Low proportions of sperm can bind to the zona pellucida of human oocytes

D.Y. Liu1, C. Garrett and H.W.G. Baker

University of Melbourne Department of Obstetrics and Gynaecology and Reproductive Services, Royal Women’s Hospital, 132 Grattan Street, Carlton, Australia 3053 and Melbourne IVF, 320 Victoria Parade, East Melbourne, Australia 3002

1 To whom correspondence should be addressed. e-mail: dyl{at}unimelb.edu.au


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Sperm binding to the zona pellucida (ZP) is required for human fertilization. Under experimental conditions not limited by ZP binding sites, the cumulative numbers of sperm binding tightly to the ZP will asymptote with time to the total number of sperm in the insemination medium capable of binding. METHODS: Numbers of ZP-bound sperm were counted after groups of 10 oocytes were incubated with 2x104 motile sperm in 20 µl droplets. The time-course of sperm binding was measured in three consecutive 2 h incubation periods using fresh oocytes for each period (n = 12). Using the kinetic theory of gases to model sperm–oocyte collision rates, the time-course results were extrapolated to give the total proportion of motile sperm capable of binding to the ZP. ZP binding of sperm after 4 h incubation was studied in 20 fertile and 20 normozoospermic subfertile men. RESULTS: The percentage of motile sperm capable of binding was for fertile men: mean 14% (range 8–25) and for the subfertile: 4.3% (range 0.1–13, P < 0.001). Sperm morphology correlated with the proportion of ZP-bound sperm. CONCLUSIONS: More than 75% of motile sperm from fertile men have no ability to bind to the ZP. This finding has important implications for improvement of semen analysis.

Key words: human sperm–oocyte interaction/semen analysis/sperm morphology/zona pellucida


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During natural human fertilization, sperm bind to the zona pellucida (ZP), undergo the acrosome reaction (AR) stimulated by ZP glycoprotein, penetrate the ZP and finally fuse with the oolemma (Overstreet and Hembree, 1976Go; Overstreet et al., 1980Go; Yanagimachi, 1994Go; Wassarman, 1999Go). Sperm capable of binding to the ZP therefore represent a subset of the sperm population satisfying a necessary condition for fertilization. Sperm–ZP binding tests correlate well with fertilization rates in vitro (Overstreet et al., 1980Go; Burkman et al., 1988Go; Liu et al., 1988aGo) and the binding process has been shown to be actively selective with respect to specific characteristics of sperm morphology, particularly in the acrosomal region (Liu and Baker, 1992aGo; Garrett et al., 1997Go). Round-headed sperm without an acrosome do not bind to the ZP or oolemma (von Bernhardi et al., 1990Go; Bourne et al., 1995Go), and sperm with small or abnormal acrosomes but otherwise normal morphology have reduced binding ability (Liu and Baker, 1992aGo,b; Menkveld et al., 1996Go). In contrast to many animal species, human sperm are morphologically highly heterogeneous, and while assessments of the percentage normal morphology have been shown to be related to IVF rates, the proportion of sperm capable of fertilization is not known (Kruger et al., 1988Go; Liu et al., 1988bGo; Ombelet et al., 1995Go; Menkveld et al., 1996Go).

Since the proportion of sperm in an ejaculate with the ability to bind to the ZP indicates the maximum proportion of the total sperm population capable of natural fertilization, it is of clinical and scientific interest to quantify the percentage of sperm in this subgroup for both fertile and subfertile men. We have modified a sperm–ZP binding assay (Liu et al., 1988aGo) to provide experimental conditions in which the number of oocyte binding sites is not a limiting factor and the opportunity for individual sperm to interact with oocytes is high. Measurement of the time-course for sperm–ZP binding under these conditions enables estimation of the proportion of sperm in the insemination medium capable of binding to the ZP. If this proportion of sperm is fixed, the depletion from the insemination population of sperm capable of binding to the ZP results in a classic exponential decrease in sperm–ZP binding rate, where the cumulative number of bound sperm asymptotes to the absolute number in that population with binding ability. If the ability of individual sperm in the population to bind to the ZP is dependent on capacitation during the incubation, the time-course will reflect a time-dependent increase in the number of sperm available for binding during that period. Similarly, details of the time-course provide information distinguishing between the possibilities that there is either a restricted number of sperm in the insemination medium capable of binding or a universal reduced capability of binding in the entire population.

Specific experiments were performed to test the influence of different experimental conditions on the experimental estimates of the proportion of motile sperm in a sample capable of ZP binding. These included tests for saturation of oocyte binding sites, loss of ZP ability to bind sperm during the incubation period, accumulation of factors in the medium which impair binding, delayed capacitation of sperm, oocyte variability and variation between semen samples from individuals.

Under the experimental conditions of this study, theoretical sperm–oocyte collision rates can be calculated by analogy with the kinetic theory of gases under the assumption that sperm swim in random directions in straight lines between collisions with no significant sperm–sperm interaction (Ojakian and Katz, 1973Go). Under these assumptions, the number of sperm swimming through a known area in a given time can be calculated and equated to the total interaction surface area of a uniform distribution of oocytes in the medium. Comparison of experimental sperm–ZP binding rates with these calculations of sperm–oocyte collision frequencies enables estimation of both the proportion of sperm in the medium capable of binding and the average probability of binding per collision. If the sperm population is sufficiently limited and the oocytes have free sperm binding sites throughout the incubation time, depletion from the insemination medium of sperm capable of binding causes an exponential decrease in the rate of sperm–ZP binding. The proportion of sperm in the droplet capable of binding affects the asymptote. The probability that sperm with binding capability do so at any collision affects the rate at which this asymptotic value is attained. This approach has also been used in modelling sperm–cervical mucus penetration, where a depletion effect shows that only a proportion of sperm are capable of penetration (Clarke et al., 1998Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
For the time-course studies, 12 semen samples from fertile or normozoospermic subfertile men were selected for sperm concentration (>=20x106/ml) and progressive motility (>40%) but a wide range of normal morphology (1–33%). Semen samples obtained from 20 fertile men whose partners were 16–32 weeks pregnant and 20 subfertile men attending our infertility clinic for routine semen analysis were also selected for normal sperm concentration and motility with a range of normal morphology (1–58%). All men were in good health generally and the subfertile had no obvious cause for their infertility.

Semen analysis and sperm preparation
Semen samples were obtained by masturbation after 3–5 days abstinence and all sperm tests were performed after liquefaction of the semen within 1 h of collection. Sperm concentration and motility were determined using standard methods (World Health Organization, 1999Go). Sperm morphology was assessed on Shorr-stained smears under oil immersion with x1000 magnification and bright-field illumination. Percentage normal sperm morphology was assessed according to strict criteria (Kruger et al., 1988Go; World Health Organization, 1999Go) on 200 sperm/sample for semen (washed with 0.9% sodium chloride) and insemination samples and >100 sperm/sample recovered after binding to the ZP.

Motile sperm were selected by swim-up technique as follows: 0.2–0.3 ml of semen was carefully added to the bottom of a test tube (12x75 mm) containing 0.7 ml human tubal fluid (HTF; Irvine Scientific, USA), supplemented with 10% heat-inactivated human serum (ICN Biomedicals; Irvine Scientific). Care was taken to avoid bubbles and not to disturb the interface between semen and the medium. After incubation for 1 h, 0.5 ml of the top layer of the medium containing motile sperm was aspirated. The motile sperm suspension was then centrifuged at 1000 g for 5 min, the supernatant removed and the sperm pellet washed again with 1 ml fresh HTF by centrifugation at 1000 g for 5 min. The washed sperm pellet was resuspended with serum-supplemented HTF to a motile sperm concentration of 2x106/ml for insemination. Only sperm samples with progressive motility >90% after swim-up were used in the experiments.

Oocytes
Immature (germinal vesicle) oocytes or oocytes which had failed to fertilize in a clinical IVF programme were used for sperm–ZP binding tests. Degenerate or morphologically abnormal oocytes or oocytes with the ZP penetrated by >10 sperm were not used. Any cumulus cells or ZP-bound sperm remaining after the IVF procedure were removed by repeated aspiration using a fine pipette. The oocytes were kept in a CO2 incubator and used within 6 days of oocyte collection.

Sperm–ZP binding
The sperm–ZP binding test of Liu et al. (1988aGo) was modified to make the number of ZP binding sites for sperm non-limiting so that depletion of sperm capable of binding to the ZP can be demonstrated. Methods for manipulation of the oocytes and sperm were as previously described (Liu and Baker, 1994, 1996). Briefly, oocytes and motile sperm were incubated in 1 ml volumes or 20 µl droplets of HTF under mineral oil at 37°C with 5% CO2 in air. After incubation, the oocytes were flushed several times to dislodge loosely adherent sperm using a pipette approximately twice the diameter of the oocyte (250 µm) in 0.5 ml phosphate-buffered saline containing 2 mg/ml bovine serum albumin. The tightly bound sperm were then removed by repeated aspiration using a narrow bore pipette with diameter slightly smaller than the oocyte (120 µm) in a small volume of medium on a slide, then smeared in a limited area and dried for counting and morphology assessment.

The modified sperm–ZP binding assay
The number of potential binding sites/oocyte was estimated experimentally by incubating a total of 40 oocytes for 2 h with large numbers of motile sperm (2x106 in 1 ml of medium) from 10 men (four oocytes/sample). To determine the experimental conditions to avoid saturation of ZP binding sites for sperm but to allow a high rate of sperm–oocyte interaction, the same sperm from 12 men and oocytes from the same IVF patients were incubated for 4 h under conditions where numbers of sperm capable of binding to the ZP would be expected to be either limiting (1x106/ml in 20 µl droplets) or non-limiting (2x106/ml in 1 ml). Based on the results of these preliminary experiments and calculated collision rates, all subsequent experiments used 10 oocytes and a total of 2x104 sperm in a 20 µl droplet.

Control experiments
To establish that the ZP did not lose ability to bind sperm during 2 h incubation periods, the number of sperm bound to a control group of 10 oocytes after 2 h continuous incubation was checked against the cumulative number of bound sperm removed from 10 oocytes incubated for two consecutive 1 h periods (n = 3). With the 1 h consecutive incubation periods, the sperm were removed for counting after the first hour of incubation and the same oocytes then returned to the original sperm suspension for the second hour.

To demonstrate that accumulation of factors in the medium does not impair sperm–ZP binding, the 10 oocytes in each of two sperm droplets were replaced after an initial 4 h incubation with fresh oocytes, with or without adding an extra 5 µl of medium containing 20 000 of the same sperm suspension which had been incubated concurrently for 4 h in the absence of oocytes. A further test for accumulation of inhibiting factors in the medium was performed by comparing the binding results of experiments using droplet volumes of 10 and 20 µl and the same numbers of sperm and oocytes (n = 7).

To establish that by commencement of incubation the insemination sperm are sufficiently capacitated to have negligible influence on the measured binding rates, sperm from the same subjects were incubated for 4 h with groups of 10 oocytes in 20 µl droplets either with or without preincubation of the sperm for 2 h before addition of the oocytes (n = 5). The effect of extended preincubation was also examined for a 20 h period before addition of the oocytes for 2 h incubation compared with a control 2 h incubation of sperm from the same subjects using groups of four oocytes with 2x106 sperm in 1 ml (n = 4).

The variability of the number of bound sperm between groups of oocytes was tested using 4 h incubations of the same sperm suspensions with two groups of 10 oocytes (n = 13). The variation in 4 h incubation results was also examined for two different semen samples from the same men (n = 3).

Time-course for depletion of sperm with ZP binding ability
Time-course experiments were performed with 12 semen samples by replacing the 10 oocytes in the insemination droplet with 10 fresh oocytes at 2 h intervals over a 6 h period, and counting the total number of ZP-bound sperm for each group of 10 oocytes. After each period of incubation, the 10 oocytes were flushed several times in the droplet to dislodge loosely adherent sperm and then collected in 2 µl of medium for counting ZP-bound sperm. Because the 10 oocytes were removed in 2 µl of medium, 10% of the sperm not bound to the ZP were removed from the droplet with each change of oocytes.

Comparison of sperm–ZP binding ability in fertile and subfertile men
Under the experimental conditions used for the time-course experiments, 90% of sperm were capable of binding within 4 h incubation. This allowed the binding results for 20 fertile and 20 normozoospermic subfertile men to be compared using a single incubation period of 4 h.

Theoretical time-course for sperm–ZP binding
Modelling the sperm–ZP binding rate based on collision frequencies enables estimates of both the proportion of sperm in the medium capable of binding and the probability of binding per collision. Assuming that sperm in the insemination medium swim in random directions in straight lines between collisions, the kinetic theory of gases can be applied to calculate the number of sperm swimming through an area A (µm2) during time {Delta}t (s) as

1/4 AvC {Delta}tx10–6

where C0 = concentration of motile sperm in the insemination medium (106/ml) and v = mean straight line swimming speed (µm/s) (Ojakian and Katz, 1973Go). Thus, allowing for there to be only a proportion n of motile sperm capable of binding and the probability p that they bind per collision, the number of sperm which bind per oocyte of radius r (µm) in time {Delta}t is

{Delta}b(t) = {pi}r2vpCn(t){Delta}tx10–6

where Cn is the residual concentration of motile sperm capable of binding in the insemination medium at time t and is in turn given by

Cn(t) = nC0b(t)Nx10–6/V

where V = volume of the insemination droplet (ml) and N = number of oocytes uniformly distributed throughout the droplet. Thus,

db(t)/dt = 3.6x10–9{pi}r2vpN/V [nVC0x106/N – b(t)]

= {alpha}vp [nVC0x106/N – b(t)](bound sperm/h)

where {alpha} = 3.6x10–9{pi}r2N/V. This differential equation has the solution

b = [nVC0x106/N][1 – exp(–{alpha}vpt)].

Thus the percentage of sperm capable of binding within t h is independent of C0 and given by

%B = 100n [1 – exp(–{alpha}vpt)]

For these experiments, r = 60, V = 0.02, N = 10, and thus {alpha} = 2.0x10–2. The average sperm swimming speed was taken as v = 72 µm/s, represented by the mean straight line velocity (VSL) measured for 22 swim-up samples using a motility analyser (IVOS 10.8; Hamilton–Thorne Research, USA) (range 54–91 µm/s). C(t) was corrected for the incidental removal of 10% of unbound sperm with each change of oocytes.

Statistical analysis
The significance of correlations between ZP binding results and sperm characteristics were examined by Spearman’s (rho) tests. A t-test and Wilcoxon rank sum (non-parametric) tests were used to examine differences between test and control experiments with different experimental conditions and also between numbers of ZP-bound sperm in fertile and subfertile men.

Ethics
The study was approved by The Royal Women’s Hospital Research and Ethics Committees and the subjects consented to the research on their gametes.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Control experiments
The performance of the modified human sperm–ZP binding assay to avoid saturation of ZP sperm binding sites was studied in detail.

ZP sperm binding sites are not saturated
The number of sperm binding sites/ZP was estimated experimentally by incubating oocytes for up to 4 h with large numbers of motile sperm (2x106 in 1 ml of medium) from 10 fertile men. An average of 910 (range 609–1524) tightly bound sperm were counted per oocyte. From a consideration of oocyte and sperm head surface areas, an average number of 910 sperm bound by their flat acrosomal region would cover about one-third of the outer surface of the ZP. In experiments using oocytes from the same IVF patients for each pair of tests on the same sperm samples, with the number of sperm available for binding being either limiting (1x106/ml incubated in 20 µl droplets) or non-limiting (2x106/ml in 1 ml), three of the 12 sperm samples had low binding (<400/ZP) under both experimental conditions. The other nine samples had an average number of sperm bound/ZP >776 (mean 1151) for the non-limiting 1 ml inseminations, consistent with the maximal sperm binding sites determined above. The corresponding results for the droplet inseminations were all <600/ZP: mean 207, range 29–446, indicating that maximum average sperm–ZP binding under these conditions remained at <600 sperm/ZP, a level that could saturate ZP binding sites for sperm. Subsequent experiments used 10 oocytes and 1x106/ml sperm in a 20 µl droplet to avoid saturation of binding sites but allow a high rate of interaction, calculated to be ~3000 collisions/ZP/h.

ZP binding ability maintained
The ZP do not lose ability to bind sperm during the 2 h incubation period. The number of sperm bound to a control group of 10 oocytes after 2 h continuous incubation is the same as the cumulative number of bound sperm removed from 10 oocytes incubated for two consecutive 1 h periods in which sperm were removed for counting after the first hour of incubation and the same oocytes then returned to the original sperm suspension for the second hour (control (%): 18.2, 6.5, 6.7; cumulative (%): 15.6, 5.6, 6.8).

Lack of evidence for accumulation of material blocking sperm–ZP binding
In the two experiments where additional sperm were added in the 4–6 h incubation interval, the numbers of ZP-bound sperm were 1085 and 1350 with the additional unexposed sperm, compared with 181 and 259 in the control droplets. No significant difference in percentages of bound sperm was found when the droplet volume was halved (11.2 ± 4.7 versus 11.6 ± 4.9, mean difference = 0.33, SD = 0.67, n = 7).

Lack of evidence for incomplete capacitation
There was no significant difference between the binding results for control sperm incubated after preparation with oocytes for 2 h (mean 11.2%, range 9.3–14.3%) and those where the same sperm suspensions were preincubated for 2 h before addition of the oocytes (mean 11.4%, range 8.8–13.2%, mean difference = 0.14, SD = 1.03, n = 5). There was a significant loss of sperm–ZP binding ability with extended preincubation. Only a mean 4.4 sperm bound/ZP in a 2 h incubation after a 20 h preincubation, compared with >100 bound sperm/ZP when there was no preincubation.

Assay reproducibility
Four hour incubations of the same sperm suspensions with two groups of 10 oocytes produced similar binding results (mean 9.3 ± 7.1% versus 9.7 ± 7.5%, mean difference = 0.35, SD = 0.81, n = 13). Results for two semen samples from the same men were in close agreement (mean difference = 0.73, SD = 3.4, n = 3) and there was also good agreement between results with 20 and 10 µl droplet volumes (mean difference = –0.33%, SD = 0.67, n = 7).

Time-course for depletion of sperm with ZP binding ability
The cumulative percentage of sperm bound to the ZP at 2, 4 and 6 h incubation is plotted in Figure 1a. The theoretical curves are based on the kinetic theory of gases and calculated as a function of the proportion of sperm capable of binding (n) and the probability of binding per oocyte collision for a sperm capable of binding (p). It can be calculated that sperm velocity would need to decline by a factor of three within the first 2 h and to near zero by 6 h to account for the observed decrease in binding with time. However, during the 6 h incubation period sperm motility decreased by <3% and velocity by <2%.



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Figure 1. (a) Cumulative percentages of zona pellucida (ZP)-bound sperm as a function of incubation time with motile sperm from 12 fertile or normozoospermic subfertile men. The fitted curves are a function of the proportion of sperm capable of ZP binding (n) and the probability of binding per collision for those sperm capable of binding (p). The results indicate that there is a wide sample variation in the percentage of sperm capable of ZP binding (mean = 0.14, SD = 0.06), but sperm capable of binding have an approximately uniform probability of binding per collision (mean = 0.43, SD = 0.11) for all samples. (b) Comparison of the model calculations with the mean experimental cumulative percentage of sperm bound to the ZP at 1, 2, 4 and 6 h, illustrating the influence of different combinations of values of n and p. The mean and range of measured numbers of ZP-bound sperm expressed as percentages of the calculated mean asymptotic value were at 1 h = 35% (24–54), 2 h = 70% (64–86), 4 h = 90% (83–98), 6 h = 94% (88–98).

 
The time-course results indicate that only small percentages of sperm in the insemination media are capable of binding to the ZP, with the fit to the theoretical binding giving a mean, time-extrapolated percentage of 14, with SD = 6 and range 3.5–26. The model calculations also suggest that sperm require approximately two collisions (p = 0.43, SD = 0.11 and range 0.30–0.68) with the ZP to bind (Figure 1a). Figure 1b illustrates the effects of varying n and p to achieve a cumulative percentage of sperm bound after 4 h incubation equal to the mean experimental value for the 12 semen samples. For a qualitative comparison, Figure 1b also includes the 4.9% binding result at 1 h, taken from the first 1 h incubation data for the three semen samples used in the control experiment involving sequential 1 h incubations.

Comparison of sperm–ZP binding ability for fertile and subfertile men
For the groups of fertile and normozoospermic subfertile men, the average results of standard semen analysis and morphology of sperm in the insemination medium and removed from the ZP are given in Table I. The subfertile men had significantly lower ZP binding results at 4 h incubation and lower normal morphology in semen and insemination medium but similar results for ZP-bound sperm to those of fertile men. Percentage normal morphology of sperm in the semen was significantly related to the percentage of motile sperm capable of binding to the ZP (Figure 2). Differences in morphology between insemination and ZP-bound sperm are illustrated in Figure 3 for two fertile men.


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Table I. Mean (range) of semen test results in fertile (n = 20) and normozoospermic subfertile (n = 20) men
 


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Figure 2. The percentage of motile sperm from 20 fertile (closed circles) and 20 normozoospermic subfertile (open circles) men which can bind to the zona pellucida after 4 h incubation, plotted as a function of % normal morphology in the semen. There is a strong positive linear relationship (Spearman’s rho = 0.60, P < 0.001).

 


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Figure 3. Light microscopy images (x1000) of stained sperm from the insemination medium (a and c) and the corresponding zona pellucida (ZP)-bound samples (b and d respectively) for two different semen samples. Sperm with normal morphology in the insemination samples are labelled at the acrosome with an asterisk. The illustrated insemination samples were assessed 43% (a) and 26% (c) normal morphology. All ZP-bound sperm illustrated in the corresponding panels b and d have normal morphology.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Two different methodologies have been developed to assess human sperm–oocyte binding: the sperm–ZP binding ratio test (Liu et al., 1988aGo) using intact oocytes and the hemizona assay (Burkman et al., 1988Go). These tests have previously been applied to prediction of fertilization in IVF, evaluation of sperm characteristics such as morphology and examination of the optimum conditions for microvolume inseminations in IVF (Liu and Baker, 1992aGo,b; Ozgur et al., 1994Go; Oehninger et al., 1997Go). We have modified the sperm–ZP binding test to enable estimation of the proportion of motile sperm which are capable of binding to the ZP. Experimental conditions were chosen so that over a measured time-course the insemination medium is depleted of sperm capable of binding and thus the cumulative number of ZP-bound sperm asymptotes toward the total number capable of binding. Tests were performed to show that the incubation of a high number of oocytes with a relatively high concentration, but low absolute number of sperm, produced binding results that were not influenced by saturation of oocyte binding sites or accumulation of factors in the medium inhibiting binding.

The theoretical time-course calculations in Figure 1b show the effects of varying n, the proportion of motile sperm capable of binding (the asymptotic variable) and p, the binding probability per collision for sperm capable of binding (the lifetime variable). The family of curves in Figure 1b are representative of different pairs of values of n and p for which the theoretical cumulative percentage of bound sperm at 4 h is equal to the mean experimental value. It is clear from the poor fit between the experimental data at other time-points and the extreme curve represented by n = 1 that the experimental results cannot be explained by a high proportion of sperm with binding capacity but limited ability to bind at each collision with the ZP. The well-defined asymptotic behaviour of the experimental time-course also excludes the operation of a time-dependent acquisition of binding ability, such as might occur if delayed capacitation of the sperm were the cause of the low binding. The lack of significant difference between binding results when sperm suspensions were preincubated for 2 h also indicates that delayed capacitation is not a significant factor influencing the time-course. The 1 h data point in Figure 1b, although not derived from the same semen samples used for the 2, 4 and 6 h data, is also consistent with no effective delay in early binding rates due to incomplete capacitation. Moreover, overnight preincubation markedly reduced the ability of sperm to bind to the human ZP, in agreement with the study by Singer (1985Go) who demonstrated no significant difference in human sperm–ZP binding results between 0 and 2 h preincubation, substantially reduced binding after 6 h preincubation, and very few sperm remaining capable of binding after preincubation of >=12 h.

The time-course results cover a range of semen samples and give a clear demonstration that only a low proportion of motile sperm from humans is capable of binding to the ZP. Direct comparison between individual experimental and theoretical time-courses gives a range of deduced values of n and p (Figure 1a) which indicate that the proportion of sperm capable of binding is universally low and highly variable between individual men (range n = 0.035–0.26). In contrast, there is relatively little variation in p (range p = 0.36–0.68), indicating that on average approximately two collisions occur between a sperm with binding ability and the ZP before strong binding is achieved.

Since the time-course data indicate that under the experimental conditions of this study 90% of sperm in the medium with binding ability have bound within 4 h incubation, the binding numbers from single 4 h incubation experiments can be extrapolated to give estimates of the percentage of motile sperm capable of binding. For all semen samples tested, the maximum percentage of motile sperm capable of binding was only 26%, with an average of 14% for semen from both the 20 fertile men and the group of 12 samples selected for good motility and concentration. The 20 subfertile men who were also selected for semen with good motility and concentration (not significantly different from the 20 fertile men) had a significantly lower mean of only 4.3% and a maximum of 13% of motile sperm capable of ZP binding.

These results are maximum estimates of the percentage of sperm capable of fertilization, as the conditions in vitro differ from those in vivo: in particular, the oocytes have no cumulus oophorus and are exposed to a high concentration of sperm. Also, only a proportion of ZP-bound sperm may undergo the subsequent events of fertilization. Our finding that only a low proportion of human sperm are able to bind to the ZP has significant implications for future assessment of semen, since the standard semen analysis variables of sperm concentration and motility are not closely related to the ability of sperm to interact with the oocyte. This study indicated that sperm morphology was significantly related to the percentage of motile sperm capable of binding to the ZP, as was previously found in similar data relating morphology to sperm ZP binding, including ZP selectivity of specific sperm morphologies (Liu and Baker, 1992aGo; Menkveld et al., 1996Go; Garrett et al., 1997Go). However, assessment of morphology in general lacks reproducibility within and between laboratories (Jorgensen et al., 1997Go; World Health Organization, 1999Go; Franken et al., 2000Go). Assessment of the proportion of sperm in a semen sample capable of ZP binding would be a valuable tool for diagnosis of male subfertility, and although routine assay of sperm–ZP binding ability is not currently possible, indirect assessment of the proportion of sperm capable of binding may be feasible by image analysis of sperm morphometry using a reference based on the specific characteristics of ZP-bound sperm (Garrett et al., 1997Go; Garrett et al., 2003Go). Alternatively, development of recombinant human ZP3 protein attached to solid beads could provide a routine test of the sperm–ZP interaction (Vazquez et al., 1989Go; van Duin et al., 1994Go; Brewis et al., 1996Go; Whitmarsh et al., 1996Go; Dong et al., 2001Go). Our determination that >75% of motile sperm in any semen sample are incapable of binding to the ZP, a critical early step in the fertilization process, is a significant scientific and clinical finding. These data support the concept that male subfertility is determined by a reduced number of capable sperm, rather than a reduced capability in all sperm.


    Acknowledgements
 
The authors thank Ming Li Liu for technical assistance, scientists in both Royal Women’s Hospital and Melbourne IVF Laboratories for collecting the oocytes, and scientists in the Andrology Laboratory, Department of Pathology, Royal Women’s Hospital for helping with sperm samples. This study was financially supported by Royal Women’s Hospital Research and Ethic Committee.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bourne, H., Liu, D.Y., Clarke, G.N. and Baker, H.W. (1995) Normal fertilization and embryo development by intracytoplasmic sperm injection of round-headed acrosomeless sperm. Fertil. Steril., 63, 1329–1332.[ISI][Medline]

Brewis, I.A., Clayton, R., Barratt, C.L., Hornby, D.P. and Moore, H.D. (1996) Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa. Mol. Hum. Reprod., 2, 583–589.[Abstract]

Burkman, L.J., Coddington, C.C., Franken, D.R., Krugen, T.F., Rosenwaks, Z. and Hogen, G.D. (1988) The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil. Steril., 49, 688–697.[ISI][Medline]

Clarke, G.N., Garrett, C. and Baker, H.W.G. (1998) Quantitative sperm mucus penetration: modified formulae for calculating penetration efficiency. Hum. Reprod., 13, 1255–1259.[Abstract]

Dong, K.W., Chi, T.F., Juan, Y.W., Chen, C.W., Lin, Z., Xiang, X.Q., Mahony, M., Gibbons, W.E. and Oehninger, S. (2001) Characterization of the biologic activities of a recombinant human zona pellucida protein 3 expressed in human ovarian teratocarcinoma (PA-1) cells. Am. J. Obstet. Gynecol., 184, 835–843; discussion 843–844.[ISI]

Franken, D.R., Barendsen, R. and Kruger, T.F. (2000) A continuous quality control program for strict sperm morphology. Fertil. Steril., 74, 721–724.[CrossRef][ISI][Medline]

Garrett, C., Liu, D.Y. and Baker, H.W.G. (1997) Selectivity of the human sperm–zona pellucida binding process to sperm head morphometry. Fertil. Steril., 67, 362–371.[CrossRef][ISI][Medline]

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Submitted on July 2, 2003; accepted on July 7, 2003.