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Research Article |
Center for Research on Reproduction and Women's Health,4 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6080
Departamento de Biología de la Reproducción,5 Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City 14000, Mexico
ABSTRACT
The initial interaction between gametes takes place at the level of the sperm surface and the zona pellucida (ZP), the extracellular matrix of the egg in mammals. Successful fertilization requires the proper molecular recognition of the ZP by the sperm. Recently, human ZP was demonstrated to be composed of four proteins: ZP1, ZP2, ZP3, and ZP4. The goals of this study were to determine the effects of recombinant human ZP2, ZP3, and ZP4 on human sperm acrosomal exocytosis and sperm motility. Exposure of sperm to ZP proteins, alone or in combination, promoted acrosomal exocytosis in a time-dependent manner. This effect occurred in parallel with a considerable decrease in progressive motility, coincident with an increase in nonprogressive sperm motility. An analysis of kinetic parameters of ZP-treated sperm demonstrated that a characteristic motility pattern could be defined by values of curvilinear velocity > 63.9 µm/s and linearity
15.5%. A strong correlation between curvilinear velocity and the amplitude of lateral head displacement was also observed. The incidence of sperm having these particular kinetic parameters increased after exposure to ZP proteins. These studies of two processes involved in sperm penetration through the ZP confirm that zona glycoproteins promote acrosomal exocytosis and now establish an additional role for these components as modifiers of sperm motility.
acrosome reaction, acrosomal exocytosis, fertilization, gamete biology, sperm capacitation, sperm motility and transport, zona pellucida
INTRODUCTION
During mammalian fertilization, but before reaching the egg plasma membrane, sperm bind to the zona pellucida (ZP), the extracellular matrix that surrounds the oocyte. The human ZP is composed of four proteins, ZP1, ZP2, ZP3, and ZP4 (ZP4 previously known as ZBP, ZP1, and ZPB) [1–5]. The initial interaction with ZP macromolecules triggers several molecular processes that stimulate sperm acrosomal exocytosis (AE), which enables the sperm to penetrate the ZP in a process that also requires sperm motility. Subsequently, the sperm then interacts and fuses with the egg plasma membrane to form the zygote.
Acrosomal exocytosis is essential for successful fertilization, and the ZP has been classically considered as the primary initiator in vivo. Nevertheless, acrosomal exocytosis can also be stimulated by other factors, such as calcium ionophore or progesterone, although the mechanisms for induction differ from those used by the ZP [6–8]. Progesterone appears to exert a priming effect on AE. When sperm are exposed first to progesterone and then to the ZP, exocytosis is enhanced to a greater degree than that seen when these agonists are presented together or in the inverse order [9]. In addition, the ZP may stimulate the completion of an exocytotic process that had already been initiated during capacitation [10, 11]. Other studies indicate that acrosomeless, round-headed human sperm do not bind to and penetrate the ZP, nor do they fuse with the oolemma of ZP-free oocytes in vitro [12]. However, some studies have demonstrated that normal rabbit and guinea pig as well as human spermatozoa are capable of adhesion to the ZP in both acrosome-intact and acrosome-reacted states [13–15]. In human and mouse sperm, internal ZP binding proteins of the acrosomal matrix or inner acrosomal membrane may be exposed after acrosomal exocytosis that allow a continuous but dynamic attachment between sperm and ZP as the male gamete traverses this extracellular matrix [16, 17].
Sperm motility also plays an essential role in events leading up to fertilization. Furthermore, the patterns of motility change according to the maturation stage of the cell. During capacitation, some sperm undergo hyperactivation, which is characterized by the transition from a moderately active, progressive (relatively linear) swimming pattern to a highly vigorous, nonprogressive, random motion, with high-curvature flagellar movements [18, 19]. Although the characterization and identification of human sperm hyperactivation has been a subject of controversy, this distinctive motion seems to parallel the capacitation process [20]. Several studies have correlated hyperactivation with the occurrence of acrosomal exocytosis, ZP binding and penetration, and in vitro fertilization success, suggesting that it could be a good marker for capacitation and a valuable tool for clinical assessment of fertility [21–24].
There are many studies related to changes in sperm function after incubation with many different reproductive factors; however, studies on human ZP-sperm interaction are severely restricted because of difficulties in obtaining human oocytes as the source of native ZP. To circumvent this limitation, several research groups have recently cloned and expressed recombinant human ZP proteins with the goal of elucidating the ZP-sperm interaction mechanisms. In this regard, recombinant human ZP3 expressed in hamster CHO cells has been shown to induce AE and to promote sperm fusion with zona-free hamster oocytes [25–27]. Similar results have been obtained with ZP4 (called ZPB by these authors) and ZP3 expressed in insect Sf21 cells [28]. Nevertheless, the utility of each cell expression system needs to be investigated further, particularly because hZP3 expressed in human 293T cells does not have any effect on AE [29].
In this study, we have used recombinant proteins expressed in Sf9 insect cells to investigate the effects of ZP2, ZP3, and ZP4 on human sperm function. Because fertilization entails a complex series of events culminating in early embryo development, it is unlikely that the evaluation of a single sperm feature or function can be predictive of the fertilization potential of the male gamete. Therefore, we evaluated some of the most remarkable variables, such as stimulation of AE and changes in sperm motility following exposure to ZP proteins. Our results demonstrate that these ZP glycoproteins are physiologically active in promoting AE and in inducing changes in motility patterns, suggesting that these recombinant proteins could be useful to study the ability of sperm to bind and penetrate ZP.
MATERIALS AND METHODS
Expression of ZP2, ZP3 and ZP4 in Sf9 Cells and Purification
Human ZP2, ZP3, and ZP4 proteins were expressed in the Spodoptera frugiperda Sf9 insect ovary cell line using the baculovirus expression system as previously described [30]. Briefly, the cDNAs coding for ZP2, ZP3, and ZP4 were cloned into the pAcHLT transfer vector (Pharmingen), and recombinant vectors were cotransfected with a linear AcNPV baculovirus into Sf9 cells using the Baculogold Transfection kit (Pharmingen) according to the manufacturer's guidelines. The resulting recombinant viruses were used for high-scale cell infection, culturing in TNM-FH medium (Invitrogen) for 3–4 days before harvesting. Although engineered to contain a signal sequence to enable the recombinant proteins to enter the secretory pathway for glycosylation, the ZP proteins, when expressed in this cell line, were not released into the culture medium, so cell pellets were analyzed by immunoblotting, as described below, to determine the presence of recombinant proteins.
The recombinant human ZP proteins were purified because of the presence of a 6-histidine tag encoded by the pAcHLT transfer vector. Purification started by solubilizing cell pellets under denaturing conditions in 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5 mM imidazole, and 6M guanidine hydrochloride at room temperature and centrifuging at 10000 x g for 30 min. The supernatant was added to pre-equilibrated Ni-NTA resin (Qiagen), which was incubated for 2 h and then washed with 5 volumes of the same buffer. The resin was suspended in 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5 mM imidazole, and 6M urea, and the bound proteins were refolded using a decreasing gradient of urea. The proteins were eluted with 500 mM imidazole and dialyzed against 20 mM Tris-HCl, pH 8.0, and 0.5 M NaCl, and the protein concentration was determined using the bicinchoninic acid assay (BCA; Pierce).
Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Analysis
The purities and molecular weights of the purified recombinant human ZP2, ZP3 and ZP4 proteins were determined by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) [31]. Briefly, 2 µl of each ZP protein was applied to a WCX2 protein chip array, which was then incubated at room temperature for 1 h. Following washes with 50 mM sodium acetate buffer (pH 4.0) and a final wash with water, the array was allowed to dry, and a saturated solution of sinapinic acid in 50% (v/v) acetonitrile and 0.5% (v/v) trifluoroacetic acid was added to each spot and dried. The protein chip array was analyzed using a SELDI ProteinChip Biomarker System (PBS-II; Ciphergen Biosystems). Spectra were collected by the accumulation of 192 shots at a laser intensity of 220 in a positive mode. The protein masses were calibrated externally using protein molecular mass standards (Ciphergen Biosystems).
Immunoblot Analysis
To confirm the presence and integrity of recombinant human ZP proteins in Sf9 infected cells before and after purification, the proteins were analyzed by immunoblotting. The cellular pellets were used for protein separation by SDS-PAGE on a 10% polyacrylamide gel [32] and electrotransferred to a polyvinyldifluoride membrane (PVDF; Millipore) [33]. After the membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBST), the recombinant human ZP proteins were probed with a 1:1000 dilution in TBST of rabbit anti-heat-solubilized pig ZP serum that cross-reacts with human ZP, and antibody binding was detected using 125I-Protein A [34].
Semen Samples and Sperm Preparation
This study and the informed consent forms signed by the semen donors were approved by the Institutional Review Board of the University of Pennsylvania. Following 2–4 days of abstinence, healthy donors (26–40 yrs) with proven fertility provided freshly ejaculated semen. The samples, collected in sterile polyethylene cups, were allowed to liquefy for at least 30 min and were assessed according to procedures and criteria established by the World Health Organization [35]. For capacitation, liquefied semen samples were centrifuged at 600 x g for 30 min through a discontinuous Percoll density gradient (Isolate: Irvine Scientific). The sperm pellets were washed with 3 ml of Sperm Washing Medium (SWM; Irvine Scientific) and suspended in equal volumes of SWM and Test Yolk Buffer (Irvine Scientific) for overnight incubation at 4°C [36]. After washing with SWM, pellets were suspended with 0.1 ml of supplemented mHTF medium [modified Human Tubal Fluid (Irvine Scientific) + 0.3% human serum albumin + 1 mM sodium pyruvate], and gently overlaid with 0.9 ml of the same medium. Following a 1-h incubation at 37°C in 5% CO2 to enable the sperm to swim up into the supernatant, the uppermost 0.9 ml with the motile sperm was carefully aspirated and reevaluated. The sperm concentration was then adjusted to 15 million per ml for subsequent experiments. In the recovered fractions, motility values were in all cases above 80%.
Sperm Function Analysis
Capacitated human sperm samples were incubated with the ZP proteins to assess effects on acrosomal exocytosis and sperm motility. After capacitation, 750000 sperm were incubated with the different ZP proteins at a final concentration of 1 µg/ml diluted in 50 µl of supplemented mHTF medium at 37°C. At the end of the incubation time, half of each sample was washed and fixed with 95% ethanol for evaluation of acrosomal status, and the other half was used to determine whether the ZP protein(s) influenced sperm motility.
Evaluation of Acrosomal Status
Duplicate smears of fixed sperm aliquots were prepared on poly-L-lysine-treated slides and air-dried. Cells were stained with 20 µg/ml of Pisum sativum agglutinin conjugated to fluorescein (FITC-PSA; Sigma) diluted in PBS and incubated for 30 min at room temperature [37]. Slides were washed three times with PBS, mounted in glycerol-based solution (VectaShield; Vector Laboratories), and scored immediately by double-blinded evaluation. At least 200 cells were evaluated for each duplicate under a fluorescence microscope (Nikon Eclipse TE2000-U; Nikon Inc.). All sperm showing acrosomal region fluorescence were considered as acrosome-intact, whereas those showing absence of fluorescence in the acrosome as well as staining at the equatorial region or patchy staining over the acrosome region were considered acrosome-reacted [38, 39]. Values of ZP-related acrosomal exocytosis were normalized by spontaneous acrosomal exocytosis observed in the negative control (sperm incubated in supplemented mHTF) under similar conditions. Sperm incubated with 10 µM calcium ionophore A23187 (Sigma) were used as positive control.
Evaluation of Sperm Motility Changes after ZP Treatment
The motility patterns of capacitated sperm were examined after ZP treatments with a double approach: 1) manual assessment (World Health Organization progression evaluation system) and 2) computer-aided sperm analysis (CASA) using an IVOS system (version 12, Hamilton-Thorne Biosciences). For manual motility measurements, 200 sperm were evaluated at each incubation time by phase contrast microscopy (Nikon Optiphot) [40, 41]. The movement of every encountered sperm was graded as a, b, c, or d, as follows: a) rapid progressive motility, b) slow or sluggish progressive motility, c) nonprogressive motility, or d) immotility [35]. The percentage of progressive motility was defined as the sum of a + b divided by the sum of the total. As a positive control, sperm were incubated with 1 mg/ml pentoxifylline [42]. For kinetic parameter evaluation, 5-µl aliquots of treated or control sperm samples were placed on a 20-µm-depth standard count analysis chamber (Leja Products), preheated at 37°C, and analyzed using the CASA system with settings according to the manufacturer's instructions. For every sample, approximately 10 randomly selected fields were evaluated, for a minimum total of 300 sperm. Average values were obtained for sperm motion parameters such as curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), amplitude of lateral displacement (ALH), linearity (LIN = VSL/VCL), and straightness (STR = VSL/VAP). Progressive motile sperm were defined by values of VAP > 20 µm/sec and STR > 80%. Rapid or slow motile sperm cells were classified according to the CASA standard cutoff values for VAP or VSL. Slow cells were defined as cells with either of the following attributes: VAP < 20 µm/sec or VSL < 30 µm/sec. For analysis and characterization of ZP motility pattern, individual sperm track trajectories generated by CASA were identified by using a semiobjective approach [20]. Stored CASA files were played back, and sperm tracks showing a characteristic pattern of motility marked by side-to-side head movements and rapid spinning in place with frequent short translational movements (biphasic pattern) were considered for further analysis and classification modeling [43].
Statistical Analysis and Modeling
Statistical analyses were carried out using the Graph Pad Statistical Package. The results are presented as the mean ± SE and were analyzed by ANOVA and Tukey HSD test for post hoc comparisons. When appropriate, P values < 0.05 were considered significant.
The motility patterns of sperm stimulated by ZP proteins were characterized by the Classification and Regression Trees software system (CART, version 4.0; Salford Systems) as previously described [43]. Technically, the CART algorithm constructs a binary decision tree wherein the relative importance of a variable/parameter is measured by the order in which it was selected in the decision tree and the number of correct predictions for which it is credited. The program constructed classification trees using the training set, and after V-fold cross-validation, the accuracy of each classification tree was then challenged with the test set. For our purpose, a multiparametric approach based on CASA analysis of two groups was considered: ZP-treated sperm tracks (40 sperm tracks from ZP incubations; CART group 1) vs. controls (43 sperm tracks from control mHTF medium incubations; CART group 0). CART estimated the threshold for each sperm kinetic parameters and the pattern combination among them that best defined the ZP-stimulated motility status. Multiple classification trees were generated using this process, and the best-performing tree was chosen for additional testing. Finally, we used the resulting variables thresholds to define the ZP proteins stimulated motility status, determining sensitivity and specificity.
RESULTS
Recombinant ZP Proteins Can Be Prepared in Sf9 Insect Cells
The full-length cDNA's encoding for human ZP2, ZP3, and ZP4 were cloned into pAcHLT vectors with the purpose of expressing these proteins as polyhistidine-tagged fusion proteins in Sf9 insect cells, as has been successfully done by others [44, 45]. The recombinant hZP proteins were purified from harvested cells by Ni-NTA affinity chromatography under denaturing conditions (Fig. 1). Purified hZPs were characterized by SELDI-TOF-MS. The purified preparations were predominantly comprised of proteins with molecular masses of 78051 for ZP2, 67239 and 57207 for ZP3, and 66795 for ZP4, and major contaminants were absent (Fig. 1A). These values approximated the values expected for the polypeptides with some attached oligosaccharide moieties (the theoretical masses for the polypeptide moieties of these proteins are ZP2, 78200; ZP3, 47000; and ZP4, 59400). The purified ZP proteins were also evaluated on immunoblots, probed with antibodies directed against native pig ZP (Fig. 1B); this antibody was previously shown to recognize native human ZP by immunohistochemistry [30]. The major constituents of the purified protein preparations were recognized by the antibody and possessed molecular weights similar to those observed by SELDI-TOF-MS. The expression of ZP3 as two molecular forms in this cell line has been previously described [30] and could be the result of differential glycosylation, as has been demonstrated for rabbit ZP1 expressed in Sf9 cells [44]. Although these results do not demonstrate that the recombinant proteins are identical to native human proteins (nor did we expect them to be, because the proteins were expressed in a heterologous system), the recombinant ZP proteins shared important properties in common with the native molecules (molecular mass and immunological reactivity).
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Recombinant Human ZP Proteins Influence Acrosomal Exocytosis and Sperm Motility
To examine the biological activities of the ZP proteins, capacitated human sperm were incubated at different intervals with purified ZP2, ZP3, and ZP4 at a final concentration of 1 µg/ml, and the effects on AE and motility were evaluated. The ZP effects on human sperm function in this study were determined primarily with a concentration of 1 µg/ml. Experiments using 5 µg/ml were also carried out, finding a similar effect with slightly higher values of AE. However, because the effects on AE were similar in both cases, the lower concentration (1 µg/ml) was considered sufficient to fulfill the purpose of the analysis, which is at the lower range (1–100 µg/ml) of baculovirus-expressed human ZPB and ZPC used to induce the acrosome reaction in capacitated human spermatozoa [28].
Incubation of sperm in the presence of ZP3 and ZP4 resulted in significant increases in the numbers of sperm undergoing AE, whereas no significant changes were observed with ZP2 (Fig. 2A). AE induction was detectable as early as 10 min, and the velocity of AE stimulation was higher at shorter incubation times, although the stimulation of AE by either ZP3 or ZP4 was statistically significant only at 20 min of incubation. Combining ZP3 with ZP4 (0.5 µg/ml each for a final concentration of 1 µg/ml ZP protein) resulted in an effect on sperm AE similar to that observed with individual proteins. Comparable results were obtained when sperm were incubated with ZP proteins in the presence of different protein concentrations (data not shown). In all cases, calcium ionophore was used as a positive control for the promotion of AE.
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Besides the stimulation of AE by ZP3 and ZP4, changes in the patterns of sperm motility occurred in sperm treated with the three recombinant glycoproteins. Individually, ZP2, ZP3, and ZP4 were able to cause a significant decrease in the proportion of progressively motile sperm while increasing the relative number of nonprogressively motile sperm compared to the negative control (Fig. 2, B and C). The effect was more pronounced when the sperm were incubated with ZP3 or ZP4 compared to ZP2. The induced AE and motility changes showed no significant differences between 20 and 120 min, indicating that the effect of these proteins on these two sperm parameters was completed by 20 min. Sperm incubations with ZP2, ZP3, and ZP4 were also performed for longer incubation times (24 h) but showed no further increase in AE. Total motility decreased, but the percentages of sperm with progressive and nonprogressive motility remained constant beyond 120 min incubation (data not shown).
Combinations of ZP Have an Additive Effect on Sperm Motility
Because the ZP is composed of multiple proteins, we hypothesized that the treatment of sperm with combinations of recombinant ZP proteins may have additive or synergistic effects. When sperm motility analysis was assessed after 20 min incubation with ZP2, ZP3, or ZP4, alone or in combination, the sperm treated with a protein mix displayed a considerable attenuation in the number of sperm with progressive motility and an increase in the number of sperm with nonprogressive motility (Fig. 3), as previously seen with individual ZP proteins. However, the combination of ZP proteins did not elicit a synergistic effect on sperm motility when compared to the ZP individual treatments. After statistical analysis, the percentage of motile cells remained stable for all treatments including the negative control, suggesting that ZP proteins influence motility patterns without promoting immotility. Treatment of sperm with pentoxifylline (PTX) as a positive control for the stimulation of sperm motility augmented the percentage of immotile cells in the samples (P < 0.05).
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Recombinant Human ZP Influences the Quality of Sperm Motility
From our manual assessment of motility, we observed that, besides the increase in nonprogressive motility after ZP treatment, there was a higher incidence of a distinct type of movement (Fig. 4A). To perform an objective evaluation, CASA was performed to delineate motility pattern changes after sperm incubation with the ZP proteins. A preliminary study was carried out considering the kinetic parameters to classify sperm movement into different types: progressive motility and rapid or slow motility, according to CASA settings. Data from the initial analysis confirmed the results obtained by our manual assessments of motility, in which ZP treatments promoted an increase in slow motility and decreases in progressive and rapid motility. Nevertheless, no significant differences were detected in the average kinetic parameters among sperm samples incubated under control conditions or with the various experimental treatments (data not shown).
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To perform a more extensive analysis of sperm motility following ZP treatment, individual tracks of ZP-treated and control sperm were selected and compared by CASA. After plotting the kinetic parameter values of individual sperm tracks in many ways, we found that the parameters of VCL, ALH, and LIN could differentiate the sperm tracks into two motility subpopulations (Fig. 4, B–D). When the previously established thresholds for hyperactivation [20]—LIN 65, ALH 
7.5, VCL 100—were applied to the subpopulation of the nonprogressive sperm treated with ZP, these cells did not fit a strict definition of hyperactivation, but did show a hyperactivated-like swimming pattern, which we term ZP-influenced motility. To perform an initial characterization of this pattern, sperm were classified into ZP-influenced or non-ZP-influenced motility groups by manual evaluation of sperm tracks registered by CASA. To refine the differentiation of ZP-influenced motility from the other patterns, we analyzed the CASA-derived values by a multiparametric classification and regression tree analysis with the CART computer program, which yielded cutoff values that were able to split the two groups. Figure 4D shows the combined CASA/CART analysis illustrating the two most suitable parameters to describe the ZP-influenced motility pattern, which was delineated by a sector bounded by VCL values greater than 63.9 µm/ml and LIN scores less than 15%. Attempts to include ALH as a value to define ZP-influenced motility (as a motion pattern defined by three parameters) made the sort too restrictive, so that many sperm that visually could have been defined as ZP-influenced motility sperm would have been rejected. The sector in Figure 4D is reasonably pure; most of the sperm with ZP-influenced motility were correctly classified, and the sector contained few dots corresponding to control tracks (sensitivity of 97.5% and specificity of 92.5%). In addition, the two-dimensional scatterplot of Fig. 4B revealed that VCL and ALH were proportional and showed similar values for control and ZP-influenced sperm, so these two variables together are not useful to characterize the observed motility. ALH and LIN values could also be used to define sperm exhibiting the ZP-influenced motility pattern (Fig. 4C), but with a lower sensitivity and specificity than that observed with VCL and LIN values.
In a different set of experiments, the previously established multiparametric CASA/CART definition (Fig. 4D) was used to sort sperm samples from different donors treated with the ZP proteins for 20 min. Figure 5 shows the occurrence of sperm meeting this definition, as a subset of the slow motility sperm detected by CASA (VSL < 30 µm/sec or VAP < 20 µm/sec). Sperm with ZP-influenced motility were considered slow sperm because they covered a very short distance (low VSL) as a consequence of the nonlinear swimming pattern (low LIN). Total sperm with slow motility and the subset sorted for ZP-influenced motility are presented as the percentage of total motile sperm meeting the appropriate criteria. In five experiments from three different donors, the relative frequency of motile cells identified as slow by CASA was similar to the frequency of nonprogressive sperm obtained by manual assessment (Fig. 3). However, the percentage of sperm with the ZP-influenced motility pattern increased for sperm treated with ZP2, ZP4/ZP3 mixture, ZP4/ZP2 mixture, or ZP2/ZP3 mixture, but not for sperm exposed to ZP4 alone, ZP3 alone, or ZP4/ZP2/ZP3 mixture.
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DISCUSSION
The composition of the human ZP and functions of its component proteins are not well known at present because of the limited availability of native human ZP. Recent advances in molecular biology provide new tools and approaches to address these issues. For example, the Human Genome Project has led to the finding that the human ZP is composed of four proteins instead of the three originally described [46–48]. For clarity and consistency in this report, we have adapted the ZP1, ZP2, ZP3, and ZP4 nomenclature recently approved by the Human Genome Organization [1–5]. However, when this study was initiated, only three human zona proteins were known and cloned, so our work presented here has addressed only the functions of ZP2, ZP3, and ZP4 (previously called ZP1 or ZPB by other investigators) [46, 49]. The absence of ZP1 in this study is probably not a critical issue because it is not very abundant in mammalian ZP [48]. Besides, ZP1 in the mouse zona is thought to function as a scaffold for the assembly of ZP2 and ZP3 into a complex macromolecular extracellular matrix [50]. Our experiments reported here used soluble, recombinant proteins.
In this study, we have demonstrated that recombinant human ZP2, ZP3, and ZP4 expressed in Sf9 insect cells are biologically active toward capacitated human sperm. Native ZP proteins are highly glycosylated, and research in various species, including human, support the concept that specific interactions between sperm and ZP are carbohydrate-mediated events. However, the state of glycosylation of the recombinant proteins expressed in insect cells is under investigation. When zona proteins are expressed in heterologous systems, varying functional results have been found. Mouse but not human sperm bind to oocytes from transgenic mice that express human ZP2 and ZP3 in place of the native murine homologues [51, 52]. These findings suggest that the species-specific recognition of the ZP is mediated, in part, by the mouse-specific glycosylation patterns regardless of the amino acid sequence. On the other hand, the polypeptide backbone could be sufficient for sperm recognition in some mammals, because nonhuman primate sperm are able to bind to homologous ZP proteins expressed in Escherichia coli, a species incapable of synthesizing glycoproteins [53]. In our studies, the ZP2, ZP3, and ZP4 expressed in Sf9 insect cells were recognizable by a heterologous antibody and were larger than the sizes predicted from the original cDNA sequences [30]. This suggests that these proteins undergo posttranslational modifications during expression, such as glycosylation. However, the molecular weights of the recombinant proteins are different from the native proteins, indicating that the type and/or extent of glycosylation produced in our expression system differ from those observed in vivo.
The recombinant proteins promoted AE and induced changes in sperm motility. In our study, only ZP3 and ZP4 could stimulate AE. These results are similar to those recently published with recombinant hZP proteins expressed in Sf21 insect cells [28]. The extent of AE induction by ZP3 and ZP4 is notable, considering that values obtained using solubilized native ZP are about 20% [38, 54]. The stimulation of AE by ZP3 expressed in mammalian cell systems has been well documented, and this ZP component has been proposed as the primary ligand involved in the recognition of ZP by sperm [25]. However, these findings should be reexamined, given the recent demonstrations by Chakravarty et al. [28] and our findings reported here that another ZP glycoprotein, ZP4, stimulates AE. Indeed, the ZPB proteins from rabbit, pig, and primates (bonnet monkey) bind to homologous sperm [55–57], and there is evidence suggesting that Sf21-expressed ZP4 (hZPB) acts on AE via a Gi-independent receptor of the sperm different from that employed by ZP3 [28]. Initial studies in the porcine system suggest that ZPB (ZP4) has sperm ligand activity [58]; however, later studies from the same group found that hetero-oligomers of ZPC-ZPB (i.e., ZP3-ZP4 in the current terminology) are responsible for binding to boar sperm-associated zona receptors [56]. Whether our finding of the ZP4 effect on AE results from specific posttranslational modifications produced in the Sf9 insect cell line, such as variations in glycosylation patterns, or is endogenous to the nature of hZP4 requires further investigation.
The nature of capacitation and its relationship to important events of fertilization are unclear. Spontaneous acrosomal exocytosis can be viewed as a true physiological event that occurs more readily during the course or as a consequence of capacitation. During fertilization, AE can then be seen as a process initiated but progressing at a very slow rate during capacitation. Upon interaction with the ZP, the rate of fusion between the sperm plasma membrane and the outer acrosomal membrane would be accelerated, leading to the completion of this exocytotic process [10]. The stimulation of AE by ZP could promote the appropriate timing of events during fertilization. Besides the action of ZP on AE, the expressed proteins were also able to induce motility changes in the sperm. The effects we have observed here are original in nature; as far as we know, there are no previous studies concerning the effects of recombinant or native human ZP proteins on sperm motility and kinematics. This could be an important finding because it may indicate something new about how sperm penetrate the zona. We also found that the CASA analyses of the percentage of progressive and slow sperm after treatment were similar to those obtained by manual assessment; these findings echoed previous studies, thereby validating our approach [40, 41].
Most sperm that have been recovered from the ampullae of naturally mated species are hyperactivated [59], suggesting that hyperactivated motility enables or prepares sperm for penetration through the cumulus mass [23, 60] and/or the ZP [20, 61]. In the present study, we have demonstrated that ZP proteins promoted a hyperactivated-like motility state, with kinetic parameters different from those used to classify hyperactivation according to criteria defined by Burkman [20]. The hyperactivated-like movements defined by CART suggested that kinetic parameters might differentiate the tracks of a population of ZP-treated sperm from other types of motility. We have found that the combination of VCL, ALH, and LIN could identify a subpopulation of sperm with a characteristic ZP-influenced motility. By CASA/CART analysis, the VCL and LIN values together could define this population with high sensitivity and specificity. ALH and LIN values could be also used to distinguish this group, but sensitivity and specificity are lower. Although the kinetic parameters of these tracks do not qualify the sperm to be classified as hyperactivated, the LIN and VCL values obtained were similar to those suggested by Burkman [20] and Robertson et al. [62] to define the "star-spin" kind of hyperactivated sperm motion. After sorting ZP-treated sperm using the cutoff values obtained by CART, sperm treated with ZP2, ZP3 plus ZP4, ZP2 plus ZP4, or ZP2 plus ZP3 displayed a higher incidence of hyperactivated-like motility. However, further investigation is required to determine whether this type of motility is truly different from hyperactivation or whether the kinetic cutoff values for the hyperactivation classification should be reevaluated to incorporate the ZP-treated sperm with the hyperactivated-like motility into this class.
Although AE acceleration and motility changes induced by ZP proteins may occur in parallel, they are not necessarily mediated by the same mechanism. For instance, sperm incubation with pertussis toxin does not affect sperm motility or binding to ZP, but it inhibits the ZP-induced AE [54]. Because the ZP ligands are not the same for each case, the sperm receptor mediating these events could be different. However, in both cases, soluble recombinant ZP proteins induce their effects very quickly, usually in a few minutes. It is possible that these proteins act in a redundant way, in view of the high homology between ZP proteins of the same species and the differences in glycosylation patterns obtained in each expression system. This observation may explain why porcine ZP proteins expressed in Sf9 cells are able to bind bovine sperm but not to boar sperm [63].
In contrast to that of many animal species, human sperm morphology is quite heterogeneous; in fact, both fertile and infertile men produce a high number of morphologically abnormal sperm [64]. In addition, sperm cells at various sites in the female reproductive tract differ in terms of their states of capacitation and patterns of motility. This observation agrees with the acrosomal exocytosis model, suggesting that, in the process leading from acrosome-intact to acrosome-reacted sperm, the existence of continuously variable intermediates as part of the acrosomal exocytosis occurs normally during the course of capacitation [11]. This concept could be valid not only for AE but also for other functional alterations happening during capacitation, such as the changes in the patterns of sperm motility.
Infertility has an important public health impact because it affects about 15% of couples of reproductive age worldwide, and the male factor accounts for up to 50% of all cases [65, 66]. In conventional in vitro fertilization, most of the conception failures attributable to sperm are derived from alterations in ZP interaction and penetration [67, 68], including disorders in ZP-induced AE [69]. To develop assays to study sperm-ZP interactions, it is necessary to have recombinant ZP proteins with biological activity. Our data demonstrated that the recombinant glycoproteins used in this investigation have physiological activity in terms of promoting AE and modifying sperm motility patterns, the two phenomena that allow sperm to penetrate the ZP and reach the oocyte. In the future, the use of recombinant human ZP1 for sperm function studies should also be considered because it may yield important information regarding ZP structure-function relationships and their roles in fertilization. The recombinant human ZP2, ZP3, and ZP4 expressed in insect Sf9 cells have biological activity, so they can be used to analyze the complex mechanism involving sperm-ZP relationships, including acrosomal exocytosis or ZP penetration under normal and abnormal conditions.
ACKNOWLEDGMENTS
The authors want to acknowledge Dr. Bayard Storey for the critical revision of this manuscript and Dr. Mariano Buffone for statistical assistance. Also, we thank all sperm donors.
FOOTNOTES
1 Supported by Contraceptive Research and Development (CONRAD) Twinning Program grants MFG-02–62 and MFG-02–63, NIH HD-41552, and the National Council of Science and Technology (CONACyT, Mexico). Also supported in part by the Bill and Melinda Gates Foundation (to X.J.F.). P.C.C. is a recipient of a training fellowship from Fundación Salud 2000 and Fundación Tambre, Madrid, Spain. M.E.G.G. and M.G.C. have been supported by CONACyT, Mexico. 
2 Correspondence: George L. Gerton, Center for Research on Reproduction and Women's Health, Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, 421 Curie Blvd., 1311 BRB II/III, Philadelphia, PA 19104-6080. FAX: 215 573 7627; gerton{at}mail.med.upenn.edu
3 These authors contributed equally to this work.
Received: 16 September 2005.
First decision: 7 October 2005.
Accepted: 4 January 2006.
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% AE) is plotted as a time function. AE after ZP4, ZP3, and calcium ionophore A23187 (CaI) treatment were higher than controls at 20 and 120 min time points (P < 0.05). B and C) Percentages of progressive (B) and nonprogressive (C) motility sperm after treatment with ZP2, ZP3 and ZP4. Results were normalized by total motile sperm. Progressive and nonprogressive motility were determined following World Health Organizations criteria (see Materials and Methods for details). Values in percentages are presented as the mean ± SEM. Motility after ZP2, ZP3, ZP4, and pentoxifylline (PTX) treatments were different from controls at 20 min and 120 min time points (P < 0.05). n = 8 experiments, done with 3 different donors.
