific sperm antigens for immunocontraceptive development Hum. Reprod., June 1, 2008; 23(6): 1324 - 1337. [Abstract] [Full Text] [PDF]
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R. K. Naz, S. K. Gupta, J. C. Gupta, H. K. Vyas, and a. G.P. Talwar Recent advances in contraceptive vaccine development: a mini-review Hum. Reprod., December 1, 2005; 20(12): 3271 - 3283. [Abstract] [Full Text] [PDF]
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C. Bohring and W. Krause Immune infertility: towards a better understanding of sperm (auto)-immunity: The value of proteomic analysis Hum. Reprod., May 1, 2003; 18(5): 915 - 924. [Abstract] [Full Text] [PDF]
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 R. K. Naz and S. C. Chauhan Human Sperm-Specific Peptide Vaccine That Causes Long-Term Reversible Contraception Biol Reprod, August 1, 2002; 67(2): 674 - 680. [Abstract] [Full Text] [PDF]
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 P. Mettens and P. Monteyne Life-style vaccines Br. Med. Bull., July 1, 2002; 62(1): 175 - 186. [Abstract] [Full Text] [PDF]
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A. J. Kouba, M. W. Atkinson, A. R. Gandolf, and T. L. Roth Species-Specific Sperm-Egg Interaction Affects the Utility of a Heterologous Bovine In Vitro Fertilization System for Evaluating Antelope Sperm Biol Reprod, October 1, 2001; 65(4): 1246 - 1251. [Abstract] [Full Text] [PDF]
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 M. Rattanachaiyanont, W. Weerachatyanukul, M.-C. Leveille, T. Taylor, D. D'Amours, D. Rivers, A. Leader, and N. Tanphaichitr Anti-SLIP1-reactive proteins exist on human spermatozoa and are involved in zona pellucida binding Mol. Hum. Reprod., July 1, 2001; 7(7): 633 - 640. [Abstract] [Full Text] [PDF]
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R. K. Naz and S. C. Chauhan Presence of antibodies to sperm YLP12 synthetic peptide in sera and seminal plasma of immunoinfertile men Mol. Hum. Reprod., January 1, 2001; 7(1): 21 - 26. [Abstract] [Full Text] [PDF]
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