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a Western Australian Institute for Medical Research & Keogh Institute for Medical Research, QEII Medical Centre, Nedlands, Perth 6009, Australia
b MRC Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh EH3 9EW, United Kingdom
d Department of Biological Sciences, University of Newcastle, NSW 2308, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The traditional approach towards the study of sperm-egg interactions has been to generate monoclonal or polyclonal antibodies that disrupt this process in vitro and then to use these reagents to identify target molecules, either by screening cDNA libraries or by immunoprecipitation and sequencing of native protein. Success with this approach has been limited to a small number of sperm antigens, very few of which are expressed on the sperm surface [6–8]. It is possible that progress in this field is about to accelerate rapidly in light of a recent report revealing the successful generation of Fab fragments recognizing human spermatozoa using a combinatorial phage display library [8]. This library was based upon RNA extracted from the lymphocytes of a vasovasotomized patient exhibiting autoimmunity to sperm antigens. This powerful technology has the potential to contribute significantly to our understanding of the immunological responses associated with human infertility and the contraceptive strategies that might flow from this information. However as a means of analyzing the molecular basis of sperm-egg interaction the approach is obviously constrained by the limited repertoire of autoantigens present on the sperm surface.
A more direct approach would be to use random peptide phage display technology to identify small linear peptides capable of binding with high affinity to the surface of human spermatozoa and disrupting sperm function [9–11]. This powerful technique permits millions of variants to be screened quickly and efficiently for their capacity to interact with the sperm surface even though the composition of the target structure(s) is largely unknown. Using 12- and 15-mer random peptide phage display libraries we have succeeded in identifying a number of peptides capable of binding to the surface of human spermatozoa, several of which exhibit homology with a domain at the N-terminus of zona glycoprotein, ZP3.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study was based upon semen samples donated by a panel of healthy normozoospermic donors after 2 or 3 days abstinence. All samples were collected in sterile containers and left for at least 30 min to liquefy before processing. The spermatozoa were isolated by discontinuous Percoll gradient centrifugation using a simple 2-step design incorporating 44% and 88% Percoll as described previously [12]. The spermatozoa recovered from the high density region of the gradient were suspended at a final concentration of 20 x 106/ml in HEPES-buffered BWW supplemented with 0.3% bovine serum albumin (BSA; fraction V, nuclease and protease free, Calbiochem, Nottingham, UK). The spermatozoa were incubated for 24 h to effect sperm capacitation after which the concentration was adjusted to 80 x 106/ml and 100-µl aliquots were transferred to the wells of a 96-well Nunc microtiter plate. The plates were centrifuged at 1000 rpm for 5 min and the supernatant was removed from each well by flicking the plate onto paper towels. The sperm pellets were allowed to dry at room temperature and then fixed for 30 min with 1% paraformaldehyde in phosphate-buffered saline (PBS). The plates were subsequently washed 3 times in PBST (PBS + 0.05% Tween-20), left to dry briefly, and stored at-20oC until used.
15-Mer Library
The phage display library used in this study was constructed in the fUSE 5 vector and displayed a foreign random 15-mer peptide on all 5 copies of capsid protein III. The bacterial strain used was K91Kan (both the library and bacterial strain were donated by G. Smith, University of Missouri, Columbia, MO).
The affinity selection procedure was based on the biopanning methods previously described by Smith and Scott [13]. The wells of Nunc microtiter plates were coated with capacitated spermatozoa as described above. The coated wells were blocked overnight at 4oC with a solution of 5% non-fat milk in Tris-buffered saline (TBS). In addition, a Nunc microtiter plate that was not sperm-coated was also blocked overnight. The next morning the uncoated plate was washed 6 times with TBS and an aliquot of the phage library corresponding to 2 x 1010 transducing units (TU) was diluted in 100 µl of TBS + 0.1% BSA and added. This was left at room temperature for 2 h to absorb those phage, which bind BSA and plastic. After 2 h, the phage + buffer were transferred to a sperm-coated well of a microtiter plate that had similarly been washed. The plate was left gently rocking at room temperature for 1 h. Following this incubation the sperm-coated well was washed 10 times with TBST (TBS + 0.5% Tween) and 100 µl of elution buffer (0.2 M glycine-HCl, pH 2.2 + 1 mg/ml BSA) was added for 10 min. The eluate was then transferred into an Eppendorf tube containing 15 µl neutralizing buffer (1 M Tris-HCl, pH 9.1). The eluate was titered as TU and amplified. Amplified eluates were subjected to two further rounds of enrichment. Sequencing of the phage clones was undertaken using primer 5'-AGTTTTGTCGTCTTTCC-3'. Selected phage were amplified as described by Smith and Scott [13].
12-Mer Library
The M13 phage display library used in this study displays a 12-mer random peptide on all copies of capsid protein III. The bacterial strain used was ER2537. The library is commercially available from New England Biolabs (Beverly, MA). Wells of Nunc microtiter plates were coated with capacitated spermatozoa or were left uncoated as described above. The wells were blocked overnight at 4oC with a solution of 0.1 M NaHCO3 (pH 8.6) + 1% non-fat milk. An uncoated well was washed 6 times with TBS and a 10-µl aliquot of the phage library corresponding to 1.4 x 1011 plaque forming units (pfu) was diluted in 100 µl of TBS + 0.1% BSA and added to it. This was left at room temperature for 2 h to absorb those phage which bind BSA and plastic. After the 2 h, phage and buffer were transferred to a blocked sperm-coated well which had been washed 6 times with TBS. The plates were left gently rocking at room temperature for 1 h. Following this incubation the sperm-coated wells were washed 10 times with TBST and 100 µl of elution buffer (0.2 M glycine-HCl, pH 2.2, + 1 mg/ml BSA) added for 10 min. The eluate was then transferred into an Eppendorf containing 15 µl neutralizing buffer (1 M Tris-HCl, pH 9.1) and titered. The eluate was stored at 4oC overnight and amplified the next day.
An overnight culture of ER2537 was diluted 1/100 in 20 ml LB broth in a 200-ml flask and the unamplified eluate was added. After vigorous shaking at 37oC for 4.5 h, the culture was centrifuged twice at 10 000 rpm at 4oC and the phage were purified from the supernatant by 2 rounds of polyethylene glycol (PEG) precipitation. The phage pellets were resuspended in 200 µl TBS and titered. The second and third rounds were carried out in the same manner. In order to sequence plaques for analysis, an overnight culture of ER2537 was diluted 1/100 in LB broth and 1-ml aliquots were added to 15-ml tubes. Plaques were stabbed with a sterile wooden stick and added to the tubes for 4.5 h at 37oC; the cultures were centrifuged and the phage DNA purified by PEG and iodide/ethanol precipitation. The DNA pellets were resuspended in TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) and used as a template for sequencing; sequencing was undertaken using primer 5'-CCCTCATAGTTAGCGTAACG-3'. For a large-scale amplification of each clone, 5 µl of each phage supernatant was added to 20 ml of a 1/100 dilution of ER2537 in LB broth and the culture was vigorously shaken at 37oC for 4.5 h. After centrifugation twice at 10 000 rpm, the supernatant was added to an early ER2537 culture in 500 ml LB broth and grown for 4.5 h. The phage were then purified from the culture using the same protocol as for the amplified eluate.
ELISA Assay
Alternate wells of a 96-well Nunc microtiter plate were coated with either spermatozoa or BWW media and blocked overnight with 0.1 M NaHCO3 (pH 8.6) + 1% non-fat milk + 0.5% Tween. The next morning, an aliquot of amplified phage of known titer from either the eluate selected clones or random control clones was diluted in ELISA buffer (TBS/1 mg/ml BSA/0.5% Tween) and left rocking at room temperature for 2 h. After this incubation, the sperm-coated, blocked plate was washed twice with TBS and aliquots of phage and ELISA buffer transferred into the appropriate wells and left gently rocking for 2 h at room temperature. The plate was washed 3 times with TBST and 3 times with TBS with 2-min intervals between washings. Two hundred microliters of a 1/5000 dilution of anti-M13/HRP conjugate (Pharmacia Biotec) in blocking buffer, was added to each well and left for 1 h at room temperature. The plate was washed as before and 200 µl of 3,3', 5'5-tetramethylbenzidine was added and left for 30 min. The reaction was stopped with the addition of 100 µl 0.5 M H2SO4 and the plate read at 450 nm on a plate reader. The experiment was repeated in triplicate.
Binding of Phage Clones to Fixed Spermatozoa
Spermatozoa were air-dried onto an 8-well chamber slide (Lab-Tek II; RS treated) and fixed for 10 min with 1% paraformaldehyde. Following 2 washes with TBS, 200 µl of blocking buffer (0.1 M NaHCO3 + 1% non-fat milk + 0.5% Tween + 1/5 dilution of goat serum) was added to the chambers and left overnight at 4oC. The wells were washed twice with TBS and then a known titer of phage was added in TBS + 1 mg/ml BSA + 0.5% Tween and left for 2 h at room temperature to bind. The wells were washed 5 times with TBST, 5 times with TBS and 200 µl of a 1/5000 dilution of rabbit anti-fd antibody in blocking buffer added for 1 h at room temperature. Following washing, 200 µl of goat anti-rabbit FITC conjugated antibody was added for 1 h at room temperature. The wells were washed and the slide was mounted in Citifluor (glycerol/PBS). The distribution of labeled peptide was then examined under an oil immersion objective (x40) using a Zeiss 510 confocal laser microscope and a filter selective for FITC fluorescence.
Peptide Synthesis
Phage fusion peptides were chemically synthesized on a MWG-Biotech peptide synthesiser to 95% purity using a C-18 reverse-phase HPLC column. The peptides were synthesized with amidation of their C-terminus. In order to preserve the binding and activity of the peptides, a linker sequence Gly-Gly-Gly-Ser was added to the C-terminus of both peptide sequences. Peptides from the 15-mer library were dissolved in 7% DMSO in H2O (v/v). Peptides synthesised from the 12-mer library were dissolved in DMSO. Fluorescein-isothiocyanate (FITC)-labeled peptides, also synthesized as above, were dissolved in DMSO.
Binding of Synthetic Peptide to Spermatozoa
Spermatozoa were air-dried onto an 8-well chamber slide and fixed as described above. Following 2 washes with TBS, 200 µl of 0.1 M NaHCO3 + 1% non-fat milk was added to the chambers and left overnight at 4oC. The slides were then washed twice with TBS, and 200 µl of 0.1 µM FITC- labeled peptide in TBS was added to individual wells and left to bind overnight at 4oC. The slides were washed gently 6 times with TBS to remove unbound peptide, mounted in Citifluor (glycerol/PBS), and then examined.
Peptide Saturation Assay
A sperm-coated plate (with spermatozoa at a concentration of 10 x 106/ml) was blocked overnight at 4oC. The wells were washed twice in TBS and incubated for 2 h at room temperature with varying concentrations of FITC-labeled M6 or MR2 peptides (0.1 µM-250 µM) in TBS. After washing 3 times with TBST and TBS, plates were read on a Wallac 1420 Manager using a filter selective for FITC fluorescence (485 nm/535 nm, 1.0 sec).
Sperm-Zona Interaction
Semen samples were prepared on a discontinuous Percoll gradient as described above, capacitated by incubation for 3 h in 3 mM pentoxifylline at 37oC in an atmosphere of 5% CO2 and finally resuspended in BWW at a concentration of 10 x 106/ml. M6 and MR2 peptides were added to 100-µl aliquots of the spermatozoa at concentrations of 100 µM, 10 µM, and 1 µM and left at 37oC for 1 h prior to the addition of washed human oocytes that had previously been stored in 1 M magnesium chloride, 0.1% dextran (w/v), and 20 mM HEPES at 4oC. After 90 min at 37oC in an atmosphere of 5% CO2 in air the oocytes were gently washed 3 times in BWW to remove loosely adherent spermatozoa and then fixed and stained with 0.65% glutaraldehyde containing Hoechst No.33342 (1 mg/ml) in 20 mM Tris (pH 8.0). An area of 125 µm2 on each zona was finally analyzed for the presence of bound spermatozoa using a Hamilton Thorn CASA system (Hamilton Thorne Research Inc. Beverly, MA).
In order to monitor zona-induced acrosome reactions spermatozoa were prepared and capacitated as above and then incubated with human zonae pellucidae for 90 min. The tightly bound spermatozoa were finally removed and assessed for acrosomal status using an FITC-labeled lectin (Arachis hypogaea, 2 mg/ml in PBS) [14, 15].
Sperm-Oocyte Fusion
Semen samples were prepared on Percoll gradients as described above and the spermatozoa were incubated for 3 h in 3 mM pentoxifylline at 37oC in an atmosphere of 5% CO2 to effect capacitation. Progesterone (5 µM) was added for the last 30 min to induce the acrosome reaction, after which the spermatozoa were pelleted at 500 x g for 5 min, resuspended in BWW to a concentration of 10 x 106/ml and subject to the analysis of sperm-oocyte fusion as described [16, 17].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A 15-mer phage library constructed from the fUSE 5 vector was biopanned against capacitated, paraformaldehyde-fixed human spermatozoa. After three rounds of affinity selection, 12 eluted phage clones were analyzed for sperm binding in an ELISA. In addition, a randomly unselected phage clone was purified and analyzed in the same ELISA in order to control for nonspecific binding. The results of this experiment are shown in Figure 1; 8 clones were considered to exhibit an affinity for the sperm surface greater than the random peptide control (M1, M2, M3, M5, M6, M7, M8, and M9).
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The sequences of the 8 fusion peptides giving highest binding were determined and these are shown in Table 1. In addition to the 12 clones described in Figure 1, we also sequenced a further 28 clones from round III to give a total of 40. The most conserved sequence, accounting for 15% of the 40 clones analyzed, displayed the sequence TLIPRSFCPTHDRDC (M1). However, clones displaying this peptide did not give the highest absorbance in the ELISA in Figure 1. Clones displaying the peptide sequence SSSSFVLWLLRPGFS (M6) accounted for only 5% of all clones sequenced and yet showed the highest overall binding. This sequence was found to exhibit significant homology with a motif at the N-terminus of ZP3 from several species (Fig. 2). Moreover elements of this sequence were detected in 3 of the other phage clones selected in this analysis (Table 1).
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Binding of Phage Clones to Human Spermatozoa
The binding site for the M6 phage clone on human spermatozoa was identified using immunohistochemistry. The M6 phage clearly localized to the sperm head, especially the equatorial segment and post-acrosomal domain, whereas the control phage clone, MR2, did not bind specifically to any region of the sperm surface (data not shown).
In order to confirm that this staining was the result of the binding of the displayed peptide to the sperm surface, M6 and MR2 peptides (MR2 random sequence—GGQMPLHTVIYALGV) were synthesised, labeled with FITC, and incubated with paraformaldehyde-fixed human spermatozoa; Figure 3 shows their distribution. Spermatozoa incubated with the control peptide, MR2, showed only background levels of staining whereas the FITC-labeled M6 peptide bound strongly to the sperm head. The staining was diffuse, covering the entire sperm head but slightly more intense over the post-acrosomal region. M6 binding to the sperm surface binding was not species specific, since caudal epididymal rat spermatozoa also bound this peptide; once again the staining was diffuse but excluded the plasma membrane covering the perforatorium (Fig. 3).
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Peptide Bioactivity
In order to test the ability of the M6 peptide to interfere with significant stages of the fertilization process, a series of bioassays was conducted. These experiments demonstrated that the M6 peptide had no significant effect on the binding of human spermatozoa to homologous zonae pellucidae at concentrations from 1 to 100 µM (Fig. 4A) or the ability of the latter to induce the acrosome reaction (Fig. 4B). However preincubation with this peptide did have a dramatic effect on the ability of acrosome-reacted spermatozoa to fuse with the vitelline membrane of the oocyte (Fig. 4C).
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12-Mer Library
A 12-mer M13 phage display library was also biopanned against paraformaldehyde-fixed spermatozoa in order to determine whether clones would be selected with a similar sequence to those selected from the 15-mer library. Table 2 shows the most frequently occurring clones from 35 plaques sequenced following round III of biopanning. The clone ‘K6’ displaying the peptide sequence-HWP-RPDDSFWRP- accounted for approximately 28% of all clones sequenced. While the next frequently encountered clone, K10, accounted for approximately 11% of the total. However, none of these clones showed any homology with the sequences selected from the 15-mer library.
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Despite its lack of homology with the 15-mer M6 peptide, K6 was also shown to possess bioactivity, significantly suppressing the ability of acrosome reacted cells to fuse with the vitelline membrane of the oocyte, in contrast to a random 12-mer peptide sequence (R1), SMATWRSTYVIP, which was biologically inactive (Fig. 4D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In view of this homology with ZP3, it was envisaged that exposure to the M6 peptide might disrupt the ability of human spermatozoa to bind to the zona pellucida or undergo an acrosome reaction at the zona surface. In the event, M6 disrupted neither of these aspects of sperm function. Such a result is in keeping with an abundance of data indicating that sperm-zona interaction is mediated by the oligosaccharide side chains of ZP3, rather than the polypeptide core [21]. Moreover, the binding sites for this peptide were particularly concentrated over the equatorial segment and post-acrosomal sheath, not on the anterior surface of the acrosome, which is the plasma membrane domain involved in sperm-zona recognition. The significance of the equatorial segment lies not in sperm-zona interaction but in recognition of the vitelline membrane and the initiation of sperm-oocyte fusion [22].
A possible role for this peptide-binding site in mediating sperm-oocyte fusion was suggested by the ability of the M6 peptide to suppress this aspect of human sperm function. The suppression of sperm-oocyte fusion in the absence of any suppressive effect on the acrosome reaction clearly suggests that the peptides are interacting with the surface of the live gamete and preventing these cells from recognizing binding sites on the vitelline membrane. These results also suggest that one of the molecules spermatozoa might be targeting on the surface of the vitelline membrane is ZP3, i.e., that this molecule plays as important a role during sperm-oocyte fusion as it does during sperm-zona binding.
ZP3 possesses a hydrophobic domain near its carboxyl terminus, downstream of a TGF-ß type III receptor-like region and immediately upstream of a short stretch of positively charged amino acids [23]. A furin cleavage site upstream of the hydrophobic domain provides a mechanism by which ZP3 can be released from the oocyte to create the zona pellucida [24, 25]. However, even in a mature ovum, ZP3 molecules are apparently expressed on the surface of the vitelline membrane [24] where they might contribute to the complex cascade of interactions associated with sperm-oocyte recognition and, ultimately, fusion.
In conclusion, phage display technology has been successfully used to select peptides capable of binding to human spermatozoa. These unique reagents should be of fundamental value in resolving the molecular architecture of the spermatozoon and in the development of new approaches to fertility control that target the male gamete. Additional studies will be needed to confirm the surface localization of the binding site and examine the bioactivity of the native ZP3 sequence covered by the selected peptides. Whatever the outcome of these studies the results already obtained indicate that random peptide phage display technology is a powerful new tool in the analysis of sperm biology that has shed a new and unexpected light on the mechanisms by which male and female gametes interact during fertilization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence: Karin A. Eidne, Western Australian Institute for Medical Research, Ground Floor, B Block, QEII Medical Centre, Nedlands, Perth 6009, Australia. FAX: 61 8 9346 1818; keidne{at}waimr.uwa.edu.au 
Accepted: May 23, 2000.
Received: October 18, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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