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Testis |
a Division of Reproductive Biology,
b Department of Obstetrics and Gynecology, and Bodega Marine Laboratory, University of California, Davis, California 95616
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acrosome reaction, fertilization, sperm, sperm capacitation, sperm motility and transport
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sperm binding to the ZP has been described as involving two phases. During the primary binding phase, one or more receptors for zona on the sperm plasma membrane interact with zona ligands to mediate sperm attachment and induction of the acrosome reaction. In the secondary binding phase, which takes place following the acrosome reaction, sperm components on the inner acrosomal membrane interact with the ZP during zona penetration [1]. The association of sperm and ZP during primary binding occurs through two successive steps, loose attachment and tight binding, the latter step being critical for zona penetration. Loose attachments were originally defined as weak bonds between sperm and zona that are easily disrupted by a serial rinsing of oocytes through narrow-bore glass pipettes [2]. Loose attachment has been observed between rodent sperm and the ZP during heterologous gamete interaction [3, 4], prompting the thinking that initial attachment of sperm to the ZP is a reversible event that is not species specific. Even under capacitating conditions with homologous gametes, a large percentage of bound sperm can be removed from the ZP by merely transferring the oocytes from one rinse bath to the next.
Sperm that remain following rinsing or repeated pipetting have been referred to as irreversibly or tightly bound [2]. In mice, capacitated sperm rapidly attach to ZP-intact oocytes in vitro, but they require 10 to 15 min before binding is tight enough to withstand rinsing [4]. When ZPs were removed from sperm suspensions (i.e., pulse) and maintained in a medium free of sperm for 10 min (i.e., chase), the number of tightly bound sperm increased in the chase drop [5]. These observations suggest that sperm require a period of time in loose, relatively nonspecific association with the ZP before tight or more-specific binding can occur, and that these different types of bindings represent different mechanisms of attachment.
Most bioassays designed to evaluate sperm-zona interaction do not include methods for evaluating sperm acrosomal status at the time of zona binding, and therefore do not allow direct observation of sperm during primary and secondary binding. Lengthy coincubations of up to several hours are commonly required before sperm develop tight binding that withstands rinsing [1], but during this time many sperm undergo the acrosome reaction [6]. Because sperm are not observed until after the coincubation period, these techniques do not resolve events associated with the transitions from loose attachment to tight binding to the acrosome reaction.
In this communication we report the methods used and the results obtained when gametes from cynomolgus macaques were observed continuously during the early stages of sperm-zona interaction. This is the first report that describes in detail the behavior of individual mammalian sperm during the processes of loose attachment, tight binding, and the zona-induced acrosome reaction. We also describe changes in the flagellar motion of sperm that underwent the acrosome reaction on the ZP. These methods were applied to investigate the initial events of sperm-zona binding in the presence of lignosulfonic acid (LSA), a sulfonated compound known to block in vitro fertilization of macaque oocytes [7].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Water of HPLC grade was obtained from Fisher Scientific (Santa Clara, CA). Fluorescein isothiocyanate (FITC)-Pisum sativum was obtained from Vector Laboratories (Burlingame, CA). An Alexa 488 protein labeling kit for the production of Alexa-SBTI (a probe for acrosin) was purchased from Molecular Probes (Eugene, OR). Dulbecco phosphate-buffered saline (DPBS) and modified Biggers, Whitten, Whittingham medium (Hepes-buffered BWW) were prepared by Irvine Scientific (Irvine, CA). All other chemicals and salts for media preparation were purchased from Sigma Chemical Company (St. Louis, MO). LSA sodium salt was purchased from Aldrich Chemical Company (St. Louis, MO) and was additionally purified to remove contaminants according to the procedures described by Higashi et al. [8]. Briefly, this procedure involved sequential solvent extraction (methylene chloride and acetonitrile) followed by dialysis (3.5-kDa cutoff) and lyophilization.
Sperm Collection, Washing, and Capacitation
Six adult male cynomolgus macaques were caged individually at the California Regional Primate Research Center (CRPRC) in compliance with the Federal Animal Welfare Act and the National Institutes of Health Guidelines for Care and Use of Laboratory Animals. The animals were maintained on a 12L:12D light cycle at 25–27°C and were fed Purina monkey chow and water ad libitum. Semen samples were collected by electroejaculation [9] into 15-ml centrifuge tubes containing 6 ml of Hepes-buffered BWW maintained at room temperature. Sperm from all six males has been shown to fertilize macaque oocytes in vitro.
Sperm were processed as described by Tollner et al. [10]. Briefly, the coagulum was removed after 15–20 min, the semen samples were then further diluted with an additional 6 ml of Hepes-buffered BWW containing 3 mg/ml BSA and washed by centrifugation at 300 x g for 10 min. The resulting sperm pellets were centrifuged through a 6 cm column of 80% Percoll at 400 x g for 25 min. The supernatant containing Percoll and any remaining seminal plasma was removed and the sperm pellet was resuspended in bicarbonate-buffered BWW [11] containing 30 mg/ml of BSA. Sperm were washed two more times by centrifugation at 300 x g for 10 min and diluted in this medium. Sperm were finally resuspended at a concentration of 10–20 x 106/ml in the bicarbonate-buffered BWW and were capacitated by a series of incubations beginning with a 24-h incubation at room temperature in 4.5% CO2. Following this room temperature incubation, sperm suspensions were incubated at 37°C in 4.5% CO2 for 2 h more, at which point the sperm concentration was adjusted to 10 x 106/ml. The sperm suspensions were incubated for an additional 1 h in media containing 1 mM caffeine and 1 mM dbcAMP (as activators). Macaque sperm capacitated according to this protocol have been used for in vitro fertilization studies and yield high rates of fertilizations after a 4 h coincubation of sperm and oocytes [7]. A 200-µl aliquot of the activated sperm suspension was added to 800 µl of Hepes-buffered BWW at 37°C with 0.3% BSA, activators, 100 µg/ml hyaluronic acid, and 2–3 µg of Alexa-SBTI. The combination of activators and hyaluronic acid has been shown to enhance the zona pellucida-induced acrosome reactions of macaque sperm [12].
Labeling of Sperm with Alexa-SBTI
The Alexa 488 reactive dye was coupled to SBTI using the Alexa 488 protein labeling kit (A-10235; Molecular Probes). The dye has a succinimidyl ester moiety that reacts with primary amines of proteins to form a stable dye-protein conjugate. Following reaction with 1 mg of SBTI, excess unbound dye was removed with a purification column provided with the kit. Depending on the batch, Alexa-SBTI was found to label sperm at concentrations ranging from 1 to 2 µg/ml. Alexa 488 is stable in the pH range between 4 and 10, and has absorption and fluorescence emission maxima of approximately 494 nm and 519 nm, respectively.
Preparation of Oocytes
Ovaries were obtained at necropsy from adult female cynomolgus macaques at the CRPRC. Intact immature oocytes and ZP shells without a vitellus were collected from the ovaries and frozen at -80°C in 2 M dimethyl sulfoxide (DMSO) in DPBS according to previously published protocols [13]. Both intact ZP and ZP shells were thawed at 22°C and rinsed through three dishes, each containing 0.5 ml of Hepes-buffered BWW medium to remove DMSO prior to experiments. ZP were incubated in Hepes-buffered BWW containing 100 µg/ml of hyaluronic acid for 1 h and then deposited onto glass slides and allowed to air-dry for 10 min. Within 5 min before sperm-zona coincubation, 2–3 µl of Hepes-buffered BWW containing activators, hyaluronic acid, and Alexa-SBTI were added to the air-dried zonae.
Video Microscopy and Assessment of Sperm-Zona Binding, Acrosomal Status, and Motility
For these experiments, sperm-zona interaction was evaluated in a 20-µm-deep binding chamber that caused sperm to align in a single focal plane. This alignment allowed a detailed analysis of flagellar movements after sperm had bound to the zona (see below). Zona shells rather than intact ZP were used for these studies because they could be more easily compressed without rupturing. Four posts of silicon grease containing 5- to 20-µm silica beads were deposited at four corners around a single zona. A 22 x 22-mm glass coverslip was carefully pressed down onto the posts until the grease was completely flattened. The slide was warmed for 5 min on a microscope stage warmer set at 37.5°C before sperm were added. A 20-µl aliquot of activated sperm diluted to 2 x 106/ml in Hepes-buffered BWW was added to the warmed slide at the edge of the coverslip. Sperm were drawn under the coverslip and around the zona by capillary action. Sperm were observed with a Lietz Laborlux S microscope equipped with 200 W mercury fluorescence vertical illuminator and a 1-Lambda Ploemopac incident light fluorescence illuminator employing an I3 filter cube with a BP 450–490 excitation filter, an RKP 0510 dichromatic mirror, an LP 515 suppression filter, and a 40x fluorescence/phase objective (JH Technologies, San Jose, CA). A charge-coupled device black-and-white video camera (Panasonic model wv-BD400; Northern Video, Sacramento, CA) attached to the microscope via an Olympus adapter (with a 3.3x ocular) (Scientific Instruments, Sunnyvale, CA) captured images of sperm at 30 frames per second. The video signal passed in series through a video time generator (VTG; model 33; For.A, Los Angeles, CA) and a 
-inch videotape recorder (Panasonic model AG6300; Northern Video).
In the first series of experiments, sperm were recorded through the following sequence: loose attachment to the zona, tight binding, and the progression of the acrosome reaction as determined by Alexa-SBTI labeling. Tight binding was defined as the nearly instantaneous immobilization of the sperm head on the zona surface. It has been demonstrated previously that the acrosome reaction can be observed in real time with the Alexa-SBTI probe when motile sperm are attached to the zona [10].
The light source was blocked shortly after tight binding and sperm were assessed for initial signs of the acrosome reaction (fluorescence of Alexa-SBTI around the apical ridge of the anterior portion of the sperm head [10]). The video camera lacked the sensitivity to capture these fluorescent images, therefore, the beginning of the acrosome reaction was indicated on video by switching the date indicator toggle on the VTG as soon as labeling was apparent. The time of the beginning of the acrosome reaction as well as the time of tight binding following loose attachment was determined from the VTG. The elapsed time between the initial signs of SBTI labeling and the completion of the acrosome reaction could not be determined with the optics used in this experiment. Images of the changes in fluorescence during SBTI labeling of acrosome-reacting sperm were obtained with an Olympus upright BH-2 microscope (Scientific Instruments) using a 60x oil immersion objective. The microscope was equipped with a Bio-Rad (Hercules, CA) MRC-600 Laser scanning confocal system, including a 15 mW krypton-argon mixed gas multiline laser. Sperm were optically sectioned (0.25 µm) and the full Z-series of images was collected and projected in order to confirm surface labeling patterns. Images were collected in real time, digitally converted with Adobe Photoshop software (Adobe Systems, San Jose, CA), and printed using dye sublimation.
For the purpose of evaluating changes in flagellar beat patterns, sperm bound to zonae were recorded from the time of loose binding until 2 min following tight binding. A 5.5x adapter ocular (Scientific Instruments) was used to enlarge the video image for manual assessment of flagellar movement. Fluorescence was applied only once for approximately 2 sec to determine whether tightly bound sperm had undergone the acrosome reaction. Video recordings of sperm were analyzed on a black-and-white 17-inch monitor (Ikegami model PM 175A). A transparent overlay (Fig. 1) generated from tracings of a micrometer recorded at the same magnification was used to measure changes in the beat amplitude of the most distal portion of the midpiece. The amplitude was measured within 1 sec of tight binding, and 30, 60, and 120 sec after tight binding. The reported amplitude was an average of the maximum amplitude of five consecutive beat cycles at each time point. The beat frequency of the flagellum was determined by counting the number of beat cycles during a 1-sec interval at the same time points.
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Removal of Sperm Bound to Zonae
The relative binding strength of sperm attached to the zona was evaluated by flushing zona binding chambers with medium following 2 to 3 min of sperm-zona coincubation. Oocytes with intact ZP were used for these experiments. Both zonae and sperm were prepared as described above. Binding chambers were modified with 300-µm beads in grease deposited on the glass slide in two parallel rows approximately 15 mm apart on opposite sides of the zona. The greater depth allowed for rapid flow of medium under the coverslip.
Sperm were first introduced into the chamber for 2 to 3 min. Just before flushing the chamber, the number of sperm displaying loose attachment and tight binding was scored separately. Four hundred microliters of Hepes-buffered BWW with 0.3% BSA were gradually deposited at one end of the chamber while immunoblot filter paper (#4; Whatman, Maidstone, U.K.) was placed against the opposite end to draw the medium through the chamber. Each flush required approximately 10 sec. After two consecutive flushes, the number of loosely attached and tightly bound sperm were counted again. Flush experiments were repeated three times; each replicate used sperm from a different male. Four to five chambers, each with one zona, were used per experiment.
Evaluation of the Effects of LSA on Sperm-Zona Interaction
We have shown previously that LSA inhibits sperm-zona interaction and blocks in vitro fertilization of macaque oocytes [7], although the specific action of LSA on sperm-zona interaction was not determined. In the present experiments we used the methods described above to characterize the effect of LSA on sperm interactions with zonae. Following collection of macaque semen and initial washing in Hepes-buffered BWW containing 3 mg/ml BSA, sperm pellets were resuspended to a sperm concentration of 100 x 106/ml. Aliquots of sperm were treated with LSA dissolved in Hepes-buffered BWW salts to obtain final concentrations of 0.25, 0.5, and 1.0 mg LSA/ml of sperm suspension. A control aliquot was treated with an equivalent volume of Hepes-buffered BWW salts. Aliquots were incubated at room temperature for 40 min and then washed through Percoll and capacitated as described above.
For zona binding assays with LSA-treated sperm, a 75-µm-deep chamber (constructed using 50- to 75-µm beads) was used to ensure rapid and even distribution of sperm around the zona. Intact ZPs were used for these assays. A 4- to 5-µl aliquot of Hepes-buffered BWW containing activators, hyaluronic acid, and Alexa-SBTI was added to each dehydrated zona before the coverslip was applied. The chamber was incubated on the microscope stage warmer at 37.5°C for 5 min before the addition of sperm. A 50-µl aliquot of activated sperm preparation was drawn by capillary action to fill the entire 22 x 22-mm space. Most eggs were sandwiched between the slide and the coverslip, and the top and bottom portions of the zona were generally inaccessible to sperm binding. Binding of sperm to the zona surface was observed with a Lietz Laborlux microscope with phase contrast optics at 400x. A timer was started at the moment the first motile sperm attached to the zona. After 3 min the total number of sperm and the number of tightly bound sperm were counted, starting at the 12 o'clock position of the zona and working clockwise to the starting point. The count required approximately 15 sec for completion. Observations were made in two different chambers, each with one zona. The experiment was replicated four times, each replicate used sperm from a different male.
The effects of LSA on the timing of tight binding and the zona-induced acrosome reaction were evaluated with video micrography as described above. Sperm were treated with 1 mg/ml LSA or with Hepes-buffered BWW salts and were washed and capacitated as described above. Sperm were recorded during the sequence of loose attachment, tight binding, and the beginning of the acrosome reaction as described above. Sperm-zona interaction in each binding chamber was observed for 5–7 min. Due to the inhibitory effects of LSA on sperm-zona binding, five to six zonae per experiment were required to collect images of tightly bound sperm that were equivalent to the number of control sperm recorded on a single zona. The experiment was replicated four times; each replicate used sperm from a different male.
Effects of LSA on sperm motility changes after tight binding to the zona were also evaluated as described above. Changes in flagellar beat patterns were measured over 2 min following tight binding in 20-µm-deep zona binding chambers.
Statistical Analysis
Statistical analyses were conducted using one-way ANOVA followed by Duncan range testing. Values are given as mean ± SEM. A P value 
0.01 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Frame by frame analysis of video recordings revealed that all sperm underwent a similar series of changes before the onset of the zona-induced acrosome reaction (Fig. 2). Initial sperm attachment was characterized by a loose association of the sperm head with the zona surface as evidenced by a rapid swiveling of the head at the point of attachment (Fig. 3A). The S-like wave form of the flagellum did not appear to change significantly from that observed for free-swimming sperm in the moments before loose attachment (Fig. 4, A and B).
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After a period of time following loose attachment (Fig. 2; 16.35 ± 3.0 sec, n = 23 sperm from 4 different males), the sperm head became tightly bound. At this time the sperm head abruptly ceased to swivel (Fig. 3, B and C; and Fig. 4C). The heads of some of these sperm were held almost completely motionless on the zona surface (Fig. 3C). In other sperm, the posterior head continued to oscillate slightly with flagellar movement, but the anterior head remained fixed in position. In all sperm bound to the zona, the middle piece of the flagellum appeared to straighten simultaneously with immobilization of the head, although the principal piece of the flagellum continued to beat vigorously (Fig. 4C). The transition from the loose attachment associated with head swiveling to the more firm attachment of tight binding required approximately 1/15 sec (data not shown). The elapsed time between tight binding and initiation of the acrosome reaction as determined by Alexa-SBTI labeling was 11.36 ± 0.6 sec (Fig. 2; n = 25 sperm from 4 males).
Acrosomal fluorescence began to concentrate in the anterior region of the sperm head starting at the apical ridge as described previously [10]. The progression of labeling with Alexa-SBTI could be observed in detail with a confocal microscope system. The serial Z-section laser-focused images were captured digitally at a rate of 30 frames/sec. Figure 5 shows the time course of the acrosome reaction of a representative sperm at several intervals over 40 sec. Labeling generally began at the apical ridge of the sperm head and continued around to the equatorial segment. The central portions of the acrosome became increasingly fluorescent until a maximum was reached after approximately 30–40 sec. The time to completion of the acrosome reaction varied from sperm to sperm but the progression of labeling from the anterior edges of the acrosome to the center was fairly consistent.
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Sperm Motility on the Zona Pellucida
Video analysis showed that macaque sperm underwent a number of changes in motility characteristics while interacting with the ZP. Initially there was a near instantaneous change in motility at the time of tight binding, which probably resulted from immobilization of the sperm head on the ZP. Tracings of the waveforms of the flagellum (Fig. 4C) were very similar to those of capacitated human sperm with heads immobilized on the tip of a fine-bore glass pipette [14]. As tight binding progressed, the midpiece appeared more rigid and the S-wave pattern associated with flagella of free swimming and loosely attached sperm was less pronounced (Fig. 4). Over the next 2 to 3 min the midpiece became more flexible and in some sperm the amplitude of displacement of the middle and principal pieces doubled. As the displacement of the flagellum increased, the wave patterns of the principal piece became less S-shaped and more C-shaped at the extreme positions of displacement (Fig. 3C). Within the first few seconds after tight binding, the flagellum moved with a combination of low-amplitude, high-frequency beats; and high-amplitude, low-frequency beats. Gradually, the high-frequency, low-amplitude beats became less frequent and after several minutes the beats were primarily symmetrical, large-amplitude lever strokes.
Flagellar motion characteristics were analyzed for 25 sperm at the time of tight binding to the zona, and at 30, 60, and 120 sec following tight binding. Frame by frame tracings of the entire flagellum did not provide a reliable measure of change because the flagellum moved out of the plane of focus several times in a single beat cycle. The midpiece of the sperm generally remained in the same plane of focus through each beat cycle, and throughout the 2- to 3-min duration of recording. A transparent overlay superimposed over the video image of sperm was used to measure the degree of displacement of the most distal portion of the midpiece (Fig. 1). The average lateral displacement of the midpiece (LDMP) did not appear to change significantly over the first minute following tight binding (Fig. 6). At 2 min following the initiation of tight binding, LDMP increased 30% from 9.2 to 13.2 µm (P 0.01). This increase in midpiece displacement is shown in the video micrograph (Fig. 3C). Flagellar beat frequency decreased after 2 min (Fig. 7), and changes in beat frequency were inversely correlated with changes in LDMP (r = 0.97). Approximately half the sperm began to detach from the zona within 3–4 min after tight binding, presumably because the torque generated by the increased LDMP disrupted binding. Many sperm that remained bound continued to display an increase in LDMP for well over 10 min. Only a small percentage of sperm became nonmotile after several minutes of tight binding.
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Removal of Zona-Bound Sperm by Flushing Medium Through Binding Chambers
Following two chamber flushes, many sperm were removed from the surface of the zona. On average, 65.2% ± 6.2% of loosely bound sperm were removed by flushing compared with 1.8% ± 1.9% of tightly bound sperm (P 0.001). From a total of 59 tightly bound sperm observed on 14 zonae, only 1 sperm became dislodged during chamber flushing. Some tightly bound sperm were immotile after flushing but remained bound to the zona.
Effect of LSA on Sperm-Zona Pellucida Interaction
The development of a chamber for making standardized observations of the initial events of sperm-zona interaction enabled us to evaluate the effect of experimental treatments on these events. Previously, we have demonstrated that treatment of sperm with LSA blocked in vitro fertilization of macaque oocytes and that this antifertility effect appeared to result from inhibition of sperm-zona binding [7]. In the present experiments LSA significantly inhibited loose attachment of sperm to the zona compared with that of controls (P 0.01; Fig. 8). Treatment with 1.0 mg/ml LSA resulted in maximum inhibition of sperm attachment to the zona (88.2%), but this was not significantly different from inhibition of binding achieved with 0.25 and 0.5 mg/ml (Fig. 8). LSA-treated sperm appeared to strike the zona with equal frequency as control sperm but were more likely to either immediately bounce off or pull away after several seconds. The latter phenomenon appeared to be due to an inhibition of tight binding. As reported previously [7], LSA had no effect on sperm motility in solution. All doses of LSA significantly inhibited tight binding (P 0.001; Fig. 9). There was 99.1% inhibition of tight binding when sperm were treated with 1.0 mg/ml LSA.
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The timing of tight binding and the onset of the acrosome reaction were evaluated following treatment with 1 mg/ml LSA or with Hepes-buffered BWW (control group). The elapsed time between loose sperm attachment to the zona and tight binding was 7.72 ± 1.5 sec (mean ± SEM; 31 sperm from 3 males) for control sperm and 27.81 ± 3.5 sec (27 sperm from the same 3 males) for LSA-treated sperm (P 0.001; Fig. 10). Zonae from a different female were used in experiments involving LSA, which may account for the differences in average time required for tight binding between control sperm in this series and those sperm reported in Figure 2. The elapsed time between tight binding and the initial signs of SBTI labeling was 10.83 ± 0.6 sec and 11.72 ± 0.6 sec for LSA-treated and control sperm, respectively (P > 0.05; Fig. 10), and was essentially the same as times reported in the first set of observations (Fig. 2).
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Sperm flagellar movements following tight binding were evaluated for a small set of LSA-treated sperm (15 sperm from 3 males) paired with untreated sperm (control; 16 sperm from the same 3 males). Both control and LSA-treated sperm had a consistent increase in displacement of the midpiece from 1 sec (8.7 ± 0.8 µm and 9.1 ± 0.6 µm, respectively) to 120 sec following tight binding (13.8 ± 0.6 µm and 12.7 ± 0.6 µm, respectively). Differences between control and LSA-treated sperm were not significant. The increase in LDMP for both controls and LSA-treated sperm was significant (P 0.01) and exhibited essentially the same outcome as nontreated sperm in the initial experiments. Thus, LSA did not appear to change the time course or the degree of change in LDMP following tight binding. Similarly, all sperm displayed a corresponding and proportional decrease with beat frequency regardless of treatment (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence indicate that tight binding, as defined in this study, is the irreversible, high-affinity, specific association between sperm and zona that is required for penetration of the ZP. Tightly bound macaque sperm remained on the ZP when medium was rapidly drawn through the binding chamber, while the majority of loosely associated sperm became detached. Even tightly bound sperm that lost motility remained on the zona following flushing.
Tight binding appears to be involved in the specific sperm-zona interactions that trigger the acrosome reaction. The acrosome reaction, as indicated by labeling with SBTI, always followed shortly after the onset of tight binding and was never observed in sperm that were loosely attached. Changes in the amplitude and frequency of midpiece movement also occurred only after sperm became tightly bound and these motility changes are likely to be the downstream results of specific signaling events. The motility of loosely attached sperm never appeared to vary from the sinusoidal wave mode seen in free-swimming sperm.
The initiation of the acrosome reaction occurred very quickly after tight binding. Alexa-SBTI labeling was first detected on the anterior head always within 17 sec after tight binding and on average between 10 and 11 sec, but the true time course may be even shorter. We have shown previously at the ultrastructural level that SBTI labels intraacrosomal contents as soon as there is evidence of membrane vesiculation [10]. However, it is reasonable to assume that there is a delay from the moment the acrosomal contents are accessible to SBTI until a perceivable fluorescence is concentrated on the sperm head.
Although a few reports have described the kinetics of the acrosome reaction of individual sperm bound to intact ZP, rapid initiation of the acrosome reaction has been shown to follow sperm challenge with other stimuli. Guinea pig sperm capacitated in calcium-free medium began to acrosome react between 30 and 60 sec after exposure to normal levels of calcium [1]. Membrane fusion between the plasma membrane and the outer acrosomal membrane of human sperm was detected as early as 10 to 15 sec following exposure to follicular fluid [18]. Similarly, individual human sperm exposed to progesterone (the active component of follicular fluid) have a near instantaneous influx of calcium (within 2–5 sec) that leads to the acrosome reaction [19]. In response to solubilized zona proteins, individual hamster sperm also exhibit a rapid rise in intracellular calcium and an acrosome reaction beginning approximately 20 sec later [20].
The progression of the acrosome reaction as detected with Alexa-SBTI suggests that membrane vesciculation began along the apical ridge of sperm bound to the zona. In more than 90% of sperm, faint labeling was first detected at the forward-most ridge of the sperm and then was propagated gradually over the entire acrosomal cap. Maximum label intensity over the acrosome required approximately 30 sec to fully develop. Confocal microscopy revealed a similar progression of these events as seen in Figure 5, and indicated that multiple fusions between membranes began along the apical ridge of the sperm. Similar patterns of membrane fusion have been observed in rabbit sperm [21], but in goats and rams, fusions appear to begin along the equatorial region of sperm [22, 23]. In human sperm treated with progesterone, a wave of calcium appears to originate at the equatorial segment [19]. On the other hand, ultrastructural analysis of human sperm induced to acrosome react with follicular fluid shows point fusions between the plasma membrane and outer acrosomal membrane that are seen initially at the most anterior region of the head [18]. In all these cases, the stimulus for the acrosome reaction was not the ZP, and it is arguable that membrane vesciculation could progress differently when sperm are exposed to these surrogate inducers. Because macaque sperm initially make zona contact with the anteriormost portion of the head [24], it is reasonable to believe that signaling, followed by membrane fusion, would begin in this region.
Changes in the waveform of sperm flagella following ZP binding as observed in the present study have been observed in other species and such changes may play a role in penetration of the ZP. Capacitated sperm of hamsters and guinea pigs have typical hyperactivated motility while swimming freely, and upon attachment to the ZP, they propagate flagellar waves that are symmetrical, mostly planar, and of similar amplitude and frequency to those of free-swimming sperm [16]. These sperm were not observed to penetrate the ZP, and such patterns are probably associated with loose attachment only [15, 25]. Following the onset of zona penetration, hamster sperm exhibit bimodal motility, with high-amplitude, low-frequency lever strokes alternating with low-amplitude, high-frequency sinusoidal waves. The asymmetric lever mode and the resulting oscillatory motions of the leading edge of the sperm head persisted during the penetration of the ZP to the perivitelline space [17]. These changes in flagellar movement may be a result of the acrosome reaction. Significant changes in the motility of free-swimming, capacitated hamster sperm have been observed following the acrosome reaction. The flagella of reacted sperm beat with less symmetry and lower frequency, and they bend into more acute curves than those of unreacted sperm [26]. Calculations of forces generated by flagella suggest that the lever mode affords significant mechanical advantages to sperm for forcing their way through the zona matrix [17]. The importance of the high-amplitude flagellar waves for ZP penetration also has been directly demonstrated. When hyperactivated motility but not capacitation is suppressed, hamster sperm cannot penetrate the ZP even though they bind and acrosome react with a frequency equal to fully hyperactivated sperm [27].
The increase in flagellar amplitude of macaque sperm on the ZP also may generate additional force to facilitate zona penetration, but in the present experiments sperm were not observed to penetrate the dehydrated/reconstituted zonae. The time required for penetration of ovulated oocytes by hamster, guinea pig, mouse, and human sperm has been observed directly and timed at 15, 20, 5, and 12 min, respectively [1]. It will be necessary to observe macaque sperm binding to the ZP of oocytes that have the ability to be fertilized [7] before we can evaluate the relationship between sperm motility changes and zona penetration capability.
The assays developed in the present study for assessment of sperm-zona binding events were applied to investigate the antifertility effects of LSA, which was previously shown to block in vitro fertilization of macaque oocytes [7]. LSA is a member of a family of related lignin-derived macromolecule (LDM) byproducts that are formed as a result of the conversion of wood pulp into paper and are generally highly sulfonated. Our previous studies on an LDM related to LSA have shown that it inhibits the sperm acrosome reaction in sea urchins [28], and we have preliminary evidence that LSA competes with the natural sulfated ligand, egg jelly, on the sperm surface [29]. Furthermore, we have observed that LSA binds to the head of capacitated macaque sperm in a location consistent with its biological activity [7]. Due to its antifertility effects combined with its lack of cytotoxicity [30–32], LSA is a strong candidate for development as a vaginal contraceptive.
In the present study we followed individual macaque sperm through a series of interactions with the zona to evaluate the specific effects of LSA on macaque gamete interaction. Previous studies had indicated that the antifertility action of LSA was at the stage of sperm-zona interaction, but the precise effect was not determined [7]. In the present study we counted the number of sperm that bound to the ZP over 3 min and determined the percentage of those sperm that developed tight binding in the same time period. We found that when sperm were exposed to high concentrations of LSA (1 mg/ml), there was inhibition of loose attachment to the ZP, and tight binding of attached sperm was nearly blocked. The interval from loose attachment to tight binding was more than three times as long for LSA-treated sperm as it was for controls. The few LSA-treated sperm that were able to bind tightly to the ZP underwent the acrosome reaction within an interval that was indistinguishable from control sperm. Based on these data, the major effect of LSA appears to be on sperm recognition of the ZP and on the transition from loose attachment to tight binding, suggesting that sulfated macromolecules on the surface of the zona are important in initial attachment of sperm.
A model is emerging that describes primary binding of sperm as requiring multiple receptors of varying affinities for the ZP. Evidence for this model has been reported in studies of a wide range of mammalian species. Two distinct plasma membrane proteins (35 and 46 kDa) in boar sperm recognize ZP fragments with high affinity, and both proteins become tyrosine phosphorylated during capacitation [33]. Dissociation of iodinated ZP3, the zona ligand for sperm recognition [34], from fixed mouse sperm plasma membranes was biphasic, suggesting both high- and low-affinity binding sites on sperm [35]. Human sperm membrane proteins in four molecular weight groups bind primarily to ZP3 (95, 63, 51, and 14–18 kDa) [36]. Furthermore, a number of low-affinity interactions between sperm lectin-like receptors and the ZP have been demonstrated. In these studies, free monosaccharides, naturally occurring polysaccharides, and synthetic oligosaccharides were shown to inhibit sperm-ZP binding, the zona-induced acrosome reaction, or both, but these molecules were not capable of inducing the acrosome reaction [37].
It has been proposed that certain receptors, potentially in a complex, may serve to stabilize or "dock" sperm on the ZP surface until sufficient numbers of receptors responsible for signaling can be aggregated [38]. Our interpretation of the effects of LSA on macaque sperm is consistent with this model. The ability of LSA to inhibit attachment and delay tight binding but not affect the acrosome reaction suggests that these functions are mediated through separate elements. LSA may selectively inhibit receptors responsible for the initial recognition of and attachment to the ZP (docking). The one or more receptors responsible for signal transduction leading to the acrosome reaction, on the other hand, do not appear to be blocked or at least not irreversibly blocked.
In conclusion, we have developed methods to assess sperm-zona interaction of macaques that allow us to observe in real time the transition from loose attachment, to tight binding, to the acrosome reaction. We envision that this system will enable us to evaluate the specific functions of sperm receptor proteins for the ZP. In particular, we should be able to dissect various receptor functions that mediate sperm-zona recognition, various binding events, and signal transduction.
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FOOTNOTES |
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2 Correspondence: James W. Overstreet, Center for Health and the Environment, One Shields Avenue, University of California, Davis, CA 95616. FAX: 530 752 5300; jwoverstreet{at}ucdavis.edu
Received: 10 July 2002.
First decision: 1 August 2002.
Accepted: 11 September 2002.
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