'ZP domain' of human zona pellucida glycoprotein-1 binds to human spermatozoa and induces acrosomal exocytosis

To clone and express ZP1_273-551aa in baculovirus, human ZP1 fragment corresponding to nucleotide (nt) 757-1653 (897 bp) was used as template which was PCR amplified from a commercially obtained adaptor-based human ovarian cDNA library (Marathon Ready cDNA library, BD Biosciences, Clontech, USA). Primers were designed based on the sequence published in the GenBank having the accession number NM_207341. The sequence for a 6× His tag was inserted in the reverse primer to facilitate easy purification of the expressed protein employing Ni-NTA affinity chromatography. The PCR amplification was performed in 50 μl reaction volume comprising of a 10× high fidelity PCR reaction buffer [600 mM Tris sulfate (pH 8.9), 180 mM ammonium sulfate], using 10-20 ng of the template DNA, 50 pmole of the forward (5'-CGGGATCCCGGCAACACAGCTACTGTC-3') and the reverse (5'-CGGAATTCTTA GTGGTGGTGGTGGTGGTGTGTAGTGCCAGTGCTACA-3) primers, 50 mM MgSO_4, 10 mM dNTPs and 1 unit (U) of Platinum Taq DNA polymerase high fidelity (Invitrogen Corp., Carlsbad, CA, USA) for extension. The template was initially denatured at 94°C for 10 min followed by amplification, which was carried out for 25 cycles of denaturation at 94°C for 60 sec, primer annealing at 52°C for 90 sec and extension at 72°C for 90 sec followed by a final extension at 72°C for 15 minutes. Subsequent cloning of the PCR amplified product into the baculovirus transfer vector pAcGP67-A (PharMingen, San Diego, CA, USA) and generation of the recombinant baculovirus expressing the above recombinant protein was performed as described previously [16]. To obtain the recombinant protein, 50 × 10⁶Spodoptera frugiperda (Sf 21) insect cells growing in a suspension culture in Spinner bottles (Thermolyne; Barnstead International, Dubuque, IA, USA) were incubated with the recombinant virus at a multiplicity of infection (MOI) of 3 at 42 rotations per minute (rpm) for 96 h after which the cells were pelleted at 1000 g for 15 min. The recombinant protein was purified under denaturing conditions using Ni-NTA resin [25] and subsequently renatured by extensive dialysis against renaturation buffer [50 mM Tris-HCl pH 8.5, 1 mM EDTA (ethylenediaminetetraacetic acid; Amresco, Solon, Ohio, USA), 0.1 mM reduced glutathione (Amresco), 0.01 mM oxidized glutathione (Amresco) and 10% sucrose (Sigma-Aldrich Inc., St. Louis, MO, USA)] for 96 h with 6 changes of the dialysis buffer with decreasing concentrations of urea (4 M, 3 M, 2 M, 1 M, 0.5 M and finally buffer without urea) to assist in removal of urea and refolding of the protein. The refolded protein was further dialyzed against 20 mM Tris pH 7.4. To purify secreted ZP1_273-551aa, culture supernatant was dialyzed against phosphate buffer (50 mM NaH₂PO4, 300 mM NaCl, pH 8.0) followed by purification under denaturing conditions using Ni-NTA affinity column essentially as described above from the cell pellet.

The purified recombinant human ZP1_273-551aa was characterized by SDS-PAGE and Western blot as described previously [11]. In brief, the protein (2-5 μg/lane) was boiled for 10 min in 2× sample buffer (0.0625 M Tris pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue) and resolved on a 0.1% SDS-10% PAGE essentially as described previously [11]. For immunoblot, the SDS-PAGE resolved protein was electrophoretically transferred overnight to a 0.45 μm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) at a constant current of 50 milliampere in Tris-Glycine buffer (25 mM of Tris-HCl and 200 mM glycine) containing 20% methanol. The membrane was then washed once with phosphate buffered saline (PBS; 50 mM phosphate and 150 mM NaCl, pH 7.4) and non-specific sites were blocked with 3% BSA in PBS for 60 min at room temperature (RT). All subsequent incubations were carried out for 1 h at RT and each incubation was followed by three washings with PBS containing 0.1% Tween-20. Post-blocking, the membrane was processed for detection of the recombinant protein by employing 1:100 dilution of mouse polyclonal antibodies against ZP1 synthetic peptide [P5; 26] followed by horse-radish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (1:2000; Pierce, Rockford, IL, USA) as secondary antibody essentially as described previously [11]. The blot was developed with 0.6% (w/v) 4-chloro-1-naphthol (Amresco) in 50 mM PBS containing 25% methanol and 0.06% H₂O₂.

To determine the glycosylation profile of recombinant human ZP1_273-551aa, a lectin-binding assay in an Enzyme Linked Immunosorbent Assay (ELISA) format, was performed [11]. As an internal control, E. coli-expressed recombinant human ZP1_273-551aa (unpublished observations) was also used. In brief, microtitration plates (Nunclon™, Rosakilde, Denmark) were coated with the recombinant proteins (500 ng/well) in 50 mM phosphate-buffered saline (PBS), pH 7.4 for 1 h at 37°C followed by overnight incubation at 4°C. All subsequent washings were done three times in 50 mM PBS with 0.05% Tween-20 (PBST). The plate was blocked with 0.1% Tween-20 in PBS (PBST, 200 μl/well) for 90 min at 37°C followed by incubation with 21 biotinylated lectins (1 μg/ml; 100 μl/well) at 37°C for 1 h. The biotinylated lectins available in the Lectin kit-I, II and III (Vector Laboratories, Burlingame, CA, USA) were used in the lectin binding assay. The bound lectins were revealed by incubating with HRP-conjugated streptavidin (1:3000; Pierce; 100 μl/well) at 37°C for 1 h. The enzyme activity was detected by adding 100 μl/well of 0.05% orthophenylenediamine and 0.06% H₂O₂ in 50 mM citrate phosphate buffer, pH 5.0 and the reaction was stopped by adding 50 μl/well of 5 N H₂SO₄. The absorbance was read at 490 nm with 630 nm as the reference filter.

The purified recombinant human ZP1_273-551aa (1 mg) was dialyzed against 0.5 M carbonate buffer (pH 9.0) and incubated with FITC Isomer I (Pierce) at a molar ratio of 1:24 for 90 min at RT with end-to-end mixing. Post-incubation, unbound FITC was removed from the labelled recombinant protein by extensive dialysis against PBS pH 7.4. All the above treatments were performed under light-protected conditions. The fluorescein/recombinant protein molar ratio (F/P) was determined by spectrophotometric analysis and calculated using the following formula: Molar F/P = (Molecular weight of the protein/389) × [(A₄₉₅/195)/A₂₈₀-{(0.35 × A₄₉₅)}/E^0.1%]

where: 389 is the molecular weight of FITC in Daltons; 195 is the absorption E^0.1% of bound FITC at 495 nm at pH 13.0; (0.35 × A₄₉₅) is the correction factor due to the absorbance of FITC at 280 nm; E^0.1% is the absorption at 280 nm of the protein at 1.0 mg/ml

All experiments using human spermatozoa were carried out under informed consent and following the clearance from the Institutional Bio-safety and Human Ethical Committee. Semen samples were collected from healthy donors and subjected to liquefaction at RT for 30 min. The motile sperm were separated by a two-step Percoll density gradient [27] and processed for capacitation in Biggers-Whitten-Whittingham (BWW) medium [28] supplemented with 2.6% bovine serum albumin (BSA, cell culture grade; Sigma-Aldrich Inc) essentially as described previously [11]. To prepare acrosome-reacted sperm, capacitated sperm were incubated with calcium ionophore A23187 (CaI; 10 μM, Sigma-Aldrich Inc) for 20 min at 37°C and 5% CO₂ in humidified air. Non-capacitated, capacitated and acrosome-reacted spermatozoa (5 × 10⁶), pre-fixed for 10 min at RT with 0.5% paraformaldehyde, were incubated with 2.5 μg of FITC-labelled recombinant ZP1_273-551aa in a reaction volume of 50 μl at 37°C and 5% CO₂ in humidified air for 60 min. In all the experiments, Fetuin (Sigma Aldrich Inc.) labelled with FITC was used as negative control. In addition, FITC-labelled baculovirus-expressed recombinant human ZP1 and ZP3 [12, 16] were also used as positive controls. Post-incubation, sperm were processed for simultaneous assessment of the status of acrosome by double labelling with 5 μg/ml tetramethyl rhodamine isothiocyanate conjugated Pisum sativum agglutinin (TRITC-PSA; Vector Laboratories Inc., Burlingame, CA, USA) and for binding of FITC-labelled human ZP1_273-551aa as described earlier [12].

Capacitated sperm (1 × 10⁶ in BWW medium and 0.3% BSA) were incubated at 37°C with 5% CO₂ in humidified air for varying times and concentrations of recombinant human ZP1_273-551aa in a total reaction volume of 100 μl and processed as described earlier [29]. Sperm incubated with BWW supplemented with 0.3% BSA alone accounted for the spontaneous induction of acrosome reaction. Baculovirus-expressed recombinant human ZP1 and ZP3 [[11, 16]; 1 μg/reaction] served as positive controls, whereas Fetuin and baculovirus-expressed recombinant human ZP2 [12] served as negative controls in all the experiments. In addition, CaI (10 μM) was also used as positive control in all the experiments. The effect of treatment of various recombinant proteins on sperm with respect to viability by eosin-nigrosin staining [30] and total motility as per the WHO guidelines [31] were assessed. After incubation with various test proteins, sperm were washed twice with 50 mM PBS pH 7.4. Subsequently, sperm pellet was resuspended in 100 μl PBS and 15 μl aliquots were spotted on poly-lysine coated slides (Sigma-Aldrich Inc.) in duplicates, air dried and fixed in chilled methanol for 30 sec. The slides were washed once with PBS and stained with 5 μg/ml TRITC-PSA for 30 min at RT. Sperm showing rhodamine fluorescence in the acrosomal region of the head were classified as acrosome intact while those that demonstrated complete loss of PSA staining in the acrosome or revealed staining at the equatorial region were denoted as acrosome-reacted. All slides were read 'blind' with coded samples. Two hundred sperm were scored for every spot and results are represented as percent stimulation of acrosome reaction which is calculated in the following manner: % stimulation of acrosome reaction = [(AR - negative control)/(CaI - negative control)] × 100

where: AR is the percent induction of acrosome reaction by various inducers, negative control is the spontaneous acrosome reaction and CaI is the percent acrosome reaction by incubation with CaI.

Various pharmacological inhibitors at different concentrations, as described previously [16], were employed to delineate the downstream signalling mechanism of the acrosome reaction induced by recombinant human ZP1_273-551aa. Among these inhibitors are the extracellular calcium chelator: EGTA; L-Type voltage operated Ca²⁺ channel (VOCC) blockers: Nifedipine & Verapamil; T-Type VOCC blockers: Amiloride & Pimozide; inhibitor of Gi protein: Pertussis toxin (PTX); all of which were pre-incubated with the sperm for 10 min (except PTX, kept for 30 min incubation) at 37°C with 5% CO₂ in humidified air prior to the addition of ZP1_273-551aa. All the above inhibitors were procured from Sigma-Aldrich Inc.

In case of binding and induction of acrosome reaction experiments, the results are expressed as mean ± SEM of 3-4 different experiments using semen samples from 3 different male donors. The statistical analysis was done by comparing the means of the negative control (Fetuin) and experimental sets by using one way analysis of variance (ANOVA) followed by Newman-Keuls Multiple Comparison Test. The statistical analysis with respect to the effect of various pharmacological inhibitors on ZP1_273-551aa-mediated induction of acrosome reaction was performed by Student's t-test. A p value of ≤ 0.05 was considered to be statistically significant.

The nucleotide (nt) sequencing of the cDNA encoding ZP1_273-551aa, PCR amplified from human ovarian cDNA library, revealed three changes at 858, 867 and 1590 nt positions as compared to human ZP1 sequence already published in Genbank (NM_207341). However, these changes did not lead to any change in the deduced aa sequence. Recombinant human ZP1_273-551aa expressed in Sf 21 insect cells was present both in the cell lysate as well as the 10× concentrated culture supernatant (Figure 1, panel b and c respectively). The Sf 21 cells infected with wild type AcNPV, used as negative control, did not show any reactivity with antibodies against ZP1 peptide. The amount of secreted ZP1_273-551aa present in the culture medium was, however lower as compared to the cell lysate. SDS-PAGE profile of the recombinant human ZP1_273-551aa, purified using Ni-NTA affinity column from the cell lysate revealed a doublet with molecular weight ranging from ~35-40 kDa (Figure 1, panel d). The recombinant protein purified from the culture supernatant also showed the same profile in Western blot as observed for the cell derived protein (data not shown).

In lectin binding ELISA, baculovirus-expressed human ZP1_273-551aa exhibited strong reactivity with Concanavalin A (ConA) and weaker reactivity with Jacalin and Wheat germ agglutinin (WGA) (Figure 2). The E. coli-expressed recombinant human ZP1_273-551aa used as an internal control, failed to show any significant binding to any of the lectins. While ConA has oligosaccharide specificity towards mannose α 1-3 or mannose α 1-6 residues, Jacalin binds to α-O glycosides of Gal or GalNac moieties. WGA has oligosaccharide specificity towards GlcNac and neuraminic acid residues. While ConA and WGA have oligosaccharide specificity towards N-linked sugar residues, Jacalin detects the presence of O-linked carbohydrate moieties.

Recombinant ZP1_273-551aa conjugated to FITC as described in Methods revealed molar F/P ratios of 1.1. The FITC-labelled recombinant protein was analyzed for binding to spermatozoa and the acrosomal status of the spermatozoa was simultaneously assessed by double labelling with TRITC-PSA. FITC-labelled recombinant human ZP1_273-551aa showed very low binding (4.01 ± 1.23%) to non-capacitated human sperm. Hence, all further studies were done using capacitated spermatozoa. The baculovirus-expressed recombinant human ZP1_273-551aa showed binding to 19.67 ± 2.40% of acrosome-intact and 15.62 ± 1.64% acrosome-reacted sperm (Table 1). Under similar experimental conditions, FITC-conjugated Fetuin, used as a negative control, bound to 3.95 ± 2.12% of acrosome-intact sperm while the positive controls, FITC-labelled baculovirus-expressed ZP3 and full length ZP1 bound to 26.08 ± 2.75% and 15.63 ± 1.88% of the acrosome-intact spermatozoa, respectively (Table 1). Binding analysis with acrosome-reacted sperm revealed that 22.65 ± 2.15% sperm bound to ZP3 and 14.40 ± 1.09% showed binding with full length ZP1 (Table 1). Careful examination of the profile of binding revealed that approximately 70% sperm showed binding of ZP1_273-551aa to the acrosomal cap (Figure 3A) and approximately 30% in the equatorial region of the acrosome-intact human sperm (Figure 3B). In acrosome-reacted spermatozoa, binding of ZP1_273-551aa to the acrosomal cap was lost and observed only in the equatorial region (Figure 3C). Further, binding of FITC-labelled baculovirus-expressed ZP3 and full length ZP1 was also observed only in the equatorial segment of the acrosome-reacted spermatozoa [12, 16]. In addition, few spermatozoa also showed binding of FITC-labelled recombinant ZP1_273-551aa to either post acrosomal region (Figure 3B) or mid-piece of acrosome-intact and acrosome-reacted spermatozoa (data not shown).

Recombinant human ZP1_273-551aa was evaluated for its ability to induce acrosomal exocytosis in capacitated human spermatozoa. No significant decrease in the viability and total motility of the sperm were observed following incubation with the recombinant protein. Capacitated sperm incubated with purified recombinant ZP1_273-551aa showed a significant (p < 0.05) increase in acrosomal exocytosis as compared to the medium control (Table 2). Baculovirus-expressed recombinant human ZP3, used as a positive control, showed a significant increase in acrosome reaction as compared to Fetuin whereas baculovirus-expressed recombinant human ZP2 [12] failed to do so (Table 2). Dose response studies with baculovirus-expressed human ZP1_273-551aa revealed a significant increase in the acrosome reaction at 500 ng/ml. The highest acrosome reaction was observed at 2 μg/ml and hence all subsequent experiments were performed using this amount of the recombinant protein. Recombinant ZP1_273-551aa purified from culture supernatant also exhibited dose dependent induction of acrosomal exocytosis comparable to cell purified recombinant protein (Data not shown). Time kinetic studies revealed that a significant increase in the induction of acrosome reaction could be seen as early as 10 min after exposure of the capacitated sperm to the recombinant ZP1_273-551aa (Figure 4). Highest level of induction of acrosome reaction was observed at 60 min.

To elucidate the downstream signalling pathway involved in acrosomal exocytosis mediated by the 'ZP domain' of ZP1, different pharmacological inhibitors were employed. The average viability in absence of pharmacological inhibitors of the capacitated sperm ranged from 85-90%. No significant change in the sperm viability was observed subsequent to incubation with various pharmacological inhibitors. No significant decrease in ZP1_273-551aa mediated induction of acrosome reaction was observed (p > 0.05) after incubation with Pertussis toxin (0.1 μg/ml; 30 min pre-incubation) pre-treated capacitated human spermatozoa (Figure 5) suggesting that activation of Gi-protein coupled receptor is not necessary for 'ZP domain' of ZP1 mediated acrosomal exocytosis. Induction of acrosomal exocytosis mediated by ZP1_273-551aa required extracellular Ca²⁺ as prior incubation of capacitated sperm in BWW medium with EGTA before exposing to recombinant protein led to a significant decrease in acrosomal exocytosis (p < 0.01, Figure 5). Both L- and T- type Ca²⁺ channels seem to play a role in ZP1_273-551aa mediated induction of acrosome reaction as inhibitors for both types of channels resulted in a significant decrease in acrosomal exocytosis (Figure 6).